Maxillary Sinus Augmentation - a histological, clinical,radiographic and patient centred assessment

Author

Alayan, Jamil

Published

2018

Thesis Type

Thesis (PhD Doctorate)

School

School of Dentistry&Oral Hlth

DOI

https://doi.org/10.25904/1912/3162

Copyright Statement

The author owns the copyright in this thesis, unless stated otherwise.

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http://hdl.handle.net/10072/381656

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Maxillary Sinus Augmentation – a

histological, clinical, radiographic

and patient centred assessment

Jamil Alayan

BSc, BDSc, MDSc (perio), FRACDS (perio), FICD

School of Dentistry & Oral Health

Faculty of Health

Griffith University

Submitted in fulfilment of the requirements of the degree of

Doctor of Philosophy

11-May-2018

i

ABSTRACT

Maxillary sinus pneumatization is a frequently encountered problem in the dental

implant rehabilitation of the posterior maxilla. Maxillary sinus augmentation

(MSA) using a lateral wall approach is a well-established and commonly utilized

surgical technique for overcoming this bone deficiency and allowing implant

placement. MSA is still in the refinement process with a large degree of variation

in all aspects of the technique including; the surgical protocol, the anatomical site,

the choice of material, the site of autogenous bone harvesting, the timing of implant

surgery and the use of barrier membranes. Generally, however, prospective

controlled clinical trials critically assessing these domains remain rare, especially

studies applying well defined success criteria for implant supported restorations

placed in sites of MSA.

The maxillary sinus grafting procedure is invasive and surgically demanding, but

appears to have limited interference with maxillary sinus physiology when

performed well. In addition, reported surgical complications are generally well

tolerated and followed by normal recovery in the vast majority of cases. Most of

this data however is derived from medium to low level evidence (clinical case

series, retrospective analyses). Patients undergoing this procedure also expect to be

counselled about their expectations regarding pain and the impact of this procedure

on their daily life in the post-operative period. Such information is not available.

There is also limited long term outcome data on implants placed in MSA.

Historically, autogenous bone grafts have been considered the gold standard due to

their inherent osteoinductivity. Their significant limitations in MSA however has

driven intense research into various bone substitutes. Anorganic bovine bone

mineral (ABBM) is a very well documented xenograft in MSA when used alone or

as a composite graft with autogenous bone (AB) (ABBM + AB). More recently,

collagen stabilized ABBM using 10% porcine type-1 collagen matrix has also

become available for use (ABBM-C) (Bio-Oss Collagen®). This formulation may

have clinical utility in its use as a sole biomaterial in MSA especially in situations

ii

of Schneiderian membrane perforation. There are however, no published studies on

this grafting material in MSA.

Whilst material selection influences the histological appearance and characteristics

of the regenerated bone, other factors can also play a role including defect anatomy

and implant surface. Chemical modification of implant surfaces has resulted in a

surface with increased surface energy and hydrophilicity (SLActive®). These

surfaces have demonstrated enhanced bone apposition during the early stages of

osseointegration and improved bone regeneration in bone defects when compared

to traditional microrough implant surfaces (SLA®). To date however, very few

studies have compared these two implant surfaces in MSA and none at the early

stages of osseointegration.

As such, this thesis aims to assess the histological, clinical (surgical), volumetric

and patient centered outcomes of ABBM-C in MSA when compared to ABBM +

AB. In addition, the influence of implant surface microtopography on early

osseointegration in MSA regenerated sites was assessed. This is continued further

to assess the outcomes of implant supported restorations placed into MSA sites

using these two biomaterials and well defined success criteria including survival,

biological and technical outcomes.

In the first part of the thesis, qualitative and quantitative histological assessment

was used to compare ABBM-C with ABBM + AB in both pre-clinical and clinical

MSA models. In the first experimental chapter (Chapter 2), a randomized

controlled trial utilized histomorphometric assessment in a pre-clinical model

(ovine) to show that both healing time and proximity to the resident sinus walls had

a positive impact on histomorphometric outcomes. Both biomaterials exhibited

very similar histomorphometric parameters in the proximal zones but the presence

of AB seemed advantageous in regions distant from resident sinus wall. In chapter

3, the histomorphometric assessment was performed in a prospective controlled

clinical trial. Both biomaterials exhibited very similar histomorphometric

parameters but the ABBM+AB group exhibited a more mature graft with a greater

proportion of lamellar bone. Based on these histological assessments, ABBM-C

appeared to be a suitable bone substitute for the purposes of MSA. In chapter 4,

iii

the influence of implant surface microtopography on early osseointegration in MSA

was assessed in a pre-clinical (ovine) model by placing implants with a hydrophilic

and hydrophobic surface in sites previously receiving the MSA procedure. Both

time and the use of a hydrophilic implant surface had a positive impact on %BIC

around implants placed into augmented maxillary sinuses. Hydrophilic implant

surfaces also had a positive impact on the surrounding tissue composition.

The second part of this thesis explores the clinical, radiographic and patient centred

outcomes of MSA using these two biomaterials, as well as the survival and success

of the implant supported restorations placed into these sites. In chapter 5, a

prospective clinical trial compared both groups of biomaterials in MSA with

material allocation based on specific clinical presentations (sinus membrane

perforation / local AB bone availability). It indicated that MSA using the lateral

wall technique is safe and associated with mild to moderate pain and restrictions to

daily activities for 48-72hrs. Patient reporting of morbidity was greater with AB

harvesting. Volumetric analysis using 3-D imaging showed that C-ABBM provides

comparable bone volume to AB + ABBM that is sufficient for placement of

implants of adequate size with no need for further vertical augmentation. Engaging

the surrounding sinus walls had a significant positive impact on graft volume. In

chapter 6, the same patient population was followed to assess implant supported

restorations after 12 months of function using well defined success criteria. Both

groups revealed high implant survival rates. Marginal bone levels & peri-implant

parameters were consistent with health in both groups. The majority of the

restorations were screw retained single crowns or small fixed partial dentures. The

incidence of mucositis was dependent on the definition threshold. Absence of peri-

implantitis and low rates of technical complications were reported in both groups.

Within the limitation of this trial, it can be concluded that collagen stabilized

ABBM can be successfully used alone for maxillary sinus augmentation and

subsequent implant rehabilitation. Its clinical utility is most relevant in cases of

sinus membrane perforation and insufficient autogenous bone in the local area.

iv

STATEMENT OF ORIGINALITY

I, Jamil Alayan declare that this work has not previously been submitted for a

degree or diploma in any university. To the best of my knowledge and belief, the

thesis contains no material previously published or written by another person

except where due reference is made in the thesis itself.

Jamil Alayan

04th May 2018

v

Table of Contents

ABSTRACT .............................................................................................................i

STATEMENT OF ORIGINALITY ............................................................................. iv

LIST OF APPENDICES .......................................................................................... vii

ACKNOWLEDGEMENTS ..................................................................................... viii

RESEARCH OUTPUTS ARISING FROM THESIS ...................................................... ix

STATEMENT OF ETHICS APPROVAL ...................................................................... x

STATEMENT OF INCLUSION OF ONLY CO-AUTHORED PAPERS ............................ xi

CHAPTER 1 – INTRODUCTION ..............................................................................1

BACKGROUND & SURGICAL TECHNIQUE ................................................................... 2

PRE-OPERATIVE ASSESSMENT ................................................................................... 4

Relevant Anatomical Features ............................................................................................ 5 Relevant Sinus Physiology ................................................................................................... 7 Common Pathological Conditions ..................................................................................... 10 Maxillary Sinus Pathology & Patient Assessment ............................................................... 11

POST-OPERATIVE CONSIDERATIONS ........................................................................11

Impact of MSA on Sinus Physiology ................................................................................... 11 Surgical & Post-operative Complications ........................................................................... 13

BONE GRAFTS & MSA ...............................................................................................16 Biomaterials – types and characteristics ........................................................................... 17 Bone Graft Healing & MSA ................................................................................................ 20 Characteristics of MSA Sites .............................................................................................. 25

DENTAL IMPLANTS & SUPPORTED RESTORATIONS IN MSA .....................................30

Dental implant surface microtopography .......................................................................... 30 Outcomes of implant supported restorations in MSA ........................................................ 33

RESEARCH HYPOTHESIS ............................................................................................38

AIMS.........................................................................................................................39

REFERENCES .............................................................................................................41

PART 1 – HISTOLOGICAL & HISTOMORPHOMETIRC ASSESSMENT OF USING

COLLAGEN STABILIZED ABBM IN MSA ...............................................................66

vi

Chapter 2 - A histomorphometric assessment of collagen stabilized anorganic

bovine bone mineral in maxillary sinus augmentation – a randomized controlled

trial in sheep. ...........................................................................................................67

Chapter 3 - A histomorphometric assessment of collagen stabilized anorganic

bovine bone mineral in maxillary sinus augmentation – a prospective clinical trial. 79

Chapter 4: Comparison of early osseointegration of SLA® and SLActive® implants in

maxillary sinus augmentation: a pilot study. ............................................................90

PART 2 – CLINICAL & RADIOGRAPHIC ASSESSMENT OF COLLAGEN STABILIZED

ABBM IN MSA .................................................................................................. 101

Chapter 5: A prospective controlled trial comparing xenograft/autogenous bone

and collagen-stabilized xenograft for maxillary sinus augmentation – Complications,

patient reported outcomes and volumetric analysis. .............................................102

Chapter 6: A prospective controlled trial comparing xenograft/autogenous bone

and collagen-stabilized xenograft for maxillary sinus augmentation – biological and

technical implant outcomes. ..................................................................................119

CHAPTER 7 – DISCUSSION & FUTURE DIRECTIONS........................................... 156

APPENDIX A ..................................................................................................... 177

vii

LIST OF APPENDICES

Appendix A: Ethics approval certificates

1. Human Ethics Approval - Dental Sciences Ethics Committee, School of

Dentistry, University of Queensland, Australia.

2. Animal Ethics approval certificate – University Animal Research

Committee, Queensland University of Technology, Australia.

3. Animal Ethics Committee – Griffith University, Australia.

viii

ACKNOWLEDGEMENTS

This doctoral thesis was carried out under the primary supervision of Prof. Saso

Ivanovski. I would like to sincerely thank Saso for the opportunity to work under

his guidance, his supervision and within his research team. It has been a wonderful

learning experience.

I would also like to acknowledge the following members of the research team for

their invaluable contribution and assistance throughout the research project:

• Dr Cedryck Vaquette for his assistance with the histological components of

this project.

• Dr Siamak Saifzadeh for his assistance with the animal model and surgery.

• Dr Dimitrios Vagenas for his assistance with the statistical analysis of this

project.

• Dr Raahib Dudhia for his assistance with the radiological component of this

study.

Furthermore, I would also like to acknowledge the love, support and sacrifice of

my family throughout this long journey.

ix

RESEARCH OUTPUTS ARISING FROM THESIS

Published Refereed Journal Articles

1. Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S. (2016) A

histomorphometric assessment of collagen stabilized anorganic bovine bone

mineral in maxillary sinus augmentation – a randomized controlled trial in

sheep. Clinical Oral Implants Research 27: 734 – 743. doi: 10.1111/clr.12652.

2. Alayan J, Vaquette C, Farah C, Ivanovski S. (2016) A histomorphometric

assessment of collagen stabilized anorganic bovine bone mineral in maxillary

sinus augmentation – a prospective clinical trial. Clinical Oral Implants

Research 27: 850 – 858. doi: 10.1111/clr.12694.

3. Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S. (2017)

Comparison of early osseointegration of SLA® and SLActive® implants in

maxillary sinus augmentation: a pilot study. Clinical Oral Implants Research

28: 1325 – 1333. doi: 10.1111/clr.12988.

4. Alayan J, Ivanovski S. (2018) A prospective controlled trial comparing

xenograft/autogenous bone and collagen-stabilized xenograft for maxillary

sinus augmentation – Complications, patient reported outcomes and volumetric

analysis. Clinical Oral Implants Research 29: 248 – 262. doi:

10.1111/clr.13107.

Articles In Final Preparations

1. Alayan J, Ivanovski S. (2018) A prospective controlled trial comparing

xenograft/autogenous bone and collagen-stabilized xenograft for maxillary

sinus augmentation – biological and technical implant outcomes. Clinical Oral

Implants Research (submitted).

x

STATEMENT OF ETHICS APPROVAL

I confirm that ethical clearance for this research was granted by the following

organisations (Appendix A):

1. Human Ethics Approval - Dental Sciences Ethics Committee, School of

Dentistry, University of Queensland, Australia (Project number: 1010).

2. Animal Ethics approval certificate – University Animal Research Committee,

Queensland University of Technology, Australia (QDPI Scientific Use

Registration Number 0052. Approval number: 1100001051).

3. Animal Ethics Committee – Griffith University, Australia (GU Ref No:

DOH/02/13/AEC).

I can confirm that research was conducted in accordance with the approved

protocols.

Jamil Alayan

04th May 2018

xi

STATEMENT OF INCLUSION OF ONLY CO-AUTHORED PAPERS

Included in this thesis are papers in Chapters 2, 3, 4, 5 and 6 which are co-authored

with other researchers. My contribution to each co-authored paper is outlined at the

front of the relevant chapter. The bibliographic details for these papers including

all authors, are:

Chapter 2: Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S. (2016)

A histomorphometric assessment of collagen stabilized anorganic bovine bone

mineral in maxillary sinus augmentation – a randomized controlled trial in sheep.

Clinical Oral Implants Research 27: 734 – 743. doi: 10.1111/clr.12652. Copyright

status: permission to reproduce granted. License number: 4342510031510.

Chapter 3: Alayan J, Vaquette C, Farah C, Ivanovski S. (2016) A

histomorphometric assessment of collagen stabilized anorganic bovine bone

mineral in maxillary sinus augmentation – a prospective clinical trial. Clinical Oral

Implants Research 27: 850 – 858. doi: 10.1111/clr.12694. Copyright status:

permission to reproduce granted. License number: 4342510448961.

Chapter 4: Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S. (2017)

Comparison of early osseointegration of SLA® and SLActive® implants in

maxillary sinus augmentation: a pilot study. Clinical Oral Implants Research 28:

1325 – 1333. doi: 10.1111/clr.12988. Copyright status: permission to reproduce

granted. License number: 4342510697065.

Chapter 5: Alayan J, Ivanovski S. (2018) A prospective controlled trial comparing

xenograft/autogenous bone and collagen-stabilized xenograft for maxillary sinus

augmentation – Complications, patient reported outcomes and volumetric analysis.

Clinical Oral Implants Research 29: 248 – 262. doi: 10.1111/clr.13107. Copyright

status: permission to reproduce granted. License number: 4342510860031.

Chapter 6: Alayan J, Ivanovski S. (2018) A prospective controlled trial comparing

xenograft/autogenous bone and collagen-stabilized xenograft for maxillary sinus

xii

augmentation – biological and technical implant outcomes. Clinical Oral Implants

Research (submitted).

Appropriate acknowledgements of those who contributed to the research but did

not qualify as authors are included in each paper.

Jamil Alayan

04th May 2018

(Countersigned) ___________________________ (Date) 16th May 2018

Supervisor: Stephen Hamlet

CHAPTER 1 – INTRODUCTION

2

BACKGROUND & SURGICAL TECHNIQUE

The rehabilitation of partial and fully edentulous patient with dental implants is

considered to be a predictable therapeutic modality with favourable long term

functional results (Buser, et al. 2012, Jung, et al. 2012, Pjetursson, et al. 2012).

Reduced alveolar bone height and bone density however, are common limitations

when considering implant rehabilitation of the posterior maxilla (Jemt & Lekholm

1995). Bone deficiency in this anatomical region is caused by post-extraction ridge

resorption and maxillary sinus pneumatization (Farina, et al. 2011). Maxillary sinus

floor elevation (MSA) using a trans-alveolar or a lateral window approach (Boyne

& James 1980, Summers 1994) are the most commonly utilized procedures to

overcome maxillary sinus pneumatization. MSA using the lateral window approach

has been shown to support predictable survival of implants placed into the

augmented bone (Bornstein, et al. 2008, Del Fabbro, et al. 2008, Pjetursson, et al.

2008, Nkenke & Stelzle 2009).

The lateral window or modified Caldwell-Luc approach has its roots in the 19th

century. George Caldwell in 1893; Scanes Spicer in 1894 and Henri Luc in 1897,

independently advocated the creation of an anterior maxillary antrostomy through

the canine fossa with nasal drainage to cure maxillary sinusitis. This became known

as the Caldwell-Luc procedure (Macbeth 1971). This procedure involved a buccal

vestibular incision form the region of the canine eminence to the first molar

(DeFreitas & Lucente 1988). The elevated mucoperiosteal flap exposed the lateral

maxillary wall and the inferior orbital nerve, which can be identified and protected.

The anterior antrostomy is placed above the roots of the maxillary teeth or it may

be created more posteriorly depending on the pathological condition. Sinus mucosa

was then removed and the amount of this removal was dictated by the extent of

disease, with healthy mucosa preserved. After the antrum is adequately cleared, an

inferior meatal antrostomy is created and the buccal flap is closed.

Philip Byone used the Caldwell-Luc technique to augment the maxillary sinus floor

with corticocancellous bone graft harvested from the iliac crest (Boyne & James

1980) After 3 months, Boyne carried out an alveolar osteoplasty of the posterior

maxilla without risk of penetrating the maxillary sinus and created the necessary

3

interarch distance in patients needing full removable dentures with advanced sinus

pneumatisation.

Subsequently, the same surgical procedure was utilised in patients with advanced

sinus pneumatisation for the purposes of placing a dental implant. Both Boyne

(Boyne & James 1980) and Tatum (Tatum 1986) used a modified Caldwell-Luc

approach to the antrum where a buccal antrostomy was created in the lateral wall

of the maxilla and the Schneiderian membrane was carefully elevated. Autogenous

particulate bone graft was subsequently placed in the sub-antral space after it was

harvested from the iliac crest. The dental implants were placed some months later

and the prosthetic reconstructions comprised of fixed and removable prostheses.

In the same period, Tatum also further developed and improved the surgical

technique. Tatum’s modification of the technique involved the creation of a trap

door from the buccal antrostomy, which was pushed into the antrum to create a new

roof. Implants were also placed simultaneously at the time of grafting. (Tatum

1977, Tatum 1986)

Since these early reports, treatment options have developed further for the

management of the deficient posterior maxilla with the advent of MSA via the

transcrestal approach and short implants (<8mm). Both options are associated with

good outcomes (Del Fabbro, et al. 2004, Esposito, et al. 2014, Schincaglia, et al.

2015). However, both of these options require a residual ridge height typically

ranging from a minimum of 4 or 5mm up to 7mm (Markovic, et al. 2016, Pohl, et

al. 2017). In contrast, MSA using the lateral window approach can achieve adequate

grafted ridge height even in cases with complete loss of an intact maxillary sinus

floor, (Cortes, et al. 2015). Furthermore, the MSA using the lateral approach is

necessary if the required gain in bone height is greater than 3-4mm in order to

accommodate the desired implant dimension (Zitzmann & Scharer 1998). A recent

systematic review comparing these options for the rehabilitation of the deficient

posterior maxilla reiterated that the three options are all viable but not strictly

equivalent, and treatment decision needs to be based on the particular features of

each individual (Corbella, et al. 2015).

4

Whilst the original Caldwell-Luc technique is based on solid principles, this

surgical technique is still being refined, with a large degree of variation in many

aspects. Some of these include the location (Tatum 1986) and method of creating

buccal antrostomy (Galindo-Moreno, et al. 2007), the use of various bone grafting

materials (Schmitt, et al. 2013), and the timing of both implant placement (Peleg,

et al. 1999).

If the initial bone height allows primary implant stability, simultaneous implant

placement can be performed (Jensen & Terheyden 2009). Several case series have

presented favourable results for simultaneous implant placement (Cordioli, et al.

2001, Mangano, et al. 2007). If primary stability cannot be achieved, then the sinus

floor should be elevated in a separate procedure followed by delayed implant

placement. A mean ridge height of 4.4mm was reported in studies using

simultaneous implant placement and 2.9mm for staged placement (Jensen &

Terheyden 2009).

The position of the antrostomy and the technique for its creation are determined by

the size and location of the maxillary sinus and anatomical features such as

vasculature, septa, lateral sinus wall thickness and internal sinus dimensions. While

rotary instruments are commonly used for window preparation the recent

development of piezoelectric surgery has specifically been adapted for sinus

surgery (Vercellotti, et al. 2001). This technology may contribute to reducing intra-

operative complications such as membrane perforation and haemorrhage (Wallace,

et al. 2007). However, a small scale clinical study with a randomised controlled

design failed to confirm this difference (Barone, et al. 2008) and further trials are

warranted on the merits of using piezoelectric surgery compared with rotary

instruments.

PRE-OPERATIVE ASSESSMENT

Post-surgical complications tend to be associated with pre-existing sinus disease or

documented susceptibility to sinus disease (Raghoebar, et al. 1999, Timmenga, et

al. 2003). Therefore, proper pre-operative evaluation of the sinus health is expected

to minimise postoperative adverse events. Sinus health assessment in the dental

setting is performed through a detailed history and a radiographic examination and

5

relies on a sound understanding of the relevant anatomy, physiology and pathology

of the region.

Relevant Anatomical Features

Osteomeatal Complex and Related Structures

The anterior osteomeatal complex (OMC) (Figure 1) is the key region to the

drainage of the anterior sinuses (frontal, anterior ethmoid and maxillary) and the

sphenoethmoidal recess drains the posterior sinuses (posterior ethmoid and

sphenoid) and is also called the posterior OMC (Naumann 1965). The anterior

OMC comprises the maxillary sinus ostia and ethmoid infundibulum, hiatus

semilunaris, middle meatus, anterior ethmoidal air cells and frontal recess (Beale,

et al. 2009).

The maxillary ostium is located in the superior-medial aspect of the maxillary sinus

(Schatz & Becker 1984). The ostium leads into the second passage, the ethmoid

infundibulum, which is a funnel shaped passage that conducts mucous from the

maxillary sinus into the middle meatus via the third passage, the hiatus semilunaris

(Roithmann, et al. 1992). A fourth passage, the frontal recess is a part of the anterior

ethmoid complex and drains the frontal sinus (Rao & El-Noueam 1994) also into

the middle meatus (Oliverio, et al. 1995). The middle meatus drains into the back

of the nasal cavity and into the nasopharynx where mucous is ultimately swallowed.

From a functional standpoint the infundibular space represents the confluence of

drainage from the frontal, anterior ethmoid and maxillary sinuses. Anatomic

abnormalities or disease states affecting any of these neighbouring structures could

obstruct this space and cause sinusitis in any or all of the adjacent structures

(Kennedy, et al. 1985).

6

Figure 1 OMC - radiographic (CT) features (from Sarna A et al. 2002)

7

Relevant Sinus Physiology

Sinus health in any patient depends on three interrelated factors (Senior & Kennedy

1996):

1. Secretion of mucous with normal viscosity, volume and composition;

2. Normal mucociliary flow to prevent mucous stasis and subsequent infection

3. Open sinus ostia to allow adequate drainage and ventilation.

In the rhino-sinus-pharyngeal district, there are three important sites from anterior

to posterior: the osteomeatal complex (OMC), the sphenoethmoidal recess and the

rhinopahrynx. The correct ventilation and effective mucociliary clearance (MCC)

of these three patho-physiologic regions maintains the healthy physiology of the

whole respiratory system (Varricchio, et al. 2010).

Schneiderian Membrane

The maxillary sinus is internally lined with a thin mucosa of ciliated respiratory

epithelium, also known as the Schneiderian membrane, that is continuous with that

of the nose (McGowan, et al. 1993) and other paranasal sinuses through their ostia.

The mucosa of the paranasal sinuses is a mucoperiosteum (Figure 2) consisting of

a layer of pseudostratified columnar ciliated epithelium with mucous secreting

goblet cells. This overlies a thin lamina propria of loose connective tissue and

elastic fibres that is continuous with the periosteum. All cells are attached on the

basal membrane. Basal cells lie on the membrane and show no contact with the

epithelial surface and utilise desmosomes for cell adhesion (Gudis & Cohen 2010).

Mucociliary Clearance (MCC)

MCC is an essential homeostatic mechanism of the respiratory system and is

designed to remove both healthy and pathological secretions from the airway. MCC

in the sinuses was demonstrated in a pre-clinical model after the removal of mucosa

from the frontal sinuses resulted in significant interference with mucociliary

transport (Hilding 1932). It was also demonstrated that each of the large sinus

cavities has distinct mucosal flow patterns (Messerklinger 1966). These

observations remain valid today. It has since been demonstrated that mucosal flow

is accomplished through the coordinated beating of cilia in a specific direction

8

resulting in organised ciliary waves propelling particulate matter out of the cavity.

The mucociliary flow from the anterior sinuses converges at the OMC before

travelling posteriorly to the nasopharynx. Once the mucous layer is in the

nasopharynx, further mucociliary action and swallowing assists its ingestion.

Microscopic cilia dysfunction at any step of this intricate transport system can result

in significant and clinically evident sinonasal pathology. It is important to

emphasize that the upper airway paranasal sinuses are almost completely dependent

on the activity of cilia for removal of mucous, whereas in the lower airway deficient

MCC may be compensated for by coughing (Wagenmann & Naclerio 1992).

Figure 2. (A) Schematic representation of the components of the Schneiderian

membrane (B) Light microscope micrograph of histological section of the

Schneiderian membrane (From Sourji et al 2009)

9

Inter-sinus Connections

Paranasal sinuses are interconnected via accessory ostia and many sinuses do not

drain directly into the nasal cavity but rather indirectly through adjacent sinuses. A

research group indicated that 37% of maxillary sinuses drained exclusively into the

middle meatus via the nasal ostium, 19% drained via the nasal ostium and ethmoid

nasal route and 52% exclusively via the ethmoid nasal route (Prott 1971, Prott 1973)

(Vogt & Schrade 1979). Similarly, nasal ostia for ethmoidal cells were seen

draining directly into the middle meatus in 15% of patients, with 33% of patients,

drainage occurred via the maxillary sinus or a shared common drainage canal with

the maxillary sinus in 19% of individuals (Prott 1971, Prott 1973, Vogt & Schrade

1979).

Ostial Patency & Ventilation

It is generally accepted fact that free air communication between the nasal cavity

and its paranasal sinus via patent ostia is a pre-requisite for physiological conditions

within the sinuses (Mann, et al. 1977). Oxygen pressure within the maxillary sinus

is dependent on the functional ostial diameter. When the ostial diameter is less than

2mm, oxygen pressure falls (Aust & Drettner 1974). This reduction of the ostial

patency leads to alterations in pO2 and pCO2 resulting in early inflammation (Aust,

et al. 1994). Pressure recordings in humans indicate identical pressure exists within

the maxillary sinus cavity and ipsilateral nasal cavity during inspiration and

expiration (Proetz 1932, Jannert, et al. 1984). Also, sinuses with patent ostia

showed pressure curves synchronous with the respiratory cycle (Scharf, et al. 1995).

Microbiological Features

Knowledge of the bacterial flora of normal sinuses is mandatory to understand the

pathogenicity of species isolated in disease sinuses (Busaba, et al. 2004). Healthy

maxillary sinuses have been considered to be sterile (Su, et al. 1983). Nonetheless,

other papers have discovered bacteria in healthy sinuses (Brook 1981, Cook &

Haber 1987, Jiang, et al. 1999). Brook et al. reported for the first time the presence

of aerobic and anaerobic bacteria in normal maxillary sinuses (Brook 1981). Also,

Jiang et al detected culturable bacteria in 46.7% from swab specimens and 41.2%

10

from mucosal specimens in healthy individuals using a puncture and aspiration

method (Jiang, et al. 1999).

Studies on this topic vary significantly in many ways, including the techniques used

for sampling and culturing, and most importantly the definition of maxillary sinus

health. While some authors based their definition of normality on the absence of

clinical symptoms (Brook 1981), others used clinical presentation, history and

radiographic appearance (Cook & Haber 1987) or normal endoscopic appearance

(Jiang, et al. 1999).

A study in 2009 (Abou-Hamad, et al. 2009) used strict criteria for defining the

normal maxillary sinus: asymptomatic patients with normal CT scan and normal

endoscopic findings. Comprehensive exclusion criteria and parameters were used

to reduce confounding factors, and included controlled sampling methods, storage

media and transportation time. The majority of sinuses were found to be sterile

(82.1%). It should be noted that all of the species recovered from healthy sinuses

(17.8%) were aerobes and no anaerobes were isolated despite the use of appropriate

methods for anaerobic detection.

Further studies with the same strict inclusion criteria and well-controlled

parameters as the study above, reported similar results for the frontal sinus, with

85.72% found to be sterile (Albu & Florian 2013). On the other hand, bacterial

growth was reported in 94.3% of normal and un-diseased anterior ethmoid sinuses

(Kirtsreesakul, et al. 2008). Anaerobes however, were not found.

Common Pathological Conditions

The maxillary sinus may become involved with several types of diseases including

inflammatory, benign and malignant neoplasms (Diecidue, et al. 1999, Sciubba

1999). Most pathologic processes in the maxillary sinus are asymptomatic and often

found incidentally. Studies report very few cases of sinus pathology that are

absolute contra-indicate MSA (Pignataro, et al. 2008, Torretta, et al. 2013). In

contrast, more than a third of patients exhibit relative contra-indications that require

further management (medical or surgical) (Beaumont, et al. 2005, Torretta, et al.

11

2013). Most frequent conditions diagnosed include chronic sinusitis, cysts,

odontogenic sinusitis and foreign bodies.

Maxillary Sinus Pathology & Patient Assessment

A key issue is interpretation of anatomical variants or radiological signs of

pathology and relating this to clinical presentation or history provided. This

important step dictates whether the MSA procedure proceeds as planned or is

contraindicated and necessitates a referral to an ENT specialist.

A prospective study assessing patients for chronic rhino-sinusitis reported that

sensitivity of symptoms alone was high for the diagnosis of CRS at 88.75. But since

CRS symptoms are non-specific and associated with many other conditions, the

positive predictive value (PPV) was only 39.9% (Bhattacharyya & Lee 2010). The

addition of endoscopy significantly increased the PPV to 66%. Relying on the

presence of symptoms alone is likely to lead to diagnostic errors. Primarily

rhinosinusitis is a clinical diagnosis that is supported by endoscopic findings.

Reliance on endoscopy however for screening of all potential patients planning to

undergo MSA is likely to lead to unnecessary referrals to ENT specialists.

Radiography however is an accessible and familiar assessment tool in the dental

setting and will assist in carrying a risk assessment of prospective patients. A

prospective study assessed the accuracy of CT in the diagnosis of CRS has shown

them to exhibit good sensitivity and above average specificity especially when

extensive or widespread opacification is seen (Bhattacharyya & Fried 2003).

POST-OPERATIVE CONSIDERATIONS

Impact of MSA on Sinus Physiology

Whilst it has been suggested that Schneiderian membrane elevation may cause

interim inhibition of ciliary activity, clinical evidence suggests that maxillary sinus

augmentation has limited effects on sinus physiology (Timmenga, et al. 1997,

Timmenga, et al. 2003). A retrospective study (Timmenga, et al. 1997) examined

the impact of MSA on sinus function. The occurrence of postoperative chronic

sinusitis was limited to patients with a predisposition for this condition. This

highlights the concept of ‘sinus compliance’ which depends on the baseline

12

anatomical and physiological condition of the sinus (Torretta, et al. 2013). The

better the health of the sinus the lower the risk of complications due to its improved

ability to regain homeostasis after MSA.

In a prospective study (Timmenga, et al. 2003), 17 patients underwent endoscopic,

microbiological and morphological examination of their maxillary sinuses after

MSA. These evaluations were performed immediately preceding MSA, and then at

3 months (at implant insertion) and 9 months (at uncovering of implants) post-

operatively. Pre-operative screening was carried out to exclude those with

maxillary sinusitis. The 3 month microbiological evaluation showed a significant

increase in bacterial growth, while the 9-months results were comparable to the pre-

operative status. Morphologically, neither fibrosis nor thickening of the epithelium

and lamina propria was observed postoperatively. The number of goblet cells in the

epithelial layer was increased. This mild inflammatory reaction observed using

endoscopic evaluation should be interpreted as a normal physiological response of

the mucosal airway. MSA does not appear to have clinical consequences in patients

without signs of pre-existing sinusitis.

A similar conclusion was reached by Sul et al. (Sul, et al. 2008), after examining

the sinus mucosa under both light and electron microscopy post-sinus membrane

elevation. The implant was placed 5mm into the sinus cavity after sinus membrane

elevation and without a bone graft. The contralateral side was left untreated and

used as a control. There were no morphologic or ultrastructural changes in the sinus

membrane between groups. Furthermore, the cilia were all synchronously arrayed,

uniformly leaning in the same direction in both sinuses. This implies that the

insertion of dental implants into the maxillary sinus cavity after elevating the

membrane without adding any graft seems to be well tolerated. An important factor

to be considered in these studies is that the membrane was not perforated in any of

these samples.

Complete regeneration of the mucous membrane including cilia occurs within five

months after total surgical removal (Selden 1974). There is also agreement that the

sinus membrane will recover form sinusitis once proper ventilation is restored

(Stammberger 1986). This was also shown by Huang et al. (Huang, et al. 2006)

13

whereby endoscopic surgery in patients with chronic maxillary sinusitis resulted in

recovery of antral mucosa and MCC with improved ventilation and drainage. In

addition, cilia were significantly regenerated compared to the pre-operative state.

Alterations in voice quality secondary to nasal cavity and paranasal sinus surgery

have been reported (Chen & Metson 1997, Hosemann, et al. 1998). A study in 2003

(Tepper, et al. 2003) examined the effects of reduction in sinus volume on voice

quality in a specific group of patients who depend on the quality of their voice for

their livelihood (e.g. speakers, singers, actors). An exhaustive list of sound / voice

parameters were analysed with no changes detected in any of the evaluated

parameters. It was concluded that sinus grafting does not appear to jeopardise the

individuals voice pattern even when the sinus volume was reduced by as much as

22%.

Surgical & Post-operative Complications

Maxillary sinus grafting is a procedure with well documented clinical success and

founded on sound biological principles. The procedure however is not free of

complications (Chanavaz 1990, Regev, et al. 1995). It is technique sensitive and

dependent on careful patient selection.

A review carried out in 2009 reported that perforation of the Schneiderian

membrane was the most common intra-operative complication (Schwartz-Arad, et

al. 2004, Chiapasco, et al. 2009). Other intra-operative complications including

excessive bleeding, displacement of implant or grafting material into the sinus

cavity, and damage to the infra-orbital nerve were reported in only a few cases

(Chiapasco, et al. 2009). The most frequent post-operative complications was

infection and /or postoperative maxillary sinusitis. Partial or total bone graft loss

occurred in less than 1% of the patients (Chiapasco, et al. 2009).

Schneiderian Membrane Perforation

Perforation of the Schneiderian membrane is the most common intra-operative

complication of sinus elevation surgery (Chiapasco, et al. 2009). The reported

14

incidence in the literature varies from 7.5% (Cortes, et al. 2015) to 35% (Jensen, et

al. 1994) and 44% (Schwartz-Arad, et al. 2004).

Few studies have reported a negative impact of perforations on implant survival,

especially in cases of larger perforations (Proussaefs, et al. 2004, Hernandez-

Alfaro, et al. 2008). In most studies however, no significant impact were seen on

implant survival rates (Barone, et al. 2006, Zijderveld, et al. 2008) even for larger

perforations (Testori, et al. 2008). Furthermore, perforations have not been shown

to negatively affect the height of regenerated bone post-augmentation (Shlomi, et

al. 2004).

Reported causes of membrane perforations include the presence of a thin

membrane, presence of septa and adhesions or previous sinus surgery (Becker, et

al. 2008, Zijderveld, et al. 2008). The angulation between the medial and lateral

walls of the maxillary sinus seems to exert an influence on the incidence of

membrane perforation during membrane elevation. A study by Cho et al. (Cho, et

al. 2001) showed a correlation between the percentage of perforation and the

anatomy of the sinus. The patient population was divided into groups based on the

internal angle of the medial and lateral sinus walls. The perforation rates were

significantly higher in the group with narrow angles. These are typically found in

the second bicuspid region (Velloso, et al. 2006).

Since the presence of septa are also associated with an increased risk of membrane

perforation, a modification of the conventional surgical technique is required (Betts

& Miloro 1994). Boyne and James recommended their cutting and removal (Boyne

& James 1980), others subdivided the trapdoor into an anterior and posterior part

(Tidwell, et al. 1992), or followed the contour of the sinus floor by making a W-

shaped preparation at smaller septa (Zijderveld, et al. 2008).

Post-grafting sinusitis & infection

When using generally accepted ENT criteria for diagnosing sinusitis, the

development of post-sinus grafting chronic maxillary sinusitis has been reported to

occur in 4.4% (2/45) of MSA cases (Timmenga, et al. 1997). Previously, maxillary

sinusitis post-sinus membrane elevation was considered to be a major drawback,

15

but many of these studies were based on poorly defined criteria for sinus

examination (Doud Galli, et al. 2001).

The occurrence of post-grafting sinusitis appears to be associated with pre-existing

structural or mucosal features that compromise the sinus ability for adequate MCC

and ventilation after the surgical challenge or due to physical obstruction caused by

endo-antral displacement of particulate bone graft into the maxillary sinus proper

(van den Bergh, et al. 2000).

Barone et al. experienced post-grafting sinusitis in 10% of their study population

(7/70 patients) (Barone, et al. 2006). These cases were treated by drainage through

the bony window and systemic antibiotics. In a small fraction of these cases

endoscopic surgery was required where infection persisted despite conservative

treatment. Endoscopy in these patients confirmed a stenotic OMC. Other case

reports of post-grafting sinusitis have confirmed overfilling of the subantral space

in-conjunction with pre-existing structural risk factors, such as pneumatization of

the middle turbinate leading to narrowing of the middle meatus (Felisati, et al.

2008).

Other reports of post-grafting sinusitis report the cause to be endo-antral

displacement of bone graft particles leading to obstruction of OMC (Regev, et al.

1995, Wiltfang, et al. 2000, Doud Galli, et al. 2001). Many of these cases required

endoscopic surgery (middle meatal antrostomy) to resolve the symptoms after

systemic antibiotics and local measures failed to manage this condition. These case

reports have been supported by Timmenga et al. (Timmenga, et al. 2001), who

reported chronic purulent maxillary sinusitis post-grafting that was non-responsive

to conservative therapy including systemic antibiotics, nasal decongestant and

corticosteroids. Functional endoscopic surgery was required. The authors

recommended surgical intervention if post-grafting sinusitis is non-responsive to

conservative measures, in order to re-establish adequate drainage and remove the

obstructing particles that may be responsible for persistent symptoms.

16

Prompt and appropriate post-operative management is required in these cases. It

also highlights the importance of adequate screening of patients to identify those

with diminished sinus compliance and the prevention of endo-antral displacement.

BONE GRAFTS & MSA

The ideal grafting material should provide an osteoconductive matrix,

osteoinductive factors and be osteogenic with viable cells contained inside the bone

graft, capable of laying new bone matrix.

Osteoinduction is a healing process in which local growth factors cause

mesenchymal cells to disaggregate, migrate, re-aggregate, proliferate and later

differentiate into chondroblasts or osteoblasts (Urist 1965, Reddi, et al. 1987).

Osteoconduction is a process where the implanted material serves as a scaffold for

ingrowth of capillaries, peri-vascular tissue and osteoprogenitor cells from the

recipient bed (Burchardt 1983). The host tissue has a specific effect on these

materials that may vary depending on the site in which they are placed (Donath, et

al. 1992) such that bone formation is seen when a biomaterial is placed into host

bone, whereas its insertion into subcutaneous tissue results only in connective tissue

encapsulation.

Consequently, the gold standard for bone grafting procedures is traditionally

considered to be autogenous bone because of its combination of osteoconduction

and osteoinduction. Despite these benefits, considerable drawbacks, such as

additional surgical procedures, donor site morbidity (Clavero & Lundgren 2003,

Cricchio & Lundgren 2003), limited availability and unpredictable resorption

(Davis, et al. 1984, Clavero & Lundgren 2003, Wiltfang, et al. 2005, Sbordone, et

al. 2013), have necessitated the pursuit of alternatives. These include bone

allografts from human donors, xenografts and various alloplastic materials (Kao &

Scott 2007). Other strategies incorporate growth factors and cell based alternatives,

used either alone or in combination with other materials (Jabbarzadeh, et al. 2008).

17

Biomaterials – types and characteristics

The ideal bone substitute must be biocompatible and allow for proliferation of new

blood vessels and ultimately the growth of new bone into the augmented area. The

ideal bone substitute maintains this biological support during healing and is

gradually replaced by newly formed bone (Jensen, et al. 2006).

From a tissue engineering perspective, (Woodruff & Hutmacher 2010) the

following characteristics are desirable for scaffolds: a three-dimensional and highly

porous structures with an interconnected pore network for cell growth and transport

of nutrients and metabolic waste, a biocompatible and bioresorbable material with

controllable degradation and resorption rate to match cell/tissue growth, suitable

surface chemistry for cell attachment, proliferation and differentiation and

mechanical properties to match those of the tissues at the site of implantation.

Although much effort has been put into the search for the ideal bone substitute

material, such a material probably does not exist. The ideal grafting material for

MSA procedures is not necessarily ideal for onlay bone grafting. For many years,

autogenous bone from the iliac crest has been considered the gold standard in

reconstructive osseous surgery. However, its pronounced resorption, resulting in

loss of almost 50% of its original volume after 6 months of healing (Johansson, et

al. 2001), implies that it cannot be considered an ideal material for MSA.

Various substitutes have been used to fill the sub-antral space after sinus membrane

elevation, including allografts, xenografts, alloplasts and composites of various

materials (Wheeler, et al. 1996, Artzi, et al. 2001, Schlegel, et al. 2003, Wiltfang,

et al. 2003, Thorwarth, et al. 2005, Schlegel, et al. 2007, Artzi, et al. 2008). To date,

a variety of bone substitute materials have been clinically demonstrated to promote

acceptable outcomes (Jensen & Terheyden 2009, Nkenke & Stelzle 2009, Klijn, et

al. 2010, Jensen, et al. 2012). The search continues for the optimal bone substitute

for sinus floor augmentation.

Anorganic Bovine Bone Mineral (ABBM)

Bio-Oss® (Geistlich AG, Wollhusen, Switzerland) is a widely used preparation of

ABBM and is a bone substitute with osteoconductive properties and high

18

biocompatibility (Jensen, et al. 1996). It is one of the best documented biomaterials,

especially in MSA.

Bio-Oss® is a xenograft consisting of deproteinised, sterilized bovine bone with

75-80% porosity and a crystal size of approximately 10 microns in the form of

cortical granules. It has a natural, non-antigenic porous matrix that is chemically

and physically identical to the mineral phase of human bone, and it has been

reported to be highly osteoconductive (Jensen, et al. 1996, Berglundh & Lindhe

1997, Haas, et al. 2002, Orsini, et al. 2005). It is available in two particle sizes, 0.25

– 1.0 mm or 1.0 – 2.0 mm.

Bio-Oss® contains macroscopic and microscopic structures with an

interconnecting pore system that serves as a physical scaffold for osteogenesis

(Tapety, et al. 2004). The pores in Bio-Oss® are of optimal size and configuration

for vascular ingrowth, which is necessary for new bone formation (Daculsi &

Passuti 1990).

Jensen et al. (Jensen, et al. 1996) examined the osteoconductive and biocompatible

features of ABBM and three other bone substitutes in a pre-clinical model. Defects

filled with Bio-Oss® exhibited normal bone healing with initial woven bone

undergoing remodeling to form haversian systems. Bio-Oss® was completely

osseointegrated with intimate contact between the surfaces of the porous system of

ABBM and the newly formed bone.

Understanding the mechanism and the rate of resorption of the different

biomaterials is clinically relevant (Artzi, et al. 2001). It has been debated whether

ABBM is truly resorbable since remnants of Bio-Oss® particles have been reported

to be present many years after their insertion, including in MSA (Piattelli, et al.

1999, Schlegel, et al. 2003).

Some studies have reported signs of resorption of the Bio-Oss® particles

(Berglundh & Lindhe 1997, Hurzeler, et al. 1997, Hammerle, et al. 1998, Yildirim,

et al. 2000) whereas others have reported a lack of resorption (Valentini & Abensur

1997, Hallman, et al. 2002, Schlegel, et al. 2003). Osteoclasts have been described

19

to be present around Bio-Oss® particles (Hurzeler, et al. 1997, Stavropoulos, et al.

2004), while others could not identify this cell type (Artzi, et al. 2001). Some

authors believe that despite the absence of osteoclasts, the ingrowth of bone and the

progressive increase in relative bone volume over a period of time could indicate a

slow resorption of the xenograft material (Sartori, et al. 2003).

A study by Orsini et al. (Orsini, et al. 2007) analyzed Bio-Oss® particles at both a

histological and ultrastructural level from samples taken at 20 months and 7 years

post-sinus grafting in a single patient. In the 7-year specimen, the Bio-Oss®

particles seemed smaller compared with the 20-month specimen at the ultra-

structural level. In contrast, human mandibular ridge defects of varying size and

parameters were assessed histologically after grafting with ABBM (Schlegel &

Donath 1998). Biopsies were taken 6 years post-grafting. The particles exhibited

no change in size or shape over the 6 years. The surface of Bio-Oss® was smooth

with no signs of resorption, not even on surfaces that contained mononuclear

macrophages and multinuclear giant cells.

Jensen et al. (Jensen, et al. 2005) demonstrated osteoclast-like multinucleated giant

cells in close approximation to where osteoblasts formed new bone on the ABBM

surface. However, the characteristic osteoclast associated resorption lacunae were

absent and no quantitative reduction of graft volume could be demonstrated. It was

suggested that the observed multinucleated giant cells had a macrophage-like role

of cleaning the surface of the graft particles, rather than resorption. Mordenfeld et

al. (Mordenfeld, et al. 2010) also evaluated histologically the long-term tissue

response to ABBM and compared particles size after 6 months and 11 years in the

same 11 patients, to determine possible resorption. After 11 years, the ABBM

particles were well integrated and surrounded by lamellar bone with no obvious

sign of resorption. There were no statistically significant differences between the

size of the particles after 11 years compared with those measured after 6 months or

when compared with sterile particles. The area fraction of the Bio-Oss® particles

(17.3%) and the individual particle area (0.063 mm2) after 11 years were similar

with results from specimens retrieved in the same patients at 6 months (14.5% and

0.066 mm2).

20

Overall the evidence seems to indicate that ABBM behaves as a semi-permanent

grafting material with no evidence of significant resorption.

Bio-Oss Collagen® is comprised of ABBM granules with the addition of 10%

highly purified porcine type-1collagen. The collagen acts, according to the

manufacturer, as a cohesive for the granules (Araujo, et al. 2010). Histological

assessment of Bio-Oss Collagen® was carried out in extraction sockets of beagle

dogs at 1 and 3 days, as well as 1, 2 and 4 weeks (Araujo, et al. 2010). The amount

of bovine bone mineral remained unchanged in the socket during the entire 4-week

healing period, whereas the collagen portion was rapidly removed. It was

hypothesized by the authors that hydrolytic enzymes (collagenase) released from

activated PMN cells were involved in the rapid degradation of the porcine collagen

portion of the graft.

Bone Graft Healing & MSA

Irrespective of the model or defect, bone formation and healing require the

recruitment, migration, and differentiation of osteogenic cells into osteoblasts,

resulting in deposition of a collagenous extracellular matrix for mineralization. The

osteoblasts are derived from progenitor cells of the mesenchymal lineage (Bruder,

et al. 1994, Bianco & Robey 2001). Mesenchymal progenitor cells can originate

from various sources such as the bone marrow, the cambrium layer of the

periosteum, and from pericytes surrounding capillaries (Bruder, et al. 1994, Bianco

& Robey 2001). Differentiation of mesenchymal progenitor cells into osteoblasts is

a multistep process, which is stimulated by local growth factors (Reddi 1998,

Gerstenfeld, et al. 2003). Members of the bone morphogenetic protein family are

likely candidates to be involved in this process as they are expressed during bone

repair and have the potential to induce bone formation at ectopic sites.

Schenk described the parameters needed for successful osteogenesis (Schenk

1987):

• Sufficient blood supply: osteoblasts require a high partial oxygen tension to

produce bone matrix. When oxygen tension is low, cells produce cartilage

or fibrous tissue

21

• Mechanical stability: this contributes to formation of stable coagulation and

granulation tissue that is rich in blood vessels.

• Solid surface: osteoblasts can only deposit lamellar bone starting from a

solid stable surface.

• Size of the defect: when a defect is overly large it will not heal through

formation of complete rapid bone bridge between defect walls. It will

instead need several months to completely fill the defect.

• Competition between cells: soft tissue cells proliferate more rapidly than

osteoprogenitor cells and can fill the defect before formation of new bone.

Fibrous tissue formed faster than bone tissue.

Neo-osteogenesis and graft consolidation

After grafts are inserted into maxillary sinus, osteogenesis is activated by surgical

trauma which ultimately causes the release of a large quantity of growth factors

(TGF-β1, aFGF, bFGF, BMP-2, BMP-7) with osteogenic effects (Joyce, et al. 1990,

Bolander 1992, Bostrom, et al. 1995, Sakou 1998, Trippel 1998). It stands to reason

that the osteogenic stimuli released by the surgical trauma and exposure of resident

sinus bone walls allows for the induction and attachment of osteoblasts leading to

deposition of osteoid on the exposed solid walls, similar to early pre-clinical models

of guided bone regeneration (Schenk, et al. 1994, Hammerle, et al. 1995).

Boyne and James (1980) showed that elevation of the Schneiderian membrane

could induce bone formation directly from the sinus floor. There is evidence that

osteogenesis extends progressively towards the centre and apical areas of the graft

and may continue for months after surgery, starting from the sinus floor and lateral

walls and creating a gradient of graft consolidation, as demonstrated in pre-clinical

models (Margolin, et al. 1998, Haas, et al. 2002, Haas, et al. 2002, Scala, et al.

2010). GBR models have suggested that gradients of graft consolidation are

specific for each biomaterial, as reflected by the osteogenic response of the host

bone and the degradation profile of the bone substitutes (Busenlechner, et al. 2008).

In 2009, a pre-clinical study (Busenlechner, et al. 2009) was carried out to assess

graft consolidation in augmented sinuses. A sequential histomorphometric analysis

22

of samples from augmented sinuses using two materials with contrasting

degradation profiles – slow (Bio-Oss®) and rapid (Ostim®) – carried out at various

distances from the resident maxillary bone wall. New bone formation increased

with time and with proximity to the resident bony wall. Residual volume of Ostim®

but not of Bio-Oss® was strongly influenced by the distance from the host bone.

The process of graft consolidation is complex and involves the interaction of the

bone substitute with cells, extracellular matrix and signalling molecules provided

by the host (Khan, et al. 2005). The microenvironment of the maxilla can influence

the behaviour of bone substitutes and the microenvironment of bone substitutes can

influence the osteogenic response of the resident maxilla.

Role of Schneiderian membrane & coagulum in MSA

In-vitro studies have demonstrated that Schneiderian membrane cells can develop

an osteogenic phenotype when cultured in the presence of osteoinductive factors

(Gruber, et al. 2004) and could be induced to express the osteogenic markers

alkaline phosphatase, BMP-2, Oteopontin, Osteonectin and Osteoclacin, in addition

to exhibiting ectopic bone formation in-conjunction with an osteoconductive

scaffold (Srouji, et al. 2009). A pre-clinical (monkey) study placed protruded

implants into the sinus cavity by 5mm, after sinus membrane elevation via a lateral

window (Boyne 1993). One side received a bone graft and the contralateral side

was allowed to fill with a coagulum. Histomorphometric evaluation indicated that

implants placed without bone grafting resulted in bone formation around the first

1-3mm of the protruding implant only. Implants that received the bone graft,

exhibited greater bone coverage around the apical portion. This suggests that the

Schneiderian membrane alone was insufficient to fully support implant integration

in the sinus and the addition of a space maintaining grafting material results in

enhanced bone volume at the implant interface.

There have however been reports of clinically successful implant integration in the

sinus floor by simply elevating the maxillary sinus membrane and simultaneously

inserting dental implants without the use of adjunctive grafting material (Lundgren,

et al. 2004, Palma, et al. 2006). Ten patients were recruited for investigating the

clinical and radiologic results of elevating the Schneiderian membrane without

23

adding any grafting materials and simultaneous implants placement (Lundgren, et

al. 2004). Implants were placed such that a minimum of 5 mm protruded into the

sub-antral space. Comparison of pre- and post-operative CT scans (6 months later)

clearly demonstrated new bone formation within the sub-antral space created by

membrane elevation only. The new bone formed however did not cover the apex of

the implant rather it was only visible around the portion of the protruding implant

proximal to the native bone wall of the maxillary sinus floor. The above technique

was subsequently repeated in monkeys (Palma, et al. 2006) with one side receiving

no grafting material, whilst the contralateral side was augmented using autogenous

bone. Histologically, the apex was in direct contact with a normal Schneiderian

membrane unless there was a thin layer of intervening bone. Below that, membrane

collapse was seen into the underlying space, irrespective of the treatment group to

form a tent shaped figure. In the mid portion of the implants, sites treated with

membrane elevation only tended to exhibit more bone at the periphery in contact

with the Schneiderian membrane. This series of studies suggests that the surgical

stimuli of sinus membrane elevation and coagulum filling the sub-antral alone leads

to bone formation. This is limited to the sinus floor due to the lack of space

maintenance and instability of the coagulum leaving the apex of the implant

supporting the membrane. This tenting effect also maintains space in the immediate

vicinity of the implant that widens gradually in the coronal direction allowing

maturation of the regenerated bone in this region. The absence of grafting material

ensures this healing process is not impeded.

In 2010, a pre-clinical study (Scala, et al. 2010) reported on the early healing after

sinus membrane elevation and simultaneous implant placement. No bone

substitutes were used and a coagulum was allowed to fill the sub-antral space. After

10 days of healing, a higher amount of woven bone was noted in continuity with

the walls of the sinus that was often preceded by a dense connective tissue layer.

The newly formed bone became more mature and organised with primary osteons

and primitive bone marrow noted. Shrinkage of the elevated area was observed

during the healing period and the formation of new bone sprouting from the resident

bony walls of the sinus, growing towards the middle of the elevated area was noted.

No bone formation from the Schneiderian membrane was identified. These findings

are in agreement with previous pre-clinical studies (Xu, et al. 2003, Xu, et al. 2004)

24

with new bone formation initiated from the walls of the sinus after maxillary sinus

elevation and not beneath the Schneiderian membrane, regardless of whether a bone

graft or a blood clot alone was used. As healing progressed, the augmented space

significantly reduced in size when using a coagulum only, corroborating other

studies showing that bone formation as a result of a coagulum is unstable (Xu, et

al. 2005).

Haas et al. (Haas, et al. 2002) also showed a tenting effect of implants placed into

the maxillary sinuses cavity after sinus membrane elevation with no additional bone

grafting in sheep. The apex of the implant was mostly in direct contact with the

Schneiderian membrane, with new bone formation noted around the part of the

implant proximal to the resident bone walls. The new bone appeared to originate

from these cortical walls. Sporadic bone islands were noted along the Schneiderian

membrane but their origin is unclear.

These studies demonstrate the importance of space maintenance in neo-

osteogenesis and that a coagulum has the potential for neo-osteogenesis if the space

can be maintained. New bone is unstable in areas where the membrane collapses

due to the instability of the blood clot and a lack of mechanical support (absence of

biomaterials and / or implant). Whilst in-vitro and some in-vivo experiments have

shown that the Schneiderian membrane may have osteogenic potential, pre-clinical

studies are not in agreement as to the role of the membrane in bone formation,

especially in the early stages of bone healing. There is overwhelming evidence that

early bone formation begins at the periphery of the defect using surrounding

resident bony walls as a stable base for neo-osteogenesis, but the osteogenic

potential of the Schneiderian membrane is questionable.

Impact of sinus pneumatization on maxillary sinus grafting

Ventilation of the maxillary sinus is accomplished by air exchange with the nasal

cavity through the sinus ostium (Scharf, et al. 1995). The presence of prolonged air

pressure has shown sinus enlargement and bony wall displacement (Zizmor, et al.

1975). As such, sinus pressure may have an impact on sinus floor location post-

grafting.

25

A study by Asai et al. (Asai, et al. 2002) investigated the impact of occluding the

maxillary sinus ostium on bone formation in a pre-clinical model. The sub-antral

space was allowed to fill with a coagulum only and initially resulted in formation

of granulation tissue with bone formation at the periphery. Occluding the ostium

resulted in regenerated bone matrix after 3 weeks which matured by week 6. Not

occluding the ostium resulted in almost complete replacement by a normal sinus

airspace after 3 weeks of healing. In a similar pre-clinical model (Xu, et al. 2004),

the same research group compared the addition of a deproteinised bone graft into

the sub-antral space with a coagulum only after sinus membrane elevation. The new

bone that had formed subsequently resorbed in the coagulum only group, but

remained in the grafted group and showed further consolidation. The authors

concluded that the coagulum did not withstand the sinus pressure during the first

several weeks, and re-pneumatization of the sinus or ‘slumping’ was noted. The

grafted sinus however maintained the space and the deproteinised bone grafts

particles resisted pressure induced bone resorption.

Characteristics of MSA Sites

Histological & histomorphometric

Large variations in the area fraction of biomaterials such as ABBM have been

reported (Valentini, et al. 2000, Yildirim, et al. 2000, Hallman, et al. 2001) and may

depend on the pressure at application of the grafting material, the initial percentage

of the material, the particle size, the biopsy technique, or the histological

preparation technique.

Biopsy harvesting techniques have been reported to have a significant impact on

histomorphometric outcomes, at least in the early phase of graft healing (Margolin,

et al. 1998, Artzi, et al. 2005). Artzi et al. histomorphometrically assessed the

variation in area fraction of residual bone substitutes and new bone in samples

harvested via a trephine from grafted maxillary sinuses (Artzi, et al. 2005). The

depth of the samples influenced both parameters. This has been corroborated by

another study reporting biopsy harvesting depth and location as important aspects

to consider, since they influence the mineralization and degree of bone formation

26

(Avila, et al. 2010). Furthermore, there is overwhelming evidence that

histomorphometric parameters vary depending on proximity to resident bony walls

(Busenlechner, et al. 2009). As such, another determining factor may be the

horizontal dimension of the lateral window and proximity of the lateral walls of the

sinus to the harvesting site (Suarez-Lopez Del Amo, et al. 2015). Further

confounding variables include the histological slice thickness (Klijn, et al. 2010)

and residual ridge height (Corbella, et al. 2015).

Haas et al. (Haas, et al. 1998) published a histological assessment of MSA sites in

an ovine model, which had simultaneous implants placement. The sinuses received

either ABBM (Bio-Oss®), AB (iliac crest) or they were left as empty controls filled

with only a coagulum. New bone formation around the ABBM particles was

observed in areas in which the material was in direct contact with local bone. None

of the specimens showed new bone formation around the particles further away

from this contact area. Great variation was noted in the degree of osseointegration

of the ABBM particles irrespective of the healing time. Grafting had no additional

osteoconductive effect in the region of the implant proximal to the resident bony

wall.

These histological changes leading to improved integration between new bone and

ABBM particles correlated well with mechanical testing carried out by the same

group (Haas, et al. 1998). The pull-out strengths of implants in the group augmented

with ABBM showed the highest values. Time also had a significantly positive

impact on pull-out strengths.

If de novo bone formation is the reference parameter however, then AB remains the

gold standard. An RCT by Schmitt et al. (Schmitt, et al. 2013) reported on the

histological and histomorphometric characteristics of samples harvested 5 months

after two stage MSA with various materials in 30 patients. The amount of newly

formed bone was significantly higher in the AB (42.74 ± 2.10 %) group when

compared to ABBM (24.90 ± 5.67 %). The measured mineralized components were

comparable between these two materials (ABBM - 46.26 ± 8.11 %) (AB - 42.74 ±

2.12 %) which is consistent with the increased fraction of residual bone substitute

27

in ABBM samples. This has been further confirmed in systematic reviews

(Corbella, et al. 2016) using comparative studies.

The composite graft of ABBM and AB has been utilized in multiple studies of

maxillary sinus floor augmentation (Maiorana, et al. 2000, Hallman, et al. 2001,

Hallman, et al. 2001, Hallman, et al. 2002, Hatano, et al. 2004, Hallman, et al. 2005,

Cannizzaro, et al. 2009, Hallman, et al. 2009, Kim, et al. 2009, Avila, et al. 2010,

de Vicente, et al. 2010, Galindo-Moreno, et al. 2010), but few comparative studies

exist with ABBM or to AB alone. The proportion of vital bone within the sinuses

after using two different ratios of ABBM and AB was evaluated in humans

(Galindo-Moreno, et al. 2011). No statistically significant differences in newly

formed bone and non-mineralized tissue were noted for 50% ABBM and 50% AB

group when compared to the 80% ABMM and 20% AB group. Additional

comparative studies also failed to show an advantage of adding AB to ABBM in

terms of %NB when analysed in a recent systematic review (Corbella, et al. 2016).

The impact of using a non-resorbing material such as ABBM is of interest when

considering the degree of bone-to-implant contact (BIC) of a dental implant. Early

reports indicated no direct contact between ABBM particles and the implant surface

(Valentini & Abensur 1997). In a pre-clinical study, Jensen et al. (Jensen, et al.

2013) examined the impact of varying the proportion of ABBM (Bio-Oss®) and

AB on BIC in mini-pigs after 12 weeks after MSA and implantation. This was

performed using (a) 100% AB, (b) 75% AB and 25% Bio-Oss®, (c) 50% AB and

50% Bio-Oss®, (d) 25% AB and 75% Bio-Oss®, and (e) 100% Bio-Oss®. Samples

were harvested 12 weeks post-grafting and BIC was calculated. The median BIC

was (a) 42.9%, (b) 37.8%, (c) 43.9%, (d) 30.2%, (e) 13.9%, respectively. BIC was

significantly higher when autogenous bone or Bio-Oss® mixed with AB was used

in varying ratios as compared to Bio-Oss® alone.

Hallman et al. (Hallman, et al. 2002) examined BIC of micro-implants placed into

augmented maxillary sinuses in humans after 6 months of healing. The sinuses were

augmented with 100% ABBM, a mixture of 80% ABMM and 20% AB and 100%

AB. No statistically significant differences in mean BIC were reported for ABBM

(mean 31.6% ± 19.1) when compared to ABBM mixed with AB (mean 54.3% ±

28

33.1) or 100% AB (mean 34.6% ± 9.5). Variations in species, model design and

healing time may explain the different results obtained in this study when compared

to the study carried out by Jensen et al.

Whilst histomorphometirc outcomes using a biopsy technique are influenced by

many factors it remains the most commonly reported method of assessing the

biological success of the graft after MSA. Whilst many pre-clinical and clinical

studies have shown the effectiveness of ABBM in MSA, a systematic review has

shown that the use of AB alone provides the greatest amount of new bone (Corbella,

et al. 2016). Furthermore, the addition of AB to ABBM yields little advantage when

compared to ABBM alone (Corbella, et al. 2016).

Impact of barrier membranes on MSA

Studies on the benefits of using barrier membranes over the buccal antrostomy once

graft placement has been completed are conflicting, with very few trials actually

carrying out controlled trials comparing the use of a membrane against no

membrane. Excluding studies using smooth surface implants, the reported survival

rates of implants placed into MSA sites with a barrier membrane appear similar

(mean 98.5% & range 92-100%) when compared to rates without the use of a

membrane (mean 98.5% & range 93-100%) (Chiapasco, et al. 2009).

Whilst earlier studies by Tarnow et al. (Tarnow, et al. 2000) and Wallace et al.

(Wallace, et al. 2005) did show improved new bone formation with the use of the

membrane, subsequent small RCTs have not replicated these results (Choi, et al.

2009, Barone, et al. 2013) with equivalent %NB reported in for both groups.

Interestingly however both of these RCTs reported significantly greater %ST when

not using a barrier membrane. Furthermore, systematic reviews using

histomorphometric data from clinical samples after MSA using AB only (Klijn, et

al. 2010) or a variety of materials (Suarez-Lopez Del Amo, et al. 2015) indicated

that the use of barrier membranes did not influence the amount of new bone

formation.

29

As discussed previously, the location and depth of the biopsy and proximity to the

lateral wall will influence histomorphometric outcomes. Since the barrier

membrane is placed on the buccal surface of the lateral wall of the maxilla and most

biopsy samples are taken from the crest, this may not be representative of its true

barrier function. Interestingly, the histological samples used in the RCT by Tarnow

et al. were taken through the lateral window. They demonstrated that the mean

percentage of new bone formation was 25.5% with and 11.9% without a covering

barrier. Overall, the evidence suggests that the presence or absence of a membrane

has no significant effect on implant survival or on %NB formation, with some

evidence of increased %ST in the absence of a barrier membrane.

Linear & volumetric changes

Re-pneumatization after MSA using a variety of grafting materials has been

reported by a variety of authors and may represent time dependent graft remodelling

leading to re-pneumatization of the maxillary sinus. One can only hypothesize as to

whether barometric pressure within sinuses exerts an effect on the remodelling

process, as was discussed earlier in the section ‘role of Schneiderian membrane &

coagulum in MSA’. Studies measuring linear changes of graft height using a variety

of bone substitutes, including AB (Wiltfang, et al. 2005, Zijderveld, et al. 2009,

Schmitt, et al. 2012), beta-tricalcium phosphate (Zijderveld, et al. 2009) and a

composite of AB and ABBM (Hatano, et al. 2004), suggest that the greatest extent

of graft height occurred early after grafting, with subsequent resorption slowing

down and eventually stabilizing. Sbordone et al. (Sbordone, et al. 2011)

hypothesized that the amount of linear graft remodelling depends on the nature of

the graft (AB or ABBM). Reduced linear changes were seen in the ABBM group

when compared to AB, especially when sourced from the iliac crest.

Considering the obvious limitations of two-dimensional radiographs, three

dimensional methods were subsequently applied to assess this remodelling

phenomenon. The earliest prospective clinical study using CT assessment of graft

volume reported a mean volume reduction using 100% AB from iliac crest of 49.5%

(31-78%) 6 months after grafting (Johansson, et al. 2001). Arasawa et al. (Arasawa,

et al. 2012) assessed the volumetric changes in 9 patients grafted with only AB

30

harvested from either the iliac crest or the mandibular ramus at 3 months and at

least 1 year after sinus augmentation. A mean volume reduction of 24.8% (SD

6.1%) was reported over this period.

A pre-clinical model examined the impact of varying the proportion of ABBM

(Bio-Oss®) and AB on bone graft volume after sinus floor elevation (Jensen, et al.

2012). CT scans of the maxillary sinus were taken immediately post-grafting and

then 12 weeks later. The volumetric reduction was significantly influenced by the

ratio of ABBM and AB, but not by the origin of the AB. The greater the proportion

of AB, the greater the reduction in volume.

A more recent systematic review of three dimensional graft volumetric change was

performed on controlled studies published up to 2013 (Shanbhag, et al. 2014).

Using a variety of bone grafting materials, weighted mean volume reductions of

63.24% (±14.29) were reported for particulate AB, 30.37% (±11.98) for allografts,

18.30% (±2.35) for bone substitutes (included: ABBM, biphasic calcium

phosphate) and 21.88% (±7.38) for composite grafts (any combination of two or

more materials). ABBM (Bio-Oss®) was the most frequently utilized material in

the reported studies. Most of these studies had short term follow up and therefore it

remains to be seen if volumetric data is similar to previous 2D linear measurements,

which indicated stabilization of the graft loss in the long term.

Overall, there is sufficient evidence to suggest that MSA using AB as the sole

material is not recommended, based on a lack of volumetric stability. In addition,

ABBM appears to exhibit superior volumetric stability.

DENTAL IMPLANTS & SUPPORTED RESTORATIONS IN MSA

Dental implant surface microtopography

Implant surface topography and chemistry affects the osseointegration process

(Gittens, et al. 2014). It has been shown to play a critical role in the predictability

of the bone response to implant placement (Junker, et al. 2009) and is a major

determinant of the rate and extent of dental implant osseointegration (Wennerberg

& Albrektsson 2009). Animal studies have shown that microrough titanium

31

implants result in superior BIC compared to minimally rough implants (Cochran,

et al. 1998), as well as superior torque removal values (Buser, et al. 1998).

Histological analysis of the sequential healing events associated with the placement

of microrough surface titanium implants showed that initial bone formation around

these implants occurs not only at the exposed bone wall of the osteotomy site, as is

the case with minimally rough surface implants, but also along an osteophylic

implant surface (Berglundh, et al. 2003). Furthermore, Abrahamsson et al.

(Abrahamsson, et al. 2004) compared the temporal healing events associated with

osseointegration to microrough and minimally rough surfaces. They demonstrated

histological evidence of superior early healing associated with the microrough

surfaces, with a higher level of wound organization as early as 4 days following

implantation, ultimately leading to greater BIC one week post-insertion.

The clinically utilized microrough SLA® surface is achieved by sand blasting

minimally rough (Ra 0.5 - 1µm) titanium with large grit followed by etching with

acid to give a microrough (Ra 1 - 2µm) surface topography. The use of such

microscale modified implant surfaces has been credited with being one of the key

factors in increasing the clinical success rate of implants, especially in areas of

compromised bone quality (Ivanovski 2010).

Chemical modification of the SLA surface to remove surface contamination

resulting in a surface with increased surface energy and hydrophilicity

(SLActive®), has further demonstrated enhanced bone apposition during the early

stages of osseointegration compared to the SLA surface (Bornstein, et al. 2008). In

vitro studies have demonstrated that the early cell response to the modified

hydrophilic surface (SLActive®) were significantly enhanced (Rausch-fan, et al.

2008). Further, Mamalis et al. (Mamalis, et al. 2011) showed that the SLActive®

surface promotes an ‘osteoprotective microenvironment’ which up-regulates

osteoblastic differentiation, whilst suppressing osteoclastogenesis. In-vivo human

studies also showed that compared with the SLA surface, SLActive® exerts a pro-

osteogenic and pro-angiogenic influence on gene expression as early as 7 days post

implantation (Donos, et al. 2011).

32

When compared with SLA, pre-clinical studies demonstrated that SLActive®

achieved significantly greater BIC at 2 weeks after placement, although

equivalence was reached at 8 weeks (Buser, et al. 2004). Similarly, Bornstein et al.

(Bornstein, et al. 2008) also showed an initially greater BIC at 2 weeks that reached

equivalence by week 4 when compared to SLA implants. SLActive® implant

surfaces have also shown significantly greater removal torque values compared to

SLA (Ferguson, et al. 2006). A histological analysis of human samples by Lang et

al. (Lang, et al. 2011) showed a superior degree of osseointegration for SLActive®

implants after 2 and 4 weeks of healing when compared to a conventional SLA

surface, but this difference was not evident by 42 days after placement.

In a review of the evidence for the chemically modified hydrophilic implant surface

(SLActive®) it was concluded that a stronger bone response was present during the

early healing. The later stages however, exhibited similar biological response to the

non-chemically modified surface (Wennerberg, et al. 2011). Nonetheless, the early

histological advantage has translated to successful earlier loading protocols, as

demonstrated by Morton et al. (Morton, et al. 2010). Loading of these implants was

achievable in 95.6% of cases at 21 days without incident. At 2 years after

placement, they reported a success rate of 97.7%. Using an identical loading

protocol, clinical parameters for SLActive implants exhibited signs consistent with

peri-implant health after 3 years of function (Bornstein, et al. 2010).

Various authors have also advocated the use of SLActive surfaces in compromised

sites, such as those experienced in diabetes and osteoporosis. Pre-clinical studies in

rabbits (Mardas, et al. 2011) and pigs (Schlegel, et al. 2013) have indicated

significantly improved BIC around the modified implant surface (SLActive®)

when compared to the conventional surface (SLA).

Studies comparing these modified implant surfaces in bone defects or grafted sites

are rare. Schwarz et al. (Schwarz, et al. 2008) evaluated such implant surfaces in

dehiscence-type defects in a canine model. At 8 weeks, the modified implants

(SLActive®) exhibited significantly greater defect bone fill and %BIC when

compared to both a standard surface (SLA) and a calcium phosphate modified dual

acid etched surface (Nanotite®) (Schwarz, et al. 2010).

33

These improvements were not however observed in a pre-clinical study assessing

the influence of this implant surface modification in grafted maxillary sinuses

(Philipp, et al. 2013). Half of the sheep received a bone graft (hydroxyapatite and

beta-tricalcium phosphate) and the other half underwent sinus floor elevation only.

Using a paired design, a conventional implant was simultaneously placed on one

side of the animal and the modified surface on the other side. The animals were

sacrificed at 12 and 26 weeks. There was no significant difference in BIC when the

two surfaces were compared in either grafted or ungrafted sites at both time points.

In light of previous studies, the sacrifice at even the earlier time-point of 12 weeks

may have been too late in the healing process to detect a significant difference.

It has been suggested that many factors influence osseointegration of implants, and

the ability to withstand functional load, in augmented maxillary sinuses (Jensen, et

al. 1998). This multitude of variables may explain the seemingly arbitrary loading

intervals reported in different studies. A recent study using a composite bone graft

(ABBM+AB 1:1) utilized a loading time of 6 months (Galindo-Moreno, et al.

2015). Another study using SLA implants placed into biphasic beta-tricalcium

phosphate or ABBM reported a mean loading time of 10 weeks (Bornstein, et al.

2008), yet a clinical trial using SLActive implants placed into ABBM or biphasic

calcium phosphate loaded at a mean of 16.8 weeks after placement (Mordenfeld, et

al. 2016). Systematic reviews report a mean loading time between 5 to 6.5 months

(Chiapasco, et al. 2009, Jensen & Terheyden 2009), irrespective of the biomaterial

used or implant surface. A review of only rough implant surfaces reported a range

of loading periods from 0.3 to 10 months with no impact on survival rates (Nkenke

& Stelzle 2009).

Outcomes of implant supported restorations in MSA

Implant Survival

Implant survival rates in MSA are enhanced when using microrough implant

surfaces, as has been reported in a number of systematic reviews (Pjetursson, et al.

2008, Chiapasco, et al. 2009, Jensen & Terheyden 2009, Nkenke & Stelzle 2009,

Rickert, et al. 2012). A median survival rate of 100% (88-100%) is reported for

microrough implant surfaces when compared to 89% (61.2 – 100%) using

34

machined implant surfaces (Jensen & Terheyden 2009). Indeed, a 100% survival

rate has been reported in a clinical study using one-piece SLA implants in MSA at

12 and 60 months after implant placement (Bornstein, et al. 2008). The enhanced

outcomes for micro-rough compared with machined implant surfaces is consistent

with superior histologically documented outcomes (Abrahamsson, et al. 2004) and

improved bone-to-implant contact (BIC) and stability (Buser, et al. 1998, Cochran,

et al. 1998). Interestingly, a survival rate of 86% was reported in a study with 10

year follow up, using commercially pure implants with a turned surface, with the

majority of their failures occurring prior to loading (Mordenfeld, et al. 2014).

The 5-year implant survival rate in MSA sites grafted using composite graft

AB+ABBM (1:1) was reported to be 92.86% (Schmitt, et al. 2015) in a small

number of patients.

Overall the survival rates for microrough implant surfaces placed into MSA sites

using ABBM or composite grafts of ABBM + AB is high even at 5 years, and is

comparable to implants placed into pristine bone.

Implant Success

Implant success is measured using a wide variety of composite or single outcomes,

with little or no consistency between studies (Tonetti, et al. 2012). The four most

frequently used parameters for assessing success are related to the implant level,

peri-implant soft tissue, prosthesis and patient’s subjective assessment

(Papaspyridakos, et al. 2012). Similarly, based on consensus reports published at

the 8th European Workshop on Periodontology, outcome measures should capture

patient reported outcomes (McGrath, et al. 2012), peri-implant health (Derks &

Tomasi 2015) and restoration outcomes (Pjetursson, et al. 2012). As such, for

implant health it was recommended to report on marginal bone levels, tissue

inflammation (BOP) and probing depth. For implant restorations, it was advised to

report on longevity of the restorations, functional / occlusal related outcomes and

technical complications. One group of researchers has recommended success

criteria report the long term outcomes of the implant-prosthetic complex as a whole

35

(Papaspyridakos, et al. 2012) rather than report each outcome separately

(Pjetursson, et al. 2012, Sanz, et al. 2012, Tonetti, et al. 2012).

Since the case definitions for peri-implant health and disease vary significantly in

terms of defined threshold (Derks & Tomasi 2015), significant variations in

reported success rates would be expected (Karoussis, et al. 2003).

The success criteria utilized in this study are based on the consensus reports

published at the 8th European Workshop on Periodontology (Pjetursson, et al. 2012,

Sanz, et al. 2012).

• Biological complications: included implants that exhibited signs of mucositis

or peri-implantitis. Mucositis was defined as any bleeding on gentle probing

without loss of supporting bone (Lang, et al. 2011). Peri-implantitis was

defined as bleeding on probing and / or suppuration with or without

concomitant deepening of peri-implant pockets, accompanied by marginal

bone level changes of greater than 1.0mm after baseline (Sanz, et al. 2012)

(Lang, et al. 2011). Furthermore, any treatment performed on the implant that

was not part of routine maintenance was by default classified as a

complication.

• Technical complications: included any implant-supported restorations that

presented with features as defined above (Pjetursson, et al. 2012). These were

divided into (1) major: such as implant fracture, loss of super-structure, (2)

medium: such as abutment fracture, veneer or framework fracture and (3)

minor: abutment and screw loosening, loss of screw hole sealing, veneer

chipping (requiring polishing) and occlusal adjustments. In addition, any

treatment performed on the restoration that was not part of routine maintenance

was by default classified as a complication. If the implant was incorporated into

an FPD, then any technical complication affecting any part of the FPD was

included in the analysis.

36

Biological Outcomes

For one-piece rough surface implants, the marginal bone remodelling process

begins immediately following implant placement and is influenced by the location

of the rough-smooth junction (Hammerle, et al. 1996, Hermann, et al. 2000,

Hermann, et al. 2001, Hermann, et al. 2001). Continued change in marginal bone

levels after this initial remodelling period has been observed in many studies. This

change may represent bone loss due to exposure to bacterial load (Lang, et al. 2011)

and masticatory forces (Marcelis, et al. 2012), rather than an adaptation to implant

placement and biologic width formation. Most studies exhibit reduced changes in

marginal bone levels after the first year of function.

A case series with a 10 year follow up (Mordenfeld, et al. 2014), reported on

marginal bone levels and pocket depths (PPD) around two-piece implants with a

turned surface and placed into MSA sites using 80% AB and 20% ABBM. Baseline

was measured at the time of abutment connection. Stable marginal bone levels were

seen after the first, third and tenth year of function with mean marginal bone loss

(ABL) of 0.4mm during the first interval (year 1-to-3) followed by a further 0.4mm

in the longer second interval (year 3-to-10). A mean pocket depth of 3.3 ±1.4mm

was reported at the 10 year follow up. No significant differences were found

between peri-implant parameters for implants placed into augmented bone or

pristine bone. Whilst the long follow up period makes this study an important one,

it does not include well defined parameters for success for marginal bone levels or

PPD. This is important as values reported in some patients for marginal bone levels

reached 5.9mm and PPD of 9mm.

Generally, there is a paucity of studies reporting on implant outcomes using well

defined success criteria when placed into the widely used composite graft of 50%

AB and 50% ABBM (Bornstein, et al. 2008, Galindo-Moreno, et al. 2015), and no

studies using collagen stabilized ABBM. Marginal bone levels ranging from 0 to

5.89mm at 18 months after loading have been reported after performing MSA using

the composite graft and subsequently placing implants with an internal or external

connection (Galindo-Moreno, et al. 2015). These as measurements however, were

taken using panoramic radiographs.

37

A 5 year prospective study reported on a range of outcomes using tissue level

(transmucosal, SLA and TPS) implants placed into MSA augmented with ABBM

or beta-tri-calcium phosphate. Stable marginal bone levels were seen with a mean

marginal bone loss of 0.25mm in the first year of function after baseline and only

0.08mm at the five year review. All patient exhibited good plaque control, a low

mean bleeding index and mean PPD of 4.14mm (± 0.11). The five year success rate

of 98% is attributed to the strict patient selection and defined conditions. A trial by

Schincaglia et al. (Schincaglia, et al. 2015) comparing two-piece (bone level),

rough surface implants placed into MSA sites using ABBM reported a mean

marginal bone loss of 0.22mm (±0.46) in the interval between implant placement

and insertion of prosthesis (baseline) and 0.39mm (±0.69) between baseline and at

the 12 months review in the MSA group. A mean PPD of 2.3mm (±1.4) was

reported in the MSA group.

Recent evidence indicates that rough implants placed into MSA sites can exhibit

high success rates with stable marginal bone levels and peri-implant parameters.

Technical Outcomes

Fixed single (SC) and fixed partial denture (FPD) implant restorations are well

documented in the literature (Pjetursson, et al. 2007, Jung, et al. 2008, Jung, et al.

2012, Pjetursson, et al. 2012). To the best of our knowledge there are no prospective

studies reporting on well-defined technical outcomes for implant supported

restorations in MSA in the partially dentate patient.

Systematic reviews have reported a 5 year survival of 96.3% for SCs and 95.4% for

FPDs (Jung, et al. 2012, Pjetursson, et al. 2012). Whilst screw and cement forms

of retention have their advantages and disadvantages (Michalakis, et al. 2003, Chee

& Jivraj 2006), more recent publications indicate a preference for screw retained

restorations based on retrievability and a lower rate of biological complications

(Gotfredsen, et al. 2012, Wismeijer, et al. 2014). Overall, the 5 year survival rates

for fixed implant supported reconstructions is 96.03% (CI: 93.85 – 97.43%) and

95.55% (CI: 92.96 – 97.19%) for screw retained and cement retained

38

reconstructions respectively (Wittneben, et al. 2014). When assessed according to

prosthesis type, the estimated 5-year survival rate of screw retained SC and FPD

reconstructions is similar to that of cemented reconstructions (Sailer, et al. 2012,

Wittneben, et al. 2014, Millen, et al. 2015).

The overall rate of technical complications of fixed implant reconstructions has

been reported to be significantly greater for cement retained when compared to

screw retained fixed restorations (Wittneben, et al. 2014, Millen, et al. 2015). When

the data is analysed according to the type of prosthesis however, screw retained SC

restorations exhibit greater rates of technical complications due to their

significantly greater rates of abutment screw loosening (Sailer, et al. 2012, Millen,

et al. 2015). Whilst an earlier review reported no significant difference in rate of

technical complications for FPDs (Sailer, et al. 2012) a more recent review reported

a significantly greater rate of technical complications for screw retained FPDs

(Millen, et al. 2015). The incidence of technical complications seems to be

dependant more upon prosthesis and retention type than prosthesis or abutment

material (Millen, et al. 2015).

The overall incidence of biological complications is significantly greater for cement

retained when compared to screw retained restorations (Wittneben, et al. 2014,

Millen, et al. 2015). No statistically significant differences were identified for

biological complications relative to retention type for SC restorations in a recent

review (Millen, et al. 2015). This review reported increased aesthetic complications

in screw retained restorations (Millen, et al. 2015). This parameter was not reported

by an earlier systematic review (Sailer, et al. 2012) and it offset the greater

incidence of bone loss (>2mm) and presence of fistula / suppuration noted in

cement retained restorations. Data for FPD’s remains scarce especially for cement

retained FPD’s hence preventing a statistical comparison.

RESEARCH HYPOTHESIS

A priori hypothesis in this project includes the demonstration of equivalence across

a range of outcomes (histomorphometric, volumetric and clinical) for the test graft

of collagen stabilized anorganic bovine bone mineral (ABBM) when compared to

39

the control graft of ABBM + autogenous bone (AB) in both a pre-clinical and

clinical model of maxillary sinus augmentation (MSA).

Furhtermore, with regard to assessing the impact of implant surfaces, we

hypothesized that modified hydrophilic implants (SLActive®) will exhibit a similar

degree of BIC and tissue compoistion when compared to a hydrophobic surface

with the same geometry and surface microroughness (SLA®).

AIMS

Broad objectives of this research:

This research aims to evaluate the performance of the collagen stabilized ABBM

grafting material in MSA under the specific clinical scenarios of Schneiderian

membrane perforation and insufficient local autogenous bone. The impact of dental

implant surface modification on early osseointegration was also assessed.

More broadly, the research also aims to report on patient reported outcome

measures (PROMs) for MSA and on the outcomes for implant supported

restorations placed in MSA sites using well defined criteria in a private practice

setting.

The thesis has been divided into two parts to address the following items:

• Part 1 – Histological & histomorphometric assessment of using collagen

stabilized ABBM in MSA (Chapters 2, 3 and 4).

• Part 2 – Clinical & radiographic assessment of using collagen stabilized

ABBM in MSA (Chapters 5 and 6).

The specific objectives of this thesis include:

• Histomorphometrically compare the use of collagen stabilized ABBM to

ABBM + autogenous bone in a pre-clinical model (ovine) of MSA. The

secondary objectives were to assess the impact of resident wall proximity

and healing time on histomorphometric outcomes (Chapter 2).

• Histomorphometrically compare the use of collagen stabilized ABBM to

ABBM + autogenous bone in MSA in a clinical model. Our secondary

40

objective was to assess the impact of the resident sinus floor proximity on

histomorphometric outcomes (Chapter 3).

• Compare the early osseointegration of implants with a hydrophilic surface

(SLActive®) to those with a hydrophobic surface with the same geometry

and surface microroughness (SLA®), in a pre-clinical model of MSA. Our

secondary objective was to characterize the tissue composition in the

immediate vicinity of the implant surface in these augmented sites (Chapter

4).

• Determine whether the use of collagen stabilized ABBM in defined clinical

scenarios (i.e. reduced local autogenous bone availability and/or sinus

membrane perforation), performs equivalently to the ABBM + autogenous

bone in a clinical model of MSA, in terms of the prevalence of surgical

complications, patient reported and volumetric outcomes (Chapter 5).

• Test whether or not implant supported restorations have similar biological

and technical outcomes when placed into previously augmented maxillary

sinuses using stabilized ABBM, as used in defined clinical scenarios (i.e.

reduced local autogenous bone availability and/or sinus membrane

perforation), when compared to those placed into a composite graft of

ABBM and autogenous bone (Chapter 6).

41

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66

PART 1 – HISTOLOGICAL & HISTOMORPHOMETIRC ASSESSMENT

OF USING COLLAGEN STABILIZED ABBM IN MSA

This section of the thesis includes the following chapter:

Chapter 2: A histomorphometric assessment of collagen stabilized anorganic

bovine bone mineral in maxillary sinus augmentation – a randomized controlled

trial in sheep.

Chapter 3: A histomorphometric assessment of collagen stabilized anorganic

bovine bone mineral in maxillary sinus augmentation – a prospective clinical trial.

Chapter 4: Comparison of early osseointegration of SLA® and SLActive® implants

in maxillary sinus augmentation: a pilot study.

67

Chapter 2 - A histomorphometric assessment of collagen stabilized anorganic

bovine bone mineral in maxillary sinus augmentation – a randomized

controlled trial in sheep.

68

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED

PAPER

This chapter includes a co-authored paper. The bibliographic details of the

co-authored paper, including all authors, are:

Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S. (2016) A

histomorphometric assessment of collagen stabilized anorganic bovine bone

mineral in maxillary sinus augmentation – a randomized controlled trial in sheep.

Clinical Oral Implants Research 27: 734 – 743. doi: 10.1111/clr.12652.

My contribution to the paper involved:

Design of the project, the provision of the data, the preliminary analysis and

categorization of the data into a usable format, the provision of direction on the

scope and structure of the analysis, the interpretation of results and drafting of the

article.

04th May 2018

Jamil Alayan

(Countersigned) 16th May 2018

Corresponding author of paper: Saso Ivanovski

(Countersigned) ___________________________ 16th May 2018

Supervisor: Stephen Hamlet

Jamil AlayanCedryck VaquetteSiamak SaifzadehDietmar HutmacherSaso Ivanovski

A histomorphometric assessment ofcollagen-stabilized anorganic bovinebone mineral in maxillary sinusaugmentation – a randomizedcontrolled trial in sheep

Authors’ affiliations:Jamil Alayan, Saso Ivanovski, School of Dentistryand Oral Health, Centre for Medicine and OralHealth, Griffith University, Southport, Qld,AustraliaCedryck Vaquette, Siamak Saifzadeh, DietmarHutmacher, Institute for Health and BiomedicalInnovation, Queensland University of Technology,Kelvin Grove, Qld, Australia

Corresponding author:Saso IvanovskiSchool of Dentistry and Oral Health, Centre forMedicine and Oral HealthGold Coast Campus Griffith UniversitySouthport, Qld 4215AustraliaTel.: +61 75 678 0741Fax: +61 75 678 0708e-mail: s.ivanovski@griffith.edu.au

Key words: animal model, bone grafting, bone regeneration, bone substitute, histomorphom-

etry, maxillary sinus augmentation

Abstract

Objective: To histomorphometrically compare the use of collagen-stabilized anorganic bovine

bone (ABBM-C) (test) to anorganic bovine bone + autogenous bone (ABBM + AB) (control) in

maxillary sinus augmentation.

Materials and methods: Nine sheep underwent bilateral sinus augmentation. Each sinus was

randomized to receive either control or test bone graft. Three animals were sacrificed at 8 weeks,

and six animals were sacrificed at 16 weeks post-grafting. The 18 sinuses were processed for

histomorphometry, which assessed the area fraction of new bone (%NB), residual graft (%RG) and

soft tissue components (% STM), as well as graft particle osseointegration (% OI), within three

zones equally distributed from the augmented sinus floor.

Results: At week 16, a significant increase in %NB was evident across all three zones in the control

group when compared to week 8. A significantly greater %NB was evident in the control group

when compared to the test group in zones 2 (P < 0.001) and 3 (P < 0.001). There was a significant

increase in %OI in week 16 when compared to week 8 across all three zones in the control group

(P < 0.001). %OI in the control group was significantly greater across all three zones when

compared to the test group at week 16 (P < 0.001). Zone was found to be a significant main effect

(P < 0.001) that was independent of time and treatment with decreasing %OI in distant zones. %

RG did not significantly change with time for both groups. There was a significant reduction in %

ST in week 16 when compared to week 8 across all three zones in the control group (P < 0.001). %

ST in the test group was significantly greater across all zones when compared to the control group

at week 16 (P < 0.001).

Conclusion: Both groups exhibited very similar histomorphometric measurements in the zones

proximal to the resident sinus wall. The % NB and % OI were greatest in the zones proximal to

resident bony walls and gradually decreased as the distance from the proximal walls increased.

There was greater % NB and % OI in the control group when compared to the test group in the

distant zone.

Implant placement in the posterior maxilla is

often prevented by insufficient bone volume

as a result of bone resorption and sinus pneu-

matization. Maxillary sinus floor elevation is

the technique most frequently used to over-

come this limitation and is considered a safe

and effective procedure with high implant

survival rates (Del Fabbro et al. 2004). Clini-

cally this surgical technique is still being

refined, with a large degree of variation in all

aspects of the procedure.

Autogenous bone, allografts, xenografts and

alloplastic materials, as well as various

mixtures of these, are currently used in sinus

grafting (Del Fabbro et al. 2004). Autogenous

bone grafts exhibit osteogenic properties, as

well as osteoconductivity and osteoinductivi-

ty, (Burchardt 1983) and have long been con-

sidered to be the gold standard grafting

material. Autogenous bone harvesting how-

ever can be associated with post-operative

patient morbidity (Clavero & Lundgren 2003;

Cricchio & Lundgren 2003) and is only pres-

ent in limited quantities intra-orally. Autoge-

nous bone grafts can also exhibit

unpredictable resorption (Davis et al. 1984;

Date:Accepted 25 May 2015

To cite this article:Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S.A histomorphometric assessment of collagen-stabilizedanorganic bovine bone mineral in maxillary sinusaugmentation – a randomized controlled trial in sheep.Clin. Oral Impl. Res. 27, 2016, 734–743doi: 10.1111/clr.12652

734 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Clavero & Lundgren 2003; Wiltfang et al.

2005; Sbordone et al. 2013) which may not

be a desirable characteristic in sinus grafting

where a slower resorbing osteoconductive

material can provide superior space mainte-

nance for de novo bone formation. These

limitations of autogenous bone have resulted

in considerable research into various alterna-

tive bone substitutes.

Bio-Oss! (Geistlich AG, Wollhusen, Swit-

zerland) is a widely utilized commercial prod-

uct consisting of anorganic bovine bone

mineral (ABBM) in the form of cortical gran-

ules. It has a natural, non-antigenic porous

matrix and is chemically and physically iden-

tical to the mineral phase of human bone. It

has been reported to be highly osteoconduc-

tive and to have a very low resorption rate

(Jensen et al. 1996; Berglundh & Lindhe

1997; Haas et al. 2002a; Orsini et al. 2005)

The pores in Bio-Oss! are of optimal size and

configuration to facilitate vascular ingrowth,

which is essential for new bone formation

(Klawitter et al. 1976; Daculsi & Passuti

1990). Bio-Oss! is one of the best docu-

mented biomaterials used in maxillary sinus

augmentation (Artzi et al. 2001; Hallman

et al. 2002b; Valentini & Abensur 2003).

The combined utilization of ABBM and

particulate autogenous bone has been

reported in multiple studies of maxillary

sinus floor augmentation (Hallman et al.

2001a,b, 2002a,b; Galindo-Moreno et al.

2010; Schmitt et al. 2014). Recent animal

studies have shown that, compared with

autogenous bone and ABBM alone, the com-

bination of autogenous bone and ABBM has

the advantages of reduced graft resorption

and increased bone-to-implant contact (Jen-

sen et al. 2012, 2013).

Bio-Oss Collagen! (ABBM-C; Geistlich

AG) is also composed of ABBM particles but

stabilized in 10% porcine type-1 collagen

matrix. ABBM-C is made available for clini-

cal use in block form, rather than as a partic-

ulate graft. The contained nature of the block

may be advantageous in maxillary sinus aug-

mentation. The impact of this altered formu-

lation of ABBM has not been investigated in

terms of bone regeneration and graft stability

in maxillary sinus augmentation. This is an

important consideration as it has been shown

that surface characteristics and the geometry

of a material has a significant impact on neo-

osteogenesis (Hing et al. 2005).

Therefore, the primary aim of this study

was to histomorphometrically compare the

use of collagen-stabilized anorganic bovine

bone (ABBM-C) to anorganic bovine

bone + autogenous bone (ABBM + AB) in an

animal (ovine) model of maxillary sinus floor

augmentation. Our secondary objectives were

to assess the impact of resident wall proxim-

ity and healing time on histomorphometric

outcomes in augmented sinuses.

Material and methods

Animal care and handling

All animal handling and surgical procedures

were conducted according to the Australian

code of practice for the care and use of ani-

mals for scientific purposes at the Medical

Engineering Research Facility (MERF),

Queensland University of Technology (QUT),

Queensland, Australia. Ethical approval was

provided by the QUT Animal Research Eth-

ics Committee (QDPI Scientific Use Registra-

tion Number: 0052). All sheep underwent a

health assessment, especially to check for

the presence of upper respiratory tract infec-

tions, and were continually monitored by a

veterinary surgeon for the duration of the

study.

The surgical procedures were performed

under general anaesthesia (2.5% pentothal

sodium I.V and halothane 2% I.V) with

multi-modal pain management including

local analgesia (bupivacaine 0.5% w/v

1 : 200,000 adrenalin), an NSAID (ketorolac,

0.5 mg/kg, SC) and an opioid analgesic (bupr-

enorphine, 0.01 mg/kg IM). During surgery,

animals received cephalothin and gentami-

cin, which were continued for 5 days after

surgery.

Bone grafting materials

Animals were randomly assigned to one of

the following groups:

• Control group – small diameter particles

(0.25–1.0 mm) of deproteinized bovine

bone mineral (Bio-Oss!, Geistlich

AG) + autogenous bone (ABBM + AB) in a

1 : 1.

• Test group – 250 mg blocks of deprotei-

nized bovine bone mineral + collagen

(ABBM-C) (Bio-Oss Collagen!, Geistlich

AG).

Animal allocation and study design

The study used nine (x9) skeletally mature

wether sheep (Ovis aries), 3–4 years old.

Each animal underwent bilateral sinus aug-

mentation procedures randomly receiving on

one side the control and the other the test

grafting material. A total of x9 sinuses

received the control graft (ABBM + AB), and

a total of x9 sinuses received the test graft

(ABBM-C).

Three animals were then sacrificed at

8 weeks, and another six animals were sacri-

ficed at 16 weeks after grafting. The 18

sinuses were then processed for histology and

histomorphometry.

Surgical procedure

All animals were treated using the same sur-

gical technique utilizing an extra-oral

approach. The anterior wall of the maxillary

sinus was exposed inferior to the lower orbi-

tal rim using an oblique sagittal skin incision

of approximately 6–8 cm followed by dissec-

tion of the masseter muscle (Fig. 1a). A full

circular antrostomy was created in the lateral

wall of the maxilla using a bone scraper

(Safescraper Twist, META, C.G.M Sp.A, Reg-

gio Emilia, Italy) and a Piezo-electric surgical

unit (Implant Center, Satelec, Acteon,

France) to gain access to the maxillary sinus

(Fig. 1b). The size of the antrostomy was

standardized using a circular sterile template

with a diameter of 2.0 cm. The sinus mem-

brane was then dissected and elevated in a

dorsocranial direction using a combination of

piezoelectric surgical tips and hand sinus

membrane elevators to create an intrasinus

space (Fig. 1c). The prepared sinus was then

randomly allocated to receive the grafting

material, which is then carefully packed into

the sub-antral space ensuring contact with as

many of the residual bony walls as possible

whilst avoiding over-packing (Fig. 1d).

The autogenous bone chips collected using

the bone scraper were used as the autogenous

bone component in the control group. To

ensure a 1 : 1 ratio was maintained, a stan-

dardized chamber was utilized to ensure that

the same volume of Bio-Oss! and autogenous

particles was used. The reconstructed sites

were covered with a resorbable porcine colla-

gen membrane (Bio-Gide!, Geistlich AG)

(Fig. 1e). The masseter muscle and skin flap

were then sutured separately using a resorb-

able polyglactin suture (Vicryl 4/0, Ethicon,

Johnson & Johnson, CA, USA) followed by a

non-resorbable polypropylene suture (Prolene

4/0, Ethicon, Johnson & Johnson) for the skin

flap to achieve primary closure (Fig. 1f).

Post-operative treatment and sacrifice

A combination analgesic was given (bupr-

enorphine, 0.01 mg/kg IM, and Ketorolac,

0.5 mg/kg, SC) for 2–3 days post-operatively

to alleviate pain.

The oral surgical wounds were checked on

a daily basis for any evidence of post-opera-

tive complications, such as failure in suture

integrity (wound dehiscence), bleeding,

inflammation and infection.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 735 | Clin. Oral Impl. Res. 27, 2016 / 734–743

Alayan et al !Bio-Oss Collagen in Sinus Augmentation

Post-operatively, each sheep was trans-

ferred to a covered pen and was cradled in a

sheep sling. To protect the operated sinus

from masticatory load immediately after the

surgery, the animals were fed on peripheral

parenteral nutrition for 24 h. Throughout the

fluid administration, the sheep remained cra-

dled in the sling and were monitored con-

stantly. From the second day after surgery,

the sheep were fed a soft diet for 1 week

post-operatively, and eating and drinking

were closely monitored following parenteral

nutrition.At the completion of the 8- and 16-week

healing periods, the animals were sacrificed

by a lethal injection of Lethabarb (320 mg/ml

Pentobarbital Sodium, 0.5 ml/kg).

Histological preparation andHistomorphometry

Blinding during histological preparation of

tissue samples was achieved by coding each

animal and by using a different operator for

the surgery (JA) and histomorphometry

(CV). Undecalcified resin embedded tissue

sections were prepared as previously

described (Donath & Breuner 1982; Boss-

hardt et al. 2011; Lang et al. 2011). The ini-

tial trimming of the fixed tissue samples

was performed to exclusively contain the

maxillary sinus. Two coronal samples (each

sample = 3 mm thick) were subsequently

obtained from the centre of each augmented

sinus by cutting perpendicular to the dorso-

ventral axis of the augmented sinus (Fig. 2a,

b). One sample was kept and not processed

further, whilst the other was dehydrated in

a graded series of ethanol and resin infil-

trated (methyl methacrylate/glycol methac-

rylate, Technovit 7200, Heraeus Kulzer,

Germany). Once cured, resin blocks were

ground using an EXAKT 400 CS microgrind-

ing system to expose the samples, glued

onto a glass slide and subsequently sec-

tioned to a thickness of 150 lm using an

EXAKT 300 cutting system (Exact Apparate-

bau, GmbH Norderstedt Germany). A single

slide per sinus was polished down to a

thickness of 20 lm using the EXAKT 400

CS microgrinding system. The thin speci-

mens were then stained with 0.1% tolui-

dine blue (Sigma Aldrich, St Louis, MO,

USA) for light microscopy.

Histomorphometric measurements were

performed blinded and carried out using a

specialized histomorphometric software pro-

gram (Aperio ImageScope version 12.1, Ape-

rio Technologies Inc©, Nussloch, Germany).

A single specimen was examined from each

augmented sinus. The sinus sample was

divided into three zones. The delineation of

the three zones was made after examining

the microscopic appearance of the whole

sample and dividing it into three regions of

equal surface area size (Fig. 3). Zone 1 repre-

(a) (b)

(c) (d)

(e) (f)

Fig. 1. A clinical photograph outlining the surgical steps of maxillary sinus augmentation in the sheep model. (a)

Extra-oral incision, (b) Full flap elevation & antrostomy preparation, (c) Schneiderian membrane elevation, (d) Bone

substitute selection and delivery, (e) Resorbable membrane placement, (f) Primary wound closure.

(a)

(b)

Fig. 2. (a) The maxilla was orientated such that coronal

cuts can be made within each sinus perpendicular to

the dorsoventral axis of the maxilla. (b) The cuts

yielded 3 mm sections that were then prepared histo-

logically. SW, superior wall (or roof) of the maxillary

sinus; MW, medial wall of the maxillary sinus; IW,

inferior wall (or floor) of the maxillary sinus; LW, lat-

eral wall of the maxillary sinus; AS, augmented sinus.

736 | Clin. Oral Impl. Res. 27, 2016 / 734–743 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al !Bio-Oss Collagen in Sinus Augmentation

sented the region immediately adjacent to

the resident wall of bone. Zone 2 represented

the middle zone and zone 3 the zone furthest

from the resident wall and adjacent to the

sinus membrane. Histomorphometric analy-

sis assessed the percentages (i.e. area fraction)

of mineralized bone, residual graft and soft

tissue components (i.e. connective tissue

and/or bone marrow) within each zone. In

addition, the degree of graft particle osseoin-

tegration in each of the three zones was also

assessed and expressed as a percentage of the

total graft particle surface area.

The definitions of each parameter are as

follows (Fig. 4):

• % NB = area of newly formed bone/total

zonal area

• % RG = area residual graft particles/total

zonal area

• % STM = area of soft tissue and marrow

spaces/total zonal area

• % OI = area ABBM particles in direct

contact with bone/total ABBM particle

area

Statistics

Quantitative data were analysed using IBM

SPSS Statistics for Windows version 21.0

(IBM Corporation 2012©, Armonk, NY,

USA). Generalized estimating equations were

used using individual animals as the cluster-

ing variable. Zone, time and treatment were

used as explanatory variables along with all

two-way interactions. A backward elimina-

tion procedure was used for arriving to the

most parsimonious model. A P ≤ 0.05 was

considered to represent statistically signifi-

cant differences.

Results

The surgical wounds in all animals healed

completely within 10–14 days with no signs

of complications.

Gross histological features

The augmented region within the intact

sinus cavity was identified by the obvious

retained volume and change in the Schneide-

rian membrane topography. The graft parti-

cles from both groups were also easily

identified (Fig. 5). Bone of varying degrees of

maturity was observed over both time points

in both treatment groups. This ranged from

woven bone to mature bone with longitudi-

nal and concentric lamellae (Fig. 5). The

presence of BMUs is also apparent in both

groups (Figs 5 and 6). Bridging between graft

particles was evident with new bone forma-

tion seen to be continuous around and

between graft particles (Fig. 6). Most samples

exhibited residual graft particles and minimal

new bone directly in contact with the Schne-

iderian membrane in both groups at both

time points (Fig. 7). Figs 8 and 9 represent

sections of the test and control groups at

weeks 8 and 16, respectively.

% New Bone (%NB)

We tested all two-way interactions and main

effects (Fig. 10a,b). Given that all two-way

interactions were significant, we subse-

quently tested the three-way interaction.

This was found to be statistically significant

and the most parsimonious model. Thus, the

final model included the three-way interac-

tion, all two-way interactions and the main

effects.

At week 8, both the control group

(ABBM + AB) and the test group (ABBM-C)

exhibited significantly decreased %NB the

further the zone is located from the resident

wall. In the control group (ABBM + AB) at

week 8, the order of presentation of %NB is

as follows: zone 1 > 2 > 3 (P < 0.001). Simi-

larly, in the test group at week 8, %NB in

zone 1 is >2 (P < 0.001), zone 1 is >3

(P < 0.001) and zone 2 is >3 (P < 0.01). A sta-

tistically significant greater %NB was noted

in the test group when compared to the con-

trol group at week 8 in zone 1 (P < 0.01).

At week 16, a significant increase in %NB

was evident across all three zones in the con-

trol group (ABBM + AB) when compared to

week 8 in zone 1 (P < 0.01), zone 2

(P < 0.001) and zone 3 (P < 0.001). This was

especially apparent in zone 3. As such, the

impact of zone is not readily apparent in this

group with only zone 2 having a greater %

NB than zone 3 (P < 0.001). In the test group

however, there was a little change in % NB

over time with only zone 2 exhibiting a sig-

nificant increase (P < 0.01). The zonal pattern

as such in the test group (ABMM-C)

remained the same, zone 1 > 2 (P < 0.05),

zone 1 > 3 (P < 0.001) and zone 2 > 3

(P < 0.001). As such, a significantly greater %

NB was evident in the control group when

compared to the test group in zones 2

(P < 0.001) and 3 (P < 0.001).

% Osseointegration (%OI)

Time*Treatment was the only significant 2-

way interaction (P < 0.001) (Fig. 10c,d). There

was a significant increase in %OI in week 16

when compared to week 8 across all three

Fig. 3. Augmented maxillary sinus divided into three

equal zones for histomorphometric assessment. Zone 1

represents the region immediately adjacent to the resi-

dent wall of bone. Zone 2 represents the middle zone,

and zone 3 is the zone furthest from the resident wall

and adjacent to the sinus membrane.

Fig. 4. A part histological image illustrating the histomorphometric method employed. This method involves out-

lining the area of new bone (%NB), residual graft particles (%RG), soft tissue/marrow spaces (%STM) and particle

osseointegration (%OI) within the entire augmented sinus, thus allowing the calculation of the relative fractions of

each parameter.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 737 | Clin. Oral Impl. Res. 27, 2016 / 734–743

Alayan et al !Bio-Oss Collagen in Sinus Augmentation

zones in the control group (ABBM + AB)

(P < 0.001). There were no significant

changes in %OI over time in the test group

(ABBM-C) (P > 0.05). %OI in the control

group was significantly greater across all

three zones when compared with the test

group at week 16 (P < 0.001) but not at week

8 (P > 0.05).

Zone was found to be a significant main

effect (P < 0.001) that was independent of

time and treatment with decreasing %OI

noted in distant zones. Zone 2 had 12.58%

("6.36) less OI than zone 1 (P < 0.05). Zone 3

had 41.25% ("6.37) less OI than zone 1

(P < 0.001). Zone 3 had 28.68% less OI than

zone 2 (P < 0.001).

% Residual Graft (%RG)

Zone*Treatment was the only significant 2-

way interaction (P < 0.01) (Fig. 11a,b). %RG

significantly increased in the test group

(ABBM-C) in the more distant zones from the

resident bone wall. At both weeks 8 and 16

in the test group, %RG in zone 3 > 2 > 1

(P < 0.01). In addition, the test group (ABBM-

C) had significantly greater % RG when com-

pared to control group in both zones 2

(P < 0.001) and 3 (P < 0.05). %RG did not sig-

nificantly change with time (P > 0.05) for

both groups.

% Soft Tissue (%ST)

Time*Treatment was the only significant 2-

way interaction (P < 0.001) (Fig. 11c,d). There

was a significant reduction in %ST in week

16 when compared to week 8 across all three

zones in the control group (ABBM + AB)

(P < 0.001). There were no significant

changes in %ST over time in the test group

(ABBM-C) (P > 0.05). %ST in the test group

was significantly greater across all three

zones when compared to the control group at

week 16 (P < 0.001) but not at week 8

(P > 0.05).

Zone was found to be a significant main

effect (P < 0.001) that was independent of

time and treatment with increasing %ST

noted in distant zones. Zone 2 had 10.01%

("4.44) more ST than zone 1 (P < 0.05). Zone

3 had 21.1% ("3.63) more ST than zone 1

(P < 0.001). Zone 3 had 11.08% more ST than

zone 2 (P < 0.001).

Discussion

The aim of this study was primarily to com-

pare the histological and histomorphometric

features of collagen-stabilized anorganic

bovine bone (test, ABBM-C) to anorganic

bovine bone + autogenous bone (control,

ABBM + AB) grafting materials in augmented

sheep maxillary sinuses. Sheep is a well-

established animal model for maxillary sinus

augmentation using an extra-oral approach

(Haas et al. 1998a, 2002a; Philipp et al. 2013).

The characteristics of the Schneiderian mem-

brane in sheep are anatomically comparable

with humans (Estaca et al. 2008), and it does

not have the disadvantage of an excessively

thick cortical bone layer such as that found

in pigs (Estaca et al. 2008).

The composite graft of anorganic bovine

bone and autogenous bone was chosen as the

control group as this combination has been

utilized in multiple studies of maxillary

sinus floor augmentation (Maiorana et al.

2000; Hallman et al. 2001a,b, 2002b; Hatano

et al. 2004; Hallman, et al. 2005; Galindo-

Moreno et al. 2010). Recent studies indicate

that this combination of autogenous bone

and Bio-Oss (ABBM) has the advantages of

Fig. 5. A histological image illustrating the varying degrees of maturity in the regenerated bone (test group week

16). Note the presence of woven bone, lamellar bone and BMUs (asterisks). ABBM, anorganic bovine bone mineral

particles; LB, lamellar bone; WB, woven bone. Toluidine blue staining.

Fig. 6. A histological image illustrating the bridging of ABBM particles by regenerated new bone (control group

week 16). ABBM, anorganic bovine bone mineral particles; NB, new bone. Toluidine blue staining.

Fig. 7. A histological image illustrating the lack of bone in the Schneiderian membrane region and the lack of

osseointegration of graft particles (test group week 16). ABBM, anorganic bovine bone mineral particles; ST, soft tis-

sue; SM, Schneiderian membrane. Toluidine blue staining.

738 | Clin. Oral Impl. Res. 27, 2016 / 734–743 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al !Bio-Oss Collagen in Sinus Augmentation

reduced graft resorption and increased bone-

to-implant contact formation (Jensen et al.

2012, 2013).

The test group received Bio-Oss Collagen!

(ABBM-C) which is comprised of ABBM gran-

ules with the addition of 10% highly purified

porcine type 1 collagen. The role of the colla-

gen is to act as a cohesive for the granules,

and the material is delivered in a block for-

mat. As such, this format may have some

advantages in sinus augmentation, especially

where a particulate material is to be avoided

such as during minor sinus membrane perfo-

ration. A histological assessment of ABBM-C

in extraction sockets of beagle dogs found

that the amount of residual bovine bone min-

eral remained unchanged in the socket dur-

ing the entire healing period, whereas the

collagen portion was rapidly removed (Araujo

et al. 2010). It was hypothesized that hydro-

lytic enzymes (collagenase) released from

activated PMN cells were involved in the

rapid degradation of the porcine collagen por-

tion of the graft. However, there are no

studies reporting on the outcome of this

material after maxillary sinus floor elevation.

The histomorphometric analysis of the

augmented sinuses was performed at various

distances from the resident maxillary bone

wall to assess the impact of resident wall

proximity on the outcomes. Further, two

time points were included to gain some

insight into the temporal healing patterns in

augmented sinuses.

A decline in new bone formation was

observed within the most distant zone away

from the resident bone walls, as evidenced by

the reduced % NB in zones 2 and 3 when

compared to zone 1 in both the test (ABBM-

C) and control groups (ABBM + AB) at week

8. New bone formation in any bone defect

including the augmented sub-antral space of

maxillary sinuses occurs via a widely con-

served process of neo-osteogenesis. This pro-

cess requires the recruitment, migration and

differentiation of osteogenic cells into osteo-

blasts, leading to synthesis and deposition

of a collagenous extracellular matrix for

mineralization (Lundgren et al. 2004). It also

requires an environment with adequate blood

supply (Ham & Harris 1971), mechanical sta-

bility and sufficient space availability

(Schenk 1987).

The process of osteogenesis is modified

with the introduction of grafting materials as

there is interaction of the inserted bone sub-

stitutes with host cells, extracellular matrix

and signalling molecules (Khan et al. 2005).

Thus, there is bidirectional interaction

between the microenvironment of the maxil-

lary sinus and the properties of a given bone

substitute which can significantly influence

the osteogenic response (Donath 1991; Busen-

lechner et al. 2009). As osteogenesis extends

progressively towards the central and distal

areas of the graft (Haas et al. 2002a,b), it is

important for the biomaterial to be highly os-

teoconductive and therefore promote the

ingrowth of capillaries, peri-vascular tissue

and osteoprogenitor cells from the recipient

bed (Burchardt 1983).

A gradient of new bone formation was

observed in this study, with more new bone

formation in regions proximal to the resident

walls and reduced new bone formation at

more distant regions proximal to the elevated

membrane. This gradient of new bone forma-

tion was also observed by a number of other

studies (Fuerst et al. 2004; Busenlechner

et al. 2009; Scala et al. 2010).

Early studies of sinus augmentation sug-

gested that the source of the osteogenic cells

and molecules could be the floor (Boyne &

James 1980) or lateral wall (Misch & Dietsh

1991) of the maxillary sinus. Our results sup-

port the premise that the process of osteogen-

esis begins and is enhanced in regions

proximal to the maxillary sinus bony walls.

This observation has been further supported

by more recent studies in mini-pigs (Fuerst

et al. 2004; Busenlechner et al. 2009), rabbits

(Xu et al. 2003, 2004), primates (Margolin

et al. 1998; Scala et al. 2010, 2012) and

humans (Boyne et al. 1997; Lundgren et al.

2004).

In this study, minimal new bone formation

was seen in direct contact with the Schneide-

rian membrane (Fig. 7). Sporadic bone islands

have been noted along the Schneiderian

membrane by previous authors, but their ori-

gin remained unclear (Haas et al. 2002a). In a

study of early healing (1 month) after sinus

membrane elevation in monkeys (Scala et al.

2010), new bone formation was seen to origi-

nate from the resident bony walls of the

sinus, but not from the Schneiderian mem-

brane even though the membrane was found

to be in direct contact with bone. These

(a)

(b)

Fig. 8. Histological images representing week 8 samples from (a) control (anorganic bovine bone mineral

(ABBM) + AB) and (b) test (ABBM-C) groups. Toluidine blue staining.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 739 | Clin. Oral Impl. Res. 27, 2016 / 734–743

Alayan et al !Bio-Oss Collagen in Sinus Augmentation

results are in contrast to in vitro and in vivo

studies, which suggest that the sinus mucosa

has osteogenic potential (Gruber et al. 2004;

Srouji et al. 2009). In a pre-clinical study in

which sinus membrane elevation and simul-

taneous implant placement were performed

in monkeys (Palma et al. 2006), the Schne-

iderian membrane was seen to be in contact

with newly formed bone after 1 month of

healing, but there was no evidence that it

participated in the early phase of hard tissue

formation.

Increased healing time improved the per-

centage of new bone formed in ABBM + AB

group in the distant zones (zones 2 and 3)

where a greater amount of new bone was

noted at week 16 when compared to week 8

in the control group. This may indicate a

rapid bone healing response in the zone prox-

imal to the resident wall (zone 1), which did

not subsequently benefit from additional

healing time. The distant zones, however,

may have experienced delayed neo-osteogene-

sis and therefore benefited from the extended

healing time and the addition of autogenous

bone. This may also explain why the

improved %NB was only seen in the

ABBM + AB in zone 3 at week 16 but not at

week 8. Notably, this improvement in new

bone formation was not seen in ABBM-C

group in the distant zone. These findings

would suggest that the nature of the grafting

material had a greater influence over the

degree of new bone formation in regions dis-

tant from the resident walls especially when

given sufficient time and had minimal influ-

ence in regions proximal to the resident wall,

where there is enhanced inherent osteogenic

potential. These findings were supported by

another study that histologically assessed

maxillary sinus elevation in sheep using

ABBM, autogenous bone or no filler (Haas

et al. 1998b) and reported that grafting had

no additional osteoconductive effect in

regions proximal to the resident bony wall.

Indeed, elevation of the Schneiderian mem-

brane alone resulted in new bone formation

in the region proximal to the resident bony

wall.

These findings for %NB are in contrast to

what was observed for % ST. Greater soft tis-

sue fractions were observed in the distant

zones in both treatment groups.

The increase in bone formation seen in the

control group (ABBM + AB) over time inver-

sely correlated with a reduction in soft tissue

seen over time in this group. This was espe-

cially so in the most distant zone.

A reduced degree of particle osseointegra-

tion was seen in the distant zones (zones 2

(a)

(b)

Fig. 9. Histological images representing week 16 samples from (a) control (anorganic bovine bone mineral

(ABBM) + AB) and (b) test (ABBM-C) groups. Toluidine blue staining.

Fig. 10. Area fraction of new bone (%NB) and degree of anorganic bovine bone mineral Particle osseointegration (%

OI) at week 8 and week 16. C, control group; T, test group; 1, zone 1; 2, zone 2; 3, zone 3; #, mean.

740 | Clin. Oral Impl. Res. 27, 2016 / 734–743 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al !Bio-Oss Collagen in Sinus Augmentation

and 3). This correlates with the decrease in

new bone formation in these zones. The

observed increase in bone formation over

time in the distant zone in the control

ABBM + AB group may have promoted fur-

ther osseointegration of particles in this

group. As this increase in bone formation

over time was not observed in the test

ABBM-C group, the degree of particle osseo-

integration similarly did not improve and

was lower than the control group. Similar

findings were reported in a study of aug-

mented sheep sinuses (Haas et al. 1998a)

where no new bone formation was found in

contact with the ABBM particles distant to

the resident bone. It is unclear whether the

reduced new bone formation and particle

osseointegration in the ABBM-C group in the

distant zones is due to an absence of autoge-

nous bone or due to the characteristics of the

material.

Large variation in the degree of particle

osseointegration was evident in the current

study, as reflected in the wide standard devia-

tions recorded for this parameter in both

groups at both time points. This may have

been exacerbated by the small number of ani-

mals used in the week 8 group.

In summary, ABBM-C exhibited very simi-

lar histomorphometric parameters to the

composite graft of ABBM + AB in regions

proximal to the resident sinus wall. The per-

centage of new bone and residual graft parti-

cle osseointegration were greatest in the

zones proximal to the resident bony walls

and gradually decreased as the distance from

the proximal walls increased. In the distant

zone however, there was significantly greater

percentage of new bone and residual graft

particles osseointegration in the ABBM + AB

group when compared to the ABBM-C group.

The clinical implications of these results

is that the choice of graft material

(ABBM + AB vs. ABBM-C) is only relevant if

the intention is to utilize the regenerated

bone in the distant third of the augmented

sinus, in which case ABBM + AB is the

superior material.

Acknowledgements: Jamil Alayan is

the recipient of a scholarship from the

National Health & Medical Research

Council of Australia (ID: APP1056633). Bio-

Oss!, Bio-Oss Collagen! and Bio-Gide! were

kindly provided by Geistlich AG, Wollhusen,

Switzerland. We would like to thank Dr

Dimitrios Vagenas (Biostatistician, Institute

for Health and Biomedical Innovation,

Queensland University of Technology,

Kelvin Grove, Australia) for his assistance

and contribution to the statistical analysis in

this project.

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Supporting Information

Additional Supporting Information may be

found in the online version of this article:

Appendix S1. CONSORT Statement 2001

Checklist.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 743 | Clin. Oral Impl. Res. 27, 2016 / 734–743

Alayan et al !Bio-Oss Collagen in Sinus Augmentation

79

Chapter 3 - A histomorphometric assessment of collagen stabilized anorganic

bovine bone mineral in maxillary sinus augmentation – a prospective clinical

trial.

80

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED

PAPER

This chapter includes a co-authored paper. The bibliographic details of the

co-authored paper, including all authors, are:

Alayan J, Vaquette C, Farah C, Ivanovski S. (2016) A histomorphometric

assessment of collagen stabilized anorganic bovine bone mineral in maxillary sinus

augmentation – a prospective clinical trial. Clinical Oral Implants Research 27: 850

– 858. doi: 10.1111/clr.12694.

My contribution to the paper involved:

Design of the project, the provision of the data, the preliminary analysis and

categorization of the data into a usable format, the provision of direction on the

scope and structure of the analysis, the interpretation of results and drafting of the

article.

04th May 2018

Jamil Alayan

(Countersigned) 16th May 2018

Corresponding author of paper: Saso Ivanovski

(Countersigned) ___________________________ 16th May 2018

Supervisor: Stephen Hamlet

Jamil AlayanCedryck VaquetteCamile FarahSaso Ivanovski

A histomorphometric assessment ofcollagen-stabilized anorganic bovinebone mineral in maxillary sinus aug-mentation – a prospective clinical trial

Authors’ affiliations:Jamil Alayan, Saso Ivanovski, School of Dentistryand Oral Health, Centre for Medicine and OralHealth, Griffith University, Southport, AustraliaCedryck Vaquette, Institute of Health andBiomedical Innovation, Queensland University ofTechnology, Kelvin Grove, AustraliaCamile Farah, UQ Centre for Clinical Research,University of Queensland, Brisbane, Australia

Corresponding author:Saso IvanovskiSchool of Dentistry and Oral HealthCentre for Medicine and Oral HealthGold Coast Campus Griffith UniversitySouthport QLD 4215AustraliaTel.: +61 75 678 0741Fax: +61 75 678 0708e-mail: s.ivanovski@griffith.edu.au

Key words: anorganic bovine bone, Bio-Oss, Bio-Oss Collagen, bone grafting, bone regenera-

tion, bone substitute, clinical trial, histomorphometry, maxillary sinus augmentation, sinus

floor elevation

Abstract

Objective: To histomorphometrically compare the use of collagen-stabilized anorganic bovine

bone (ABBM-C) (test) to anorganic bovine bone + autogenous bone (ABBM + AB) (control) in

maxillary sinus augmentation.

Materials and Methods: Forty (n = 40 sinuses) patients underwent sinus augmentation and

received either control (20 sinuses) or test bone graft (20 sinuses). Bone samples were harvested

from the augmented sinuses 5 months postgrafting. The samples were processed for

histomorphometry, which assessed within the primary region of interest (ROI-1), the area fraction

of new bone (%NB), graft particle osseointegration (% OI), residual graft (%RG), and soft tissue

components (% STM). The same analysis was also carried out in a second region of interest (ROI-2)

located in a zone 1 mm proximal to the previous maxillary sinus floor.

Results: In both ROI-1 and ROI-2, the mean % NB, %RG, and %STM in the control group were

similar to mean values in the test group. The % OI was significantly greater in the control group

(42.0 +/! 26.8) when compared to the test group (19.6 +/! 27.3) in ROI-2 (P < 0.05). No statistically

significant differences were seen when ROI-1 and ROI-2 were compared except for improved %OI

in ROI-2 in the control group. The mean proportion of lamellar bone to woven bone in the control

group (1.22 " 1.48) was significantly greater than the test group (0.38 " 0.29) (P < 0.05).

Conclusion: ABBM-C exhibited very similar histomorphometric parameters to the composite graft

of ABBM + AB. The ABBM + AB group was more mature as indicated by the significantly greater

proportion of lamellar bone when compared to the ABBM-C. Improved % OI was seen in the zone

proximal to the resident bony floor in the ABBM + AB group. Based on histological assessment,

ABBM-C is a suitable bone substitute for the purposes of maxillary sinus augmentation. Its clinical

utility may be indicated in cases of sinus membrane perforation and insufficient autogenous bone

in the local area.

Implant placement in the posterior maxilla is

often prevented by insufficient bone volume

as a result of bone resorption and sinus

pneumatization. Maxillary sinus floor eleva-

tion is the technique most frequently used to

overcome this limitation and is considered a

safe and effective procedure with high

implant survival rates (Del Fabbro et al.

2004; Chiapasco et al. 2009). Nevertheless,

clinically this surgical technique is still being

refined, with a large degree of variation in

many aspects of the procedure.

To date, a variety of bone substitute mate-

rials have demonstrated clinically acceptable

outcomes (Froum et al. 2006; Cordaro et al.

2008; Nkenke & Stelzle 2009; Klijn et al.

2010). However, despite the established clini-

cal efficacy of the technique, the search con-

tinues for a bone substitute with optimal

properties including rapid osteogenesis as

well as long-term stability and space mainte-

nance.

If de novo bone formation is the reference

parameter, then autogenous bone remains

the gold standard (Klijn et al. 2010; Schmitt

et al. 2013). Autogenous bone harvesting,

however, can be associated with postopera-

tive patient morbidity (Clavero & Lundgren

2003; Cricchio & Lundgren 2003) and pro-

longed surgical time and is only present in

Date:Accepted 16 August 2015

To cite this article:Alayan J, Vaquette C, Farah C, Ivanovski S. Ahistomorphometric assessment of collagen-stabilizedanorganic bovine bone mineral in maxillary sinusaugmentation – a prospective clinical trial.Clin. Oral Impl. Res. 27, 2016, 850–858doi: 10.1111/clr.12694

850 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

limited quantities. Autogenous bone grafts

can also exhibit unpredictable resorption

(Davis et al. 1984; Clavero & Lundgren 2003;

Wiltfang et al. 2005; Sbordone et al. 2013),

which may not be a desirable characteristic

in sinus grafting. Indeed, sinuses augmented

using autogenous bone as the sole grafting

material have been shown to undergo signifi-

cant re-pneumatization and loss of aug-

mented volume (Johansson et al. 2001;

Arasawa et al. 2012). These limitations of

autogenous bone have resulted in consider-

able research into alternative bone substi-

tutes. It has been proposed that a slower

resorbing osteoconductive material may pro-

vide superior space maintenance for de novo

bone formation.

Anorganic deproteinized bovine bone min-

eral (ABBM) (Bio-Oss!; Geistlich AG, Woll-

husen, Switzerland) is a widely utilized

commercial product in the form of cortical

granules. It has a natural, non-antigenic por-

ous matrix and is chemically and physically

identical to the mineral phase of human

bone. It has been reported to be highly

osteoconductive and to have a very low

resorption rate (Jensen et al. 1996; Berglundh

& Lindhe 1997; Haas et al. 2002; Orsini

et al. 2005). The pores in Bio-Oss! are of

optimal size and configuration to facilitate

vascular ingrowth, which is essential for

new bone (NB) formation (Klawitter et al.

1976; Daculsi & Passuti 1990). Bio-Oss! is

one of the best-documented biomaterials

used in maxillary sinus augmentation (Artzi

et al. 2001; Hallman et al. 2002; Valentini

& Abensur 2003).

ABBM is also available in a block form that

is stabilized in 10% porcine type-1 collagen

matrix (ABBM-C) (Bio-Oss Collagen!; Geis-

tlich AG). The contained nature of the block

may be advantageous in maxillary sinus aug-

mentation when the common surgical com-

plication of Schneiderian membrane

perforation occurs (Chiapasco et al. 2009).

The impact of this altered formulation of

ABBM has not been investigated in terms of

bone regeneration in maxillary sinus aug-

mentation. This is an important considera-

tion because it has been shown that surface

characteristics and the geometry of a material

have a significant impact on neo-osteogenesis

(Hing et al. 2005).

Therefore, the primary aim of this con-

trolled clinical trial was to histomorphomet-

rically compare the use of collagen-stabilized

anorganic bovine bone (ABBM-C) vs. anor-

ganic bovine bone + autogenous bone

(ABBM + AB) in maxillary sinus floor aug-

mentation. Our secondary objective was to

assess the impact of the resident sinus floor

proximity on histomorphometric outcomes.

Material and methods

Ethics and consent

The University of Queensland School of Den-

tistry Dental Sciences Research Ethics Com-

mittee provided ethical approval for this

project (Project Number: 1010). The project

complies with the provisions contained in

the National Statement on Ethical Conduct

in Research Involving Humans and complies

with the regulations governing experimenta-

tion on humans.

All patients provided informed consent

prior to enrolling in the study.

Inclusion criteria

To be considered eligible for the study, the

following inclusion criteria were applied:

• Males and females, over 21 years of age at

the time of surgery

• Must present with a bone deficiency in

the maxillary sinus region, which necessi-

tates sinus floor augmentation to facili-

tate the placement of one or more dental

implants.

• If bilateral sinus augmentation was

required, only one sinus was used for the

purposes of this study.

• Residual alveolar bone height of the eden-

tulous maxilla below the floor of the

maxillary sinus ≤5 mm and ≥ 1 mm, as

measured on a CT scan in the deepest

floor of the sinus

• Residual alveolar bone width of ≥6 mm as

measured on a CT scan

• Absence of sinus infection

• Generally fit and healthy and able to

undergo oral surgical procedures under

local anesthesia

• Patients with a history of chronic peri-

odontitis were required to be treated first

and subsequently enrolled in a supportive

maintenance program

• Teeth at the surgical site which required

removal were extracted a minimum of

8 weeks prior to sinus floor elevation

Exclusion criteria

Patients were considered ineligible if any of

the following criteria were met:

• Any systemic medical condition that

could interfere with the surgical proce-

dure

• Current pregnancy at the time of recruit-

ment

• Physical handicaps that would interfere

with the ability to perform adequate oral

hygiene

• Alcoholism or chronic drug abuse

• Patients who smoke more than 10 cigar-

ettes per day

• Medications which interferes with bone

formation

• Mucosal diseases such as erosive Lichen

Planus

• History of local radiation therapy

• Severe bruxism or clenching habits

• Signs and symptoms consistent with

acute sinus infections

• Previous guided bone regeneration (bone

graft) or dental implant treatment in the

posterior segments of the maxilla

• Benign or malignant tumors of the maxil-

lary sinus

• Pathological conditions that severely

impact mucociliary clearance including;

chronic sinusitis, primary ciliary dyskine-

sia, or cystic fibrosis.

Bone graft materials and dental implants

Patients were randomly assigned to one of

the following groups:

• Control group – small diameter particles

(0.25–1.0 mm) of deproteinized bovine

bone mineral (Bio-Oss!; Geistlich

AG) + autogenous bone (ABBM + AB) in a

1 : 1 ratio, as determined using a stan-

dardized 2 cc bone collecting chamber.

• Test group – 250 mg blocks of depro-

teinized bovine bone mineral + collagen

(ABBM-C) (Bio-Oss Collagen!, Geistlich

AG).

All patients’ received a single Straumann

(Institut Straumann AG, Basel, Switzerland)

tissue level dental implant 5 months post-

grafting. The implant had a standard micro-

roughened sandblasted and acid-etched (SLA)

surface. The dimensions of the implants were

chosen according to the specific site charac-

teristics.

Participant allocation and study design

The study included 42 (n = 42) patients that

underwent the informed consent process

after meeting the outlined criteria. It was cal-

culated that a sample size of 16 patients per

group was required to detect a 10% difference

in the means of the primary parameter (%

NB), based on 95% confidence level, 80%

power and a predicted variance of 100 (10%

deviation from mean). Twenty-one patients

were recruited per group to allow for possible

dropouts. All patients were recruited prospec-

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 851 | Clin. Oral Impl. Res. 27, 2016 / 850–858

Alayan et al #Bio-Oss collagen in human sinus grafting

tively from the School of Dentistry at the

University of Queensland and from a special-

ist private practice from July 2010 to March

2014. All patients were referred specifically

for replacement of teeth with implant-sup-

ported restorations.

Allocation of each patient to either the test

(ABBM-C) or the control group (ABBM + AB)

was based on two clinical parameters; the

presence of Schneiderian membrane perfora-

tion and/or availability of adequate autoge-

nous bone in the vicinity of the surgical site.

ABBM-C was selected if mild (≤3 mm) sinus

membrane perforations occurred (four cases)

or if there was insufficient autogenous bone

available for harvesting from the buccal

aspect of the posterior maxilla within the

same surgical region. ABBM + AB was alter-

natively used if the Schneiderian membrane

remained intact and if there was sufficient

autogenous bone that could be harvested

from the buccal wall of the maxilla.

Five months later, all participants under-

went surgical implant placement whereby

they received a single implant in the aug-

mented site. At this time, a single bone

biopsy was harvested during the osteotomy

preparation and collected from a total of 40

patients (920 from test group and 920 from

the control group). These samples were used

for the histological assessment.

The same clinician (JA) performed all of

the surgical procedures. While clinician

blinding was not possible at the time of the

augmentation surgery, it was possible during

the data collection and analysis stages of this

project.

Presurgical procedures

Prior to surgery 3-dimensional CT scanning

was carried on all patients to determine the

site anatomy and to identify patients with

significant sino-nasal pathology. All partici-

pants started systemic antibiotic therapy

(1000 mg amoxycillin or 300 mg clin-

damycin) 24 h prior to surgery. A 0.2%

chlorhexidine mouth rinse was utilized for

60 s immediately prior to grafting surgery.

Surgical procedure – sinus floor elevation

All patients were treated with the same sur-

gical technique consisting of sinus floor ele-

vation via a lateral wall (modified Caldwell-

Luc) approach. After elevation of a full buccal

mucoperiosteal flap, a full antrostomy was

created in the lateral wall of the maxilla

using a bone scraper (Safescraper Twist,

META, C.G.M Sp.A, Reggio Emiia, Italy) and

a Piezo-electric surgical unit (Implant Center,

Satelec, Acteon, France) to gain access to the

maxillary sinus. The size, shape, and location

of the antrostomy were variable and tailored

to the site and patient’s characteristics. The

only consistent feature of the antrostomy

was the superior osteotomy, which was

located 10 mm superior/apical to the alveolar

bony crest. The sinus membrane was then

dissected and elevated in all directions to cre-

ate an intra-sinus space that received one of

the two grafting materials (Fig. 1). The mem-

brane was elevated superiorly to allow for

grafting material packing up to 10 mm apical

to the alveolar bone crest. This was achieved

using standard sinus membrane elevator

spoons. Sufficient material was placed until

the desired 10 mm of vertical height was

achieved. The material was carefully packed

to ensure maximum contact with resident

bony walls, with care taken to avoid over-

packing.

Autogenous bone chips collected using a

bone scraper were used for the autogenous

bone component needed for the control

group. If additional autogenous bone was

required, it was harvested from the zygo-

matic buttress within the same surgical field.

The reconstructed sites were covered with a

porcine collagen membrane (Bio-Gide; Geis-

tlich AG). The mucoperiosteal flaps were

then sutured using a non-resorbable thermo-

plastic polyether-ester monofilament suture

(Dyloc 5/0; Dynek, Adelaide, Australia) to

achieve primary closure.

A postoperative regimen of amoxycillin

500 mg t.d.s for 7 days (or if allergic 150 mg

clindamycin q.i.d for 6 days) was prescribed,

as was paracetamol 500 mg, ibuprofen

200 mg and 0.2% chlorhexidine mouth rinse

b.d for 7 days. Patients were reviewed at 1

and 2 weeks postoperatively.

Surgical procedure – dental implant placementand biopsy

A preimplant placement CT scan was taken

5 months later when dental implant place-

ment was performed. All participants began

systemic antibiotic therapy (1000 mg amoxy-

cillin or 300 mg of clindamycin) 60 min prior

to surgery. A 0.2% chlorhexidine mouth

rinse was utilized for 60 s immediately prior

to surgery. A single bone biopsy was har-

vested from each participant during the

osteotomy preparation using a standard tre-

phine bur with an internal/external diameter

of 2.0/3.0 mm, respectively. Drilling with the

trephine was taken as far apically as possible

without perforating the Schneiderian mem-

brane, as predetermined on the preimplant

CT scan. The trephine was directed in the

exact three dimensional position/axis as the

dental implant osteotomy. The osteotomy

preparation was then completed, and the

implant was delivered. The mucoperiosteal

flap was then sutured using a non-resorbable

thermoplastic polyether-ester monofilament

suture (Dyloc 5/0; Dynek) to achieve primary

closure.

A postoperative regimen of amoxycillin

500 mg three times daily for 7 days was pre-

scribed, together with paracetamol 500 mg,

ibuprofen 200 mg, and 0.2% chlorhexidine

mouth rinse twice daily for 7 days. Patients

were reviewed 1-week postoperatively.

The bone samples were immediately stored

in paraformaldehyde until further histological

processing.

Histological preparation and histomorphometry

Blinding during histological preparation of

tissue samples was achieved by coding each

sample and using a different operator for the

surgery (JA) and histomorphometry (CV).

Undecalcified resin embedded tissue sections

were prepared as previously described

(Donath & Breuner 1982; Schlegel & Donath

1998; Schmitt et al. 2015). Each sample was

fixed in 4% paraformaldehyde phosphate-buf-

fered formalin. The samples were cut along

the long axis of the biopsy and divided into

two. One sample was kept and not processed

further, while the other was dehydrated in a

graded series of ethanol and resin infiltrated

(Methyl Methacrylate/Glycol Methacrylate,

Fig. 1. Clinical photographs showing the surgical place-

ment of the test group grafting material (ABBM-C). (a)

An ABBM-C block. (b) The ABBM-C blocks after they

were delivered into the maxillary intra-sinus space.

852 | Clin. Oral Impl. Res. 27, 2016 / 850–858 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al #Bio-Oss collagen in human sinus grafting

Tecknovit 7200, Heraeus Kulzer, Hanau, Ger-

many). Once cured, resin blocks were ground

using an EXAKT 400 CS microgrinding sys-

tem to expose the samples, glued onto a glass

slide and subsequently sectioned to a thick-

ness of 150 lm using an EXAKT 300 cutting

system (Exact Apparatebau GmbH, Norderst-

edt, Germany). A single slide per sample was

polished down to a thickness of 20 lm using

the EXAKT 400 CS microgrinding system.

The specimens were then stained with 0.1%

toluidine blue (Sigma-Aldrich, St Louis, MO,

USA) for light microscopy.

Histomorphometric measurements were

performed blinded and carried out using a

specialized histomorphometric analysis soft-

ware program (Aperio ImageScope version

12.1; Aperio Technologies Inc©, Nussloch,

Germany) (Fig. 2) by the same operator (JA).

Two regions of interest (ROI) were delineated

after carefully studying the microscopic

appearance of the whole biopsy. The first

region of interest (ROI-1) represented the area

from the junction of the preexisting or native

bone (i.e., the former floor of the sinus cav-

ity) to where the biopsy broke during the har-

vesting procedure (Fig. 2). The second region

of interest (ROI-2) represented the area from

the junction of the preexisting or native bone

(i.e., the former floor of the sinus cavity) and

ended 1 mm apically into the sample (Fig. 2).

Histomorphometric analysis assessed the

percentages (i.e., area fraction) of mineralized

bone (% NB), residual graft (%RG), and soft

tissue components (i.e., connective tissue

and/or bone marrow) (%STM) within each

ROI. In addition, the degree of graft particle

osseointegration (%OI) in each ROI was also

assessed and expressed as a percentage of the

total graft particle surface area (Fig. 3).

The definitions of each parameter are as

follows (Fig. 3):

• % NB = area of newly formed bone/area

ROI.

• % RG = area residual graft particles/area

ROI.

• % STM = area of soft tissue and marrow

spaces/area ROI.

• % OI = area particles in direct contact

with bone/total particles area ROI.

In addition, the ratio of lamellar bone to

woven bone was compared between control

and test groups.

Statistics

Area fractions and the ratio of lamellar bone

to woven bone were analyzed using IBM

SPSS Statistics for Windows version 21.0

(IBM Corporation 2012©, Armonk, NY,

USA). A two-sample, two-tailed t-test was

performed after assessing normality of the

data set using histogram, skewness, and kur-

tosis. Homogeneity of variance was assessed

using the Levene’s test. Mean difference, con-

fidence intervals, and P-values were adjusted

if equal variance could not be assumed. Simi-

larly, baseline parameters, including residual

radiological bone height and the area of ROI,

were compared between test and control

groups using a t-test. If data were not found

to be normally distributed, then a Mann–

Whitney U-test was used. The null hypothe-

sis was that of no difference between control

and test groups for all histomorphometric

parameters. A P ≤ 0.05 was considered to rep-

resent statistically significant differences

between test and control groups. All nor-

mally distributed data were expressed as

mean " standard deviation. The median was

reported for values calculated using a Mann–

Whitney U-test. In addition, mean difference

and the 95% confidence interval of the differ-

ence are provided where applicable for com-

parative analysis (Tables 1 and 2).

The primary outcome measure includes

the area fractions in control and test samples

in ROI-1. The secondary outcome measure

includes the area fractions in control and test

samples in ROI-2.

Results

Study population

Forty-two (n = 42) patients were enrolled in

this study and underwent sinus augmenta-

tion. Prior to sinus grafting, the mean resi-

dent bone ridge height in the deepest portion

of the maxillary sinuses floor was

2.4 " 0.75 mm in the control group and

2.2 " 0.87 mm in the test group. The mean

difference was 0.19 mm (95% CI !0.34–0.71)

and was not significant (P ≤ 0.49). A total of

40 patients (n = 40) underwent sample har-

vesting and histomorphometric analysis (con-

trol group n = 20) (test group n = 20) with

one sample harvested per sinus and a single

dental implant placed in each site (n = 20

control group) (n = 20 test group). Two

patients could not undergo sample harvesting

in the implant site due to limited access.

The mean patient age in the control group

was 57.70 years with a female/male ratio of

2.3. The mean patient age in the test group

was 54.60 years with a female/male ratio of

3.0. Wound healing proceeded as expected

with no signs of impaired wound healing or

wound dehiscence. A total of four membrane

perforations occurred in the test group of

patients. These perforations were small

(≤3 mm in diameter) and repaired by creating

a pouch using the placement of a resorbable

Fig. 2. Histological images outlining the histomorpho-

metric analysis after each parameter of interest was

delineated such as the residual graft (RG) particles, new

bone, and RG particle osseointegration. (a) First region

of interest (ROI-1) represents the area from the junction

of the preexisting or native bone (i.e., the former floor

of the sinus cavity) to where the biopsy broke during

the harvesting procedure. (b) Second region of interest

(ROI-2) represents the area from the junction of the pre-

existing or native bone (i.e., the former floor of the

sinus cavity) and ends 1 mm apically into the sample.

Fig. 3. A high magnification part histological image highlighting the area of new bone (%NB), residual graft parti-

cles (%RG), soft tissue/marrow spaces (%STM), and particle osseointegration (%OI) within a region of interest

(ROI) allowing then the calculation of the relative fractions of each parameter.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 853 | Clin. Oral Impl. Res. 27, 2016 / 850–858

Alayan et al #Bio-Oss collagen in human sinus grafting

porcine collagen membrane (Bio-Gide; Geis-

tlich AG) as per Proussaefs & Lozada (Prous-

saefs & Lozada 2003). Symptoms consistent

with postoperative sinusitis were observed in

one patient in the control group, which

resolved spontaneously within 48 h after

sinus grafting.

Gross histological features

Common histological features observed in

both groups included readily identifiable RG

particles, presence of NB around and

between graft particles, and a significant

non-mineralized (soft tissue/marrow) tissue

fraction (Fig. 4). Bone of varying degrees of

maturity was observed in both treatment

groups (Fig. 5). This ranged from woven

bone to mature bone with longitudinal and

concentric lamellae (Fig. 5). The presence of

bone multicellular units (BMUs) was also

apparent in both groups. The mean propor-

tion of lamellar bone to woven bone in the

control group (1.22 " 1.48) was significantly

greater than the test group (0.38 " 0.29).

The mean difference was 0.84 (95% CI

0.14–1.54) and represented a significant dif-

ference (P ≤ 0.021).

Comparison between ROI-1 and ROI-2

The mean area for ROI-1 in the control group

(6.92 " 4.18 mm2) was similar to the ROI-1

in the test group (7.73 " 3.53 mm2) (Fig. 6

and Table 1). The mean difference was

!0.81 mm2 (95% CI !3.29–1.67) and was not

significant (P ≤ 0.51). The mean area for ROI-

2 in the control group (2.09 " 0.82 mm2) was

similar to the ROI-2 in the test group

(1.95 " 0.41 mm2). The mean difference was

0.14 mm2 (95% CI !0.28–0.56) and was not

significant (P ≤ 0.49).

In the control group, ROI-2 had greater %

NB (ROI-1: 29.06 " 13.51 vs. ROI-2:

34.03 " 13.05), greater %OI (ROI-1:

26.34 " 14.30 vs. ROI-2: 42.03 " 26.83),

lower %RG (ROI-1: 19.11 " 8.87 vs. ROI-2:

15.59 " 9.93), and lower %STM (ROI-1:

51.82 " 10.98 vs. ROI-2: 50.38 " 10.96)

when compared to ROI-1 (Fig. 6). The mean

differences were not statistically significant,

except for the %OI (P ≤ 0.03) (Table 1).

In the test group, ROI-2 had greater %NB

(ROI-1: 30.71 " 12.24 vs. ROI-2:

39.68 " 17.36) and lower %OI (ROI-1: 25.93

vs. ROI-2: 4.49), lower %RG (ROI-1: 14.11 vs.

ROI-2: 3.86) and lower %STM (ROI-1:

54.18 " 10.53 vs. ROI-2: 49.82 " 20.47)

when compared to ROI-1 (Fig. 6). These dif-

ferences were not statistically significant

except for the %OI (P ≤ 0.05) and %RG

(P ≤ 0.05) (Table 1).

Comparison between test and control groups

In ROI-1, the control and test groups had

very similar mean values for all parameters:

%NB (control: 29.06 " 13.51 vs. test:

30.71 " 12.24), %OI (control: 26.34 " 14.30

vs. test: 26.57 " 18.14), %RG (control:

19.11 " 8.87 vs. test: 15.11 " 9.65), and %

STM (control: 51.82 " 10.98 vs. test:

54.18 " 10.53) (Fig. 6 and Table 2). These

mean differences were not statistically signif-

icant (Table 2).

Similarly, in ROI-2, the control and test

groups had very similar mean values for the

following parameters: %NB (control:

34.03 " 13.05 vs. test: 39.68 " 17.36) and %

STM (control: 50.38 " 10.96 vs. test:

49.82 " 20.47) (Fig. 6). These mean differ-

ences were not statistically significant

(Table 2). The %OI and %RG, however, were

greater in the control group when compared

to the test group (%OI control: 48.62 vs. test:

4.49) and (%RG control: 13.21 vs. test: 3.86)

(Fig. 6). These differences were statistically

significant (P ≤ 0.05) (Table 2).

Discussion

The aim of this prospective clinical trial

was primarily to compare the histological

and histomorphometric features of colla-

gen-stabilized anorganic bovine bone (test,

ABBM-C) and anorganic bovine bone + au-

togenous bone (control, ABBM + AB) graft-

ing materials in augmented maxillary

sinuses. There are no reported studies

assessing collagen-stabilized anorganic

bovine bone (test, ABBM-C) in maxillary

sinus augmentation.

Table 1. Outcomes of histomorphometric evaluation comparing ROI-1 (total sample area) vs. ROI-2(area proximal to resident wall) within samples taken from grafted maxillary sinuses

Parameter P-value Mean difference

95% Confidenceinterval of themean difference

ROI-1 vs. ROI-2 Group: control (ABBM + AB) (n = 20)

% NB 0.24 !4.97 !13.48–3.53% OI 0.03 !15.69 !29.59–(!1.78)% RG 0.24 3.52 !2.5–9.55% STM 0.68 1.44 !5.57–8.47

ROI-1 vs. ROI-2 Group: test (ABBM-C) (n = 20)

% NB 0.07 8.96 !18.58–0.65% OI* 0.014 NA NA% RG* 0.02 NA NA% STM 0.40 4.36 !6.18–14.89

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM-C, collagen-stabilized anor-ganic bovine bone mineral; ROI-1, border of previous sinus floor-to-most apical portion of the regionof the sample; ROI-2, border previous sinus floor-to-1 mm apical into the sample; NB, new bone; RG,residual graft particles; STM, soft tissue/marrow spaces; OI, particle osseointegration; SD, standarddeviation; P, P-value for inter-group comparison after applying a t-test.*Mann–Whitney U-test was carried out on these parameters.

Table 2. Outcome of histomorphometric evaluation comparing control (ABBM + AB) vs. test(ABBM-C) samples taken from grafted maxillary sinuses

Parameter P-value Mean difference

95% Confidenceinterval of themean difference

Control vs. Test groups ROI-1 (n = 20) total sample area

% NB 0.69 !1.65 !9.90–6.60% OI 0.96 !0.23 !10.69–10.22% RG 0.18 4.01 !1.93–9.94% STM 0.49 !2.35 !9.24–4.53

Control vs. test groups ROI-2 (n = 20) area proximal to resident wall

% NB 0.25 !5.65 !15.48–4.18% OI* 0.01 NA NA% RG* 0.03 NA NA% STM 0.92 0.55 !10.06–11.17

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM-C, collagen-stabilized anor-ganic bovine bone mineral; ROI-1, border of previous sinus floor-to-most apical portion of the regionof the sample; ROI-2, border previous sinus floor-to-1 mm apical into the sample; NB, new bone; RG,residual graft particles; STM, soft tissue/marrow spaces; OI, particle osseointegration; SD, standarddeviation; P, P-value for inter-group comparison after applying a t-test.*Mann–Whitney U-test performed on these parameters.

854 | Clin. Oral Impl. Res. 27, 2016 / 850–858 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al #Bio-Oss collagen in human sinus grafting

The composite graft of anorganic bovine

bone and autogenous bone was chosen as the

control group as this combination has been

utilized in multiple studies of maxillary

sinus floor augmentation (Maiorana et al.

2000; Hallman et al. 2001a,b, 2002, 2005,

2009; Hatano et al. 2004; Cannizzaro et al.

2009; Kim et al. 2009; Avila et al. 2010;

Galindo-Moreno et al. 2010; de Vicente et al.

2010). While the use of autogenous bone pro-

vides the unique benefits of osteoinductivity

and osteogenic potential, extensive remodel-

ing may occur when it is used as the sole

material for sinus augmentation (Schlegel

et al. 2003). This has been demonstrated to

lead to undesirable volumetric changes after

grafting in the first year (Schmitt et al. 2015).

Recent animal studies indicate that the

combination of autogenous bone and ABBM

(Bio-Oss!) has the advantages of reduced graft

resorption yet increased bone-to-implant con-

tact formation (Jensen et al. 2012, 2013).

Human studies, however, showed no signifi-

cant differences with respect to NB formation

when directly comparing trephine samples

from sinuses grafted with ABBM and

ABBM + AB (Schmitt et al. 2015). In this

study, the use of autogenous bone in combina-

tion with ABBM led to a more mature regener-

ated bone matrix on the basis of a significantly

greater mean proportion of lamellar bone to

woven bone when compared to the test group.

The test group received ABBM-C (Bio-Oss

Collagen!), which is comprised of ABBM

granules with the addition of 10% highly

purified porcine type-1 collagen. A histologi-

cal assessment of ABBM-C in extraction

sockets of beagle dogs found that the amount

of residual bovine bone mineral remained

unchanged in the socket during the entire

healing period, whereas the collagen portion

was rapidly removed (Araujo et al. 2010). It

was hypothesized that hydrolytic enzymes

(collagenase) released from activated PMN

cells were involved in the rapid degradation

of the porcine collagen portion of the graft.

However, there are no studies reporting on

the outcome of this material after maxillary

sinus floor elevation.

The histomorphometric analysis of the aug-

mented sinuses was performed at two ROIs.

The first (ROI-1) represented an assessment of

the entire sample. The second region of inter-

est (ROI-2) represented the area from the

junction of the preexisting or native bone

(i.e., the former floor of the sinus cavity) and

ended 1 mm apically into the sample. This

second analysis was carried out to assess the

impact of resident wall (floor) proximity on

histological outcomes. There were no signifi-

cant differences in % NB and % STM when

comparing ROI-1 to values seen in ROI-2 in

both the control and test groups. The impact

of the resident wall was discernable on the %

OI. A positive influence was seen in the con-

trol group with significant increase seen in

ROI-2, but a significant reduction was seen in

the test group in ROI-2. These differences

may be related to the block form of collagen-

stabilized ABBM and the ability to place a

block substitute in proximity to the resident

wall of bone. This is supported by the signifi-

cant reduction in %RG seen in ROI-2 when

compared to ROI-1.

Comparable NB percentage fractions were

obtained in our study for both collagen-stabi-

Fig. 4. Representative histological images (a) control

(ABBM + AB) and (b) test (ABBM-C) groups, indicating a

similar histological appearance between groups. Resid-

ual graft particles are readily identifiable, presence of

new bone around and between graft particles, and a sig-

nificant non-mineralized (soft tissue/marrow) tissue

fraction is apparent. Toluidine blue staining.

Fig. 5. Representative histological images (a) control

(ABBM + AB) and (b) test (ABBM-C) groups illustrating

the varying degrees of maturity in regenerated bone.

Note the presence of woven bone, lamellar bone, and

anorganic bovine bone mineral particles; ABBM, anor-

ganic bovine bone mineral; LB, lamellar bone; WB,

woven bone. Toluidine blue staining.

Fig. 6. Box and whisker plot of histomorphometric outcomes. (a) Percentage new bone formation (%NB). (b) Per-

centage graft particle osseointegration (% OI). (c) Percentage residual graft particles (% RG). (d) Percentage of soft

tissue and marrow (% STM). First region of interest (ROI-1) represents the area from the junction of the preexisting

or native bone (i.e., the former floor of the sinus cavity) to where the biopsy broke during the harvesting procedure.

Second region of interest (ROI-2) represents the area from the junction of the preexisting or native bone (i.e., the for-

mer floor of the sinus cavity) to 1 mm apically into the sample.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 855 | Clin. Oral Impl. Res. 27, 2016 / 850–858

Alayan et al #Bio-Oss collagen in human sinus grafting

lized ABBM (30.71 " 12.24%) and

ABBM + AB (29.06 " 13.51%) to a recent

study of maxillary sinus grafting in humans

which used ABBM + AB bone also in a 1 : 1

ratio (27.50 " 6.31%) and utilized a similar

biopsy harvesting technique (Schmitt et al.

2015).

Harvesting of autogenous bone can be asso-

ciated with increased postoperative patient

morbidity (Clavero & Lundgren 2003; Cric-

chio & Lundgren 2003) and prolonged surgical

time. These disadvantages are exaggerated if a

second intra-oral surgical site has to be

accessed to gain sufficient autogenous bone

volume. As such, the use of bone substitutes

solely has a distinct clinical advantage. Based

on the histomorphometric outcomes of this

study, the use of collagen-stabilized anorganic

bovine bone mineral (ABBM-C) alone is suit-

able in such clinical situations.

The impact of proximity to the sinus floor

on NB formation was not discernable in this

study as the %NB in ROI-1 and ROI-2 were

not significantly different in both groups. A

recent animal study (Alayan et al. 2015) by

our group noted a positive histomorphometric

impact of proximity to the resident bony

walls on parameters such as %NB and %OI.

This gradient of bone formation has also been

reported in other studies (Haas et al. 1998;

Busenlechner et al. 2009). The differences in

outcomes compared to the current study may

have been due to different sampling tech-

niques. The animal study by Alayan et al.

(Alayan et al. 2015) assessed entire sinus sam-

ples, whereas this study assessed trephine/

biopsy samples. The biopsy harvesting tech-

nique has been reported to have a significant

impact on histomorphometric outcomes at

least in the early phases of graft healing (Mar-

golin et al. 1998; Artzi et al. 2005). Artzi

et al. (2005) histomorphometrically assessed

the variation in area fraction of residual bone

substitutes and NB in samples harvested via a

trephine from grafted maxillary sinuses. The

depth of the samples influenced both the area

fraction of NB and RG particles. As such, the

limitations of the biopsy technique may pre-

vent observation of a gradient of graft consoli-

dation by not being fully representative of the

entire maxillary sinus environment.

Additional sources of variations in the area

fraction of biomaterials such as ABBM have

been reported (Valentini et al. 2000; Yildirim

et al. 2000; Hallman et al. 2001b) and may

depend on the density of the grafting material

at application, the initial percentage of the

material, the particle size, and the histologi-

cal preparation technique. In this study, cer-

tain factors were controlled, as the same

clinician carried out all surgical procedures,

the particle size was identical in all cases

and the proportion of ABMM to autogenous

bone was maintained at 1 : 1 using a stan-

dardized bone collection chamber.

A significant difference was seen in the

degree of particle OI with the control group

showing improved %OI in ROI-2 when com-

pared to the test group. While this mean dif-

ference was statistically significant, a large

variation in the degree of particle OI between

samples was noted. This was reflected in the

wide confidence intervals. This wide varia-

tion in %OI was also seen in a recent animal

study by our group (Alayan et al. 2015), has

also been reported in previous animal studies

(Haas et al. 1998), and may have been influ-

enced by the sample size. These histological

variations may influence the available bone

volume for implantation and bone-to-implant

contact. It would also imply that clinicians

should aim to maximize the regenerative

potential of the procedure by maximizing the

bony walls involved in the healing process

and ensuring intimate contact between the

graft particles and resident walls.

The role of the collagen in the test grafting

material (ABBM-C) is to cohesively bind the

ABBM granules, and hence, the material is

delivered in a block format. As such, it may

have some advantages in sinus augmentation,

especially where a particulate material is to be

avoided such as during sinus membrane perfo-

ration. This is the most frequently reported

surgical complication during maxillary sinus

augmentation and according to a recent review

has a mean occurrence rate of 10% (4.8–58%)

(Chiapasco et al. 2009). This complication

occurred in 9.5% of cases in this study (4 of

42), with all of these allocated to the test

group. All of these perforations were small in

diameter (≤ 3 mm) and were repaired with a

resorbable porcine collagen membrane using a

previously described technique (Proussaefs &

Lozada 2003). ABBM-C is advantageous in

these cases because of its block format incor-

porating stabilized ABBM particles. As such, it

would be less likely to migrate into the maxil-

lary sinus cavity proper and obstruct the osteo-

meatal complex, thereby minimizing the

occurrence of postgrafting sinusitis. Previous

studies have cited particulate migration as a

source of postoperative sinusitis (Quiney et al.

1990; Wiltfang et al. 2000; Doud Galli et al.

2001) which may require endoscopic removal

and re-establishment of drainage (Doud Galli

et al. 2001; Timmenga et al. 2001). The use of

ABBM-C in this manner had no impact on his-

tomorphometric outcomes. The significance

of sinus membrane perforations on implant

survival or graft success remains contentious

with some studies reporting a negative impact

especially in large perforations (Proussaefs

et al. 2004; Hernandez-Alfaro et al. 2008),

while others showed no differences (Becker

et al. 2008).

In summary, ABBM-C exhibited very simi-

lar histomorphometric parameters to the com-

posite graft of ABBM + AB. Improved % OI

was seen in the zone proximal to the resident

bony floor in the anorganic bovine bone + au-

togenous bone group (ABBM + AB). In addi-

tion, the ABBM + AB group was more mature

as indicated by the significantly greater pro-

portion of lamellar bone when compared to

the ABBM-C. It can be concluded that from a

histological standpoint ABBM-C is a suitable

bone substitute for the purposes of maxillary

sinus augmentation. Its clinical utility may be

in cases of sinus membrane perforation and

insufficient autogenous bone in the local area.

This material could potentially be a useful

addition to the armamentarium of clinicians

carrying out maxillary sinus augmentation.

Acknowledgements: This project was

funded by a grant from the Australian Dental

Research Foundation (RM#: 2010001012).

Bio-Oss!, Bio-Oss Collagen! & Bio-Gide!

were kindly provided by Geistlich AG,

Wollhusen, Switzerland. We would like to

thank Dr Dimitrios Vagenas and Lee Jones

(Biostatisticians, Institute for Health and

Biomedical Innovation, Queensland

University of Technology, Kelvin Grove,

Australia) for their assistance and

contribution to the statistical analysis in this

project.

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Supporting Information

Additional Supporting Information may be

found in the online version of this article:

Appendix S1. CONSORT 2010 checklist of

information to include when reporting a ran-

domised trial.

858 | Clin. Oral Impl. Res. 27, 2016 / 850–858 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al #Bio-Oss collagen in human sinus grafting

90

Chapter 4: Comparison of early osseointegration of SLA® and SLActive®

implants in maxillary sinus augmentation: a pilot study.

91

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED

PAPER

This chapter includes a co-authored paper. The bibliographic details of the

co-authored paper, including all authors, are:

Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S. (2017) Comparison

of early osseointegration of SLA® and SLActive® implants in maxillary sinus

augmentation: a pilot study. Clinical Oral Implants Research 28: 1325 – 1333. doi:

10.1111/clr.12988.

My contribution to the paper involved:

Design of the project, the provision of the data, the preliminary analysis and

categorization of the data into a usable format, the provision of direction on the

scope and structure of the analysis, the interpretation of results and drafting of the

article.

04th May 2018

Jamil Alayan

(Countersigned)16th May 2018

Corresponding author of paper: Saso Ivanovski

(Countersigned) ___________________________ 16th May 2018

Supervisor: Stephen Hamlet

Jamil AlayanCedryck VaquetteSiamak SaifzadehDietmar HutmacherSaso Ivanovski

Comparison of early osseointegrationof SLA! and SLActive! implants inmaxillary sinus augmentation: a pilotstudy

Authors’ affiliations:Jamil Alayan, Saso Ivanovski, Authors’Affiliations:School of Dentistry and Oral Health,Centre for Medicine and Oral Health, MenziesHealth Institute Queensland, Griffith University,Southport, AustraliaCedryck Vaquette, Siamak Saifzadeh, DietmarHutmacher, Institute for Health and BiomedicalInnovation, Queensland University of Technology,Kelvin Grove, Australia

*Corresponding author:Saso IvanovskiSchool of Dentistry and Oral Health, Centre forMedicine and Oral HealthGold Coast Campus Griffith UniversitySouthport, QLD 4215, AustraliaTel.: +61 75 678 0741Fax: +61 75 678 0708e-mail: s.ivanovski@griffith.edu.au

Key words: animal model, bone grafting, bone regeneration, bone substitute, dental implant,

histomorphometry, maxillary sinus augmentation

Abstract

Objective: To assess the impact of a hydrophilic implant surface (SLActive!) placed into

augmented maxillary sinuses on bone-to-implant contact (BIC) and surrounding tissue composition

when compared to a hydrophobic surface (SLA!).

Material and methods: Four sheep underwent bilateral sinus augmentation. Each sinus received

anorganic bovine bone mineral + autogenous bone (ABBM + AB). Sixteen implants were

subsequently placed 12 weeks postgrafting with each sinus receiving a control (SLA!) and test

implant (SLActive!). Two animals were sacrificed at 2 weeks and another two animals were

sacrificed at 4 weeks postimplantation. The eight sinuses and 16 implants were processed for

histomorphometry, which assessed bone-to-implant contact (%BIC) and tissue elements (woven

bone – WB, lamellar bone – LB, soft tissue – ST) in the interthread region of implants within the

augmented sinus.

Results: There was a statistically significant increase in %BIC at week 4 compared to the week 2

animals in both test (P < 0.005) and control (P < 0.005) groups. There was a statistically significant

greater %BIC around test implants when compared to control implants in both week 2 (P < 0.05)

and week 4 animals (P < 0.05). Greater %WB (11.17% !6.82) and %LB (11.06% !3.67) were seen

in the test implants when compared to the control implants independent of time. This was only

statistically significant for %LB (P < 0.05). A statistically significant reduction of 16.78% (!6.19) in

%ST was noted in test implants when compared to control implants (P < 0.05) independent of

time.

Conclusion: Both time and the use of hydrophilic implant surface had a positive impact on %BIC

around implants placed into augmented maxillary sinuses. Hydrophilic implant surfaces also had a

positive impact on surrounding tissue composition. Larger trials are needed to better assess and

detect differences between these two surfaces in augmented maxillary sinuses.

Implant surface topography and chemistry

affect the osseointegration process (Gittens

et al. 2014). It has been shown to play a criti-

cal role in the predictability of the bone

response to implant placement (Junker et al.

2009) and is a major determinant of the rate

and extent of dental implant osseointegration

(Wennerberg & Albrektsson 2009).

The SLA! surface is a moderately rough

surface which is commonly utilized clini-

cally and widely documented in the litera-

ture. It is formed by coarse grit blasting with

0.25–0.5 mm corundum followed by acid

etching with sulphuric and hydrochloric acid

(Zhao et al. 2005). Animal studies have

shown that these surface results in superior

bone-to-implant contact compared to

minimally rough implants (Cochran et al.

1998), as well as superior torque removal val-

ues (Buser et al. 1998). Histological analysis

of the sequential healing events associated

with the placement of SLA titanium

implants showed that initial bone formation

around these implants occurs not only at the

exposed bone wall of the surgically created

recipient site (distance osteogenesis), as is

the case with turned minimally rough sur-

face implants, but also along the implant sur-

face (contact osteogenesis; Abrahamsson

et al. 2004).

The SLActive! surface is produced with the

same sandblasting and acid etching technique

as SLA!, but differs in that the SLActive!

implants are rinsed under nitrogen protection

Date:Accepted 7 September 2016

To cite this article:Alayan J, Vaquette C, Saifzadeh S, Hutmacher D, Ivanovski S.Comparison of early osseointegration of SLA! and SLActive!

implants in maxillary sinus augmentation: a pilot study.Clin. Oral Impl. Res. 00, 2016, 1–9doi: 10.1111/clr.12988

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1

to prevent exposure to air and are then stored

in a sealed glass tube containing isotonic

NaCl solution as opposed to dry storage (Zhao

et al. 2005). This method allows the

SLActive! implant to have a higher surface

energy and be more hydrophilic in nature than

the SLA! implant (Rupp et al. 2006).

Preclinical studies showed that SLActive!

implants achieved up to 60% more bone-to-

implant contact at 2 weeks after placement

(Buser et al. 2004), had greater removal tor-

que values (Ferguson et al. 2008), and

demonstrated faster and more mature bone

formation in comparison with SLA! surfaces

(Schwarz et al. 2008, 2009). Similarly, a

human study indicated a superior degree of

osseointegration for SLActive! implants

after 2 and 4 weeks of healing when com-

pared to SLA! implants, but this difference

was not evident by 6 weeks postplacement

(Lang et al. 2011). This supports the hypoth-

esis of accelerated bone healing on

SLActive! at least in the early stages. This

histologically demonstrated advantage has

translated to successful earlier loading proto-

cols (Morton et al. 2010).

Studies on modified hydrophilic implants

in bone defects or grafted sites are rare. Modi-

fied implants have been shown to have sig-

nificantly greater bone regeneration in

dehiscence defects (Schwarz et al. 2008) and

circumferential defects in canines (Lai et al.

2009). To date, however, there is only one

study comparing SLA! and SLActive! in

grafted maxillary sinuses. This animal study

failed to show any significant difference in

BIC between the two types of implants at 12

and 26 weeks postplacement in augmented

maxillary sinuses (Philipp et al. 2014). This

may be because SLActive! has been shown

to have a beneficial effect during early

osseointegration (2–4 weeks), with this effect

not evident at later time points (Buser et al.

2004; Lang et al. 2011).

The primary objective of this in vivo study

was to compare the early osseointegration of

implants with a hydrophilic surface

(SLActive!) to those with a hydrophobic sur-

face with the same geometry and surface

microroughness (SLA!), in augmented maxil-

lary sinuses. Our secondary objective was to

characterize the tissue composition in the

immediate vicinity of the implant surface in

these augmented sites.

Material and methods

Animal care and handling

The ARRIVE guidelines on reporting of

in vivo experiments in animal research were

adhered to in this study (Kilkenny et al.

2010). All animal handling and surgical pro-

cedures were conducted according to the

Australian code of practice for the care and

use of animals for scientific purposes at the

Medical Engineering Research Facility

(MERF), Queensland University of Technol-

ogy (QUT), Queensland, Australia. This facil-

ity is a GLP (good laboratory practice)-

accredited large animal research facility. Eth-

ical approval was provided by the QUT Ani-

mal Research Ethics Committee (QDPI

scientific use registration number: 0052). The

animal experiments were carried out between

January and September 2012. The experimen-

tal animals were sourced from the ex-Univer-

sity of Queensland agricultural research farm

at Mount Cotton, Queensland, Australia.

The sheep were housed either in the QUT

MERF building (animal house) at Chermside,

Queensland, Australia, immediately prior to

and after surgery or the QUT MERF agist-

ment farm in Pinjarra Hills, Queensland,

Australia for the remainder of the study.

All sheep underwent a health assessment,

especially to check for the presence of upper

respiratory tract infections, and were contin-

ually monitored by a veterinary surgeon for

the duration of the study.

The surgical procedures were performed

under general anaesthesia (5 mg/kg propofol

I.V and 2–2.5% isoflurane) with multimodal

pain management including local analgesia

(bupivacaine 0.5% w/v 1:200,000

adrenaline), an NSAID (ketorolac, 0.5 mg/

kg, SC) and an opioid analgesic (buprenor-

phine, 0.01 mg/kg IM). During surgery ani-

mals received cephalothin and gentamicin,

which were continued for 3 days after sur-

gery.

Animal allocation and study design

The study used four skeletally mature wether

sheep (Ovis aries), 3–4 years old. Every ani-

mal underwent bilateral sinus augmentation

procedures, and hence, a total of eight

sinuses received the graft (ABBM + AB).

Twelve weeks following the sinus augmenta-

tion procedure, a control (SLA!) and a test

implants (SLActive!) were placed in each of

the grafted sinuses. A total of two implants

were placed in each sinus with the caudal/

cranial position determined by a coin toss. A

total of 94 implants were therefore placed in

each animal, and hence, a total of 16

implants were placed.

Two animals were then sacrificed at

2 weeks, and another two animals were sacri-

ficed at 4 weeks after implant placement. The

eight sinuses and 16 implants were then pro-

cessed for histology and histomorphometry.

Bone grafting materials

All animals received small diameter particles

(0.25–1 mm) of deproteinized bovine

bone mineral (Bio-Oss!, Geistlich AG,

Wollhusen, Switzerland) + autogenous bone

(ABBM + AB) in a 1 : 1 ratio.

(a) (b)

(c) (d)

Fig. 1. A clinical photograph outlining the surgical steps of maxillary sinus augmentation in the sheep model. (a)

Line demarcating the extra-oral incision, (b) after antrostomy preparation and Schneiderian membrane elevation,

the bone substitute selection and delivery, (c) implant placement 12 weeks later, and (d) harvested sample of

implant containing augmented sinus.

2 | Clin. Oral Impl. Res. 0, 2016 / 1–9 © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al "SLA! and SLActive! implants in maxillary sinus augmentation

Maxillary sinus augmentation

All animals were treated using the same sur-

gical technique utilizing an extra-oral

approach (Fig. 1a), as previously described

(Alayan et al. 2016). The anterior wall of the

maxillary sinus was exposed inferior to the

lower orbital rim using an oblique sagittal

skin incision of approximately 6–8 cm fol-

lowed by dissection of the masseter muscle.

A full circular antrostomy was created in the

lateral wall of the maxilla using a bone scra-

per (Safescraper Twist, META, C.G.M Sp.A,

Reggio Emiia, Italy) and a piezoelectric

surgical unit (Implant Center, Satelec,

Acteon, France) to gain access to the maxil-

lary sinus (Fig. 1b). The size of the antros-

tomy was standardized using a circular

sterile template with a diameter of 2 cm.

The sinus membrane was then dissected and

elevated in a dorso-cranial direction using a

combination of piezoelectric surgical tips and

hand sinus membrane elevators to create an

intra-sinus space. The prepared sinus then

received the grafting material, which was

carefully packed into the subantral space

ensuring contact with as many of the

residual bony walls as possible whilst avoid-

ing over-packing (Fig. 1b).

The autogenous bone chips collected using

the bone scraper were used as the autogenous

bone component in the control group. To

ensure a 1 : 1 ratio was maintained, a stan-

dardized 2 cc chamber was utilized to ensure

that the same volume of Bio-Oss! and auto-

genous particles was used. The reconstructed

sites were covered with a resorbable porcine

collagen membrane (Bio-Gide!, Geistlich

AG, Wolhusen, Switzerland). The masseter

muscle and skin flap were then sutured sepa-

rately using a resorbable polyglactin suture

(Vicryl 4/0, Ethicon, Johnson & Johnson) fol-

lowed by a nonresorbable polypropylene

suture (Prolene 4/0, Ethicon, Johnson & John-

son) for the skin flap to achieve primary clo-

sure.

Dental implant placement

Twelve weeks following the sinus augmenta-

tion procedure, two endosseous dental

implants were placed in each of the grafted

sinuses (Fig. 1c). One control implant had a

standard SLA! surface (SLA-sandblasted and

acid-etched; Institut Straumann AG, Basel,

Switzerland), and the second test implant

had a modified hydrophilic SLA surface

(SLActive!; Institut Straumann AG, Basel,

Switzerland). All implants were bone level in

design with a diameter of 3.3 and 8 mm in

height. A total of 16 implants were placed.

Implant osteotomy preparation was per-

formed using the universally accepted proto-

col of stepwise bone excavation using

progressively increasing diameter drills and

copious sterile physiologic saline cooling.

This protocol was carried out with sharp

drills, light pressure, and intermittent drilling

thereby ensuring minimal bone trauma. The

implants were then submerged under the

mucocutaneous flap and were therefore not

exposed to the external environment.

Postoperative treatment and sacrifice

Practices and procedures used for the care

and management of animals in this study

were based on current best practice, as per

Australian code for the care and use of ani-

mals for scientific purposes (https://www.

nhmrc.gov.au/_files_nhmrc/publications/at

tachments/ea28_code_care_use_animals_1312

09.pdf). This takes into consideration the rel-

evant aspects of species-specific biology,

physiology, and behaviour. It also includes

the potential adverse impact of conditions

and procedures on the well-being of the ani-

mals, and strategies to minimize adverse

impacts on the animals after surgery.

(a)

(b)

(c)

Fig. 2. A histological image illustrating the histomorphometirc method employed. (a) The method excluded the

region of the implant in contact with resident bone. (b) This highlighted region in green represents the implant sur-

face within the augmented sinus. The regions highlighted in red represent those regions of implant surface in con-

tact with bone and within the augmented sinus. (c) Magnified histological image corresponding to the demarcated

region illustrating this methodology.

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 3 | Clin. Oral Impl. Res. 0, 2016 / 1–9

Alayan et al "SLA! and SLActive! implants in maxillary sinus augmentation

Postoperatively, each sheep was transferred

to a covered pen and was cradled in a sheep

sling. A combination analgesic was given

(buprenorphine, 0.01 mg/kg IM, and ketoro-

lac, 0.5 mg/kg, SC) for 2–3 days postopera-

tively to alleviate pain. The animals were

housed indoor and monitored three times per

day over the first week and two times daily

thereafter until the end of the experimental

period. Monitoring observations were re-

corded. Interventions were implemented

when signs of discomfort were detected.

Suitable dressing was used postoperatively

to protect the surgical wound, and acute

postoperative monitoring (3 times/day) for

the first seven took place. The extra-oral sur-

gical wounds were checked for any evidence

of postoperative complications, such as fail-

ure in suture integrity (wound dehiscence),

bleeding, inflammation, and infection.

To protect the operated sinus from masti-

catory load immediately after the surgery,

the animals were maintained NPO (nil per

os) with parenteral support for 24 h.

Throughout the fluid administration, the

sheep remained cradled in the sling and were

monitored constantly. From the second day

after surgery, the sheep were fed a soft diet

for 1 week postoperatively, and eating and

drinking were closely monitored following

parenteral nutrition.

At the completion of the 2 and 4 week

healing periods, the animals were sacrificed

by a lethal injection of Lethabarb (320 mg/ml

pentobarbital sodium, 0.5 ml/kg).

Histological preparation and histomorphometry

Blinding during histological preparation of tis-

sue samples was achieved by coding each ani-

mal and by using a different operator for the

surgery (JA) and histological processing (CV).

Undecalcified resin-embedded tissue sections

were prepared as previously described (Donath

& Breuner 1982; Schlegel et al. 1998, 2006;

Bosshardt et al. 2011; Lang et al. 2011). The

initial trimming of the fixed tissue samples

was performed to exclusively contain the

implants within the maxillary sinus (Fig. 1d).

The samples were further sectioned longitudi-

nally to the implant long axis leaving a 1-mm-

thick tissue border around the implant and

subsequently dehydrated in a graded series of

ethanol and resin infiltrated (methyl

methacrylate/glycol methacrylate, Technovit

7200, Heraeus Kulzer, Germany). Once cured,

resin blocks were ground using an EXAKT 400

CS microgrinding system until the threads of

the implant was exposed, glued onto a glass

slide, and subsequently sectioned to a thick-

ness of 150 lm using an EXAKT 300 cutting

system (Exact Apparatebau, GmbH Norderst-

edt Germany). A single slide per implant was

polished down to a thickness of 20 lm using

the EXAKT 400 CS microgrinding system. A

single sample from the central aspect of each

implant was then stained with 0.1% toluidine

blue (Sigma Aldrich, St Louis, MO, USA) for

light microscopy and analysis.

Histomorphometric measurements were

performed blinded and carried out by a single

experienced operator (JA) using a specialized

histomorphometric software program (Aperio

ImageScope version 12.1, Aperio Technolo-

gies Inc.©). Histomorphometric analysis was

carried out as previously described (Bosshardt

et al. 2011; Lang et al. 2011) and the follow-

ing parameters were assessed (Fig. 2): (a) the

proportion of mineralized bone in contact

with the surface of the implants contained

within the augmented sinus and (b) the tis-

sue elements occupying the interthread

region including the proportions of woven

bone, lamellar bone, nonmineralized struc-

tures (residual tissue), and residual graft par-

ticles.

The definitions of each parameter are as

follows and were measured only within the

augmented sinus (Fig. 2):

• % WB = area of woven bone interthread

regions/total interthread area

• % LB = area of lamellar bone interthread

regions/total interthread area

• % RG = area residual graft particles inter-

thread regions/total interthread area

• % STM = area of soft tissue interthread

regions/total interthread area

• % BIC = length implant in contact with

bone/total implant length within aug-

mented sinus

(a)

(b)

Fig. 3. Magnified histological images illustrating peri-implant tissue of (a) control (SLA!) implant after 4 weeks of

healing. New bone, mostly woven bone is seen on both the existing regenerated bone and on residual ABBM. (b)

Further magnification of highlighted area (star) showing woven bone trabeculae extending from the ERB towards

the implant surface. Secondary osteons as part of a BMU (asterisks) are seen indicative of ongoing remodeling.

ABBM, anorganic bovine bone mineral particles; ERB, existing regenerated bone; LB, lamellar bone; NB, new bone;

WB, woven bone. Toluidine blue staining.

4 | Clin. Oral Impl. Res. 0, 2016 / 1–9 © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al "SLA! and SLActive! implants in maxillary sinus augmentation

Statistics

Quantitative data were analysed using IBM

SPSS Statistics for Windows version 21.0

(IBM Corporation 2012©, Armonk, NY, US).

A generalized linear model was used using

individual animals as the clustering variable.

Time and implant surface were used as

explanatory variables. A backward elimina-

tion procedure was used for arriving to the

most parsimonious model. A P ≤ 0.05 was

considered to represent statistically signifi-

cant differences. Our primary outcome mea-

sure was %BIC and our secondary outcomes

measures included tissue composition in the

interthread regions.

The null hypothesis is no difference in degree

of BIC or tissue composition around an

SLActive! surface implant when compared to

an SLA! surface in augmented maxillary

sinuses.

Results

The surgical wounds in all animals healed

completely within 10–14 days with no signs

of complications.

Gross histological features

The augmented region within the intact sinus

cavity was identified by the obvious retained

residual graft particles and change in the Sch-

neiderian membrane topography. Bone of vary-

ing degrees of maturity was observed over both

time points in both treatment groups (Figs 3,

4, 5). This ranged from woven bone to mature

bone with longitudinal and concentric lamel-

lae in addition to the presence of BMUs,

indicative of ongoing bone maturation and

remodeling. Bridging between graft particles

was evident with new bone formation seen to

be continuous around and between graft parti-

cles (Figs 3, 4). A predominance of woven

bone, however, was noted in areas proximal to

the implant surface in both groups (Fig. 4).

Graft particles were located at varying dis-

tances from the implant surface but not seen

in direct contact with the implant surface

except in some areas where the pitch of the

implant thread was in direct contact with

residual ABBM particles. Some of the gaps

between the host bone and the implant surface

were partly filled with debris resulting from

the surgical osteotomy preparation. New bone

was seen in the form of a thin coating on the

implant surfaces, the existing regenerated bone

(ERB), and the residual ABBM particles (ABBM;

Figs 3, 4). In addition, projections of woven

bone trabeculae were seen extending from the

ERB towards the implant surface (Fig. 4).

The impact of time (Figs 6 and 7)

There was a statistically significant increase

in %BIC at week 4 compared to the week 2

animals for both test (SLActive!; P < 0.005;

from 16.48 ! 6.13 at week 2 to 37.78 ! 12 at

week 4; mean ! SD) and control (SLA!)

implants (P < 0.005; from 14.63 ! 8.52 at

week 2 to 26.4 ! 12.67 at week 4;

mean ! SD). The impact of time was indepen-

dent of implant surface with implants in the

week 4 group having 13.23% (!3.92) greater %

BIC than those in the week 2 group.

Greater %WB (9.02% !6.82) and %LB

(6.35% !3.67) was seen in week 4 samples

compared to week 2. Further an 8.59%

(!5.48) reduction in %ST was seen in week 4

samples. Very little difference was noted in

%RG (<1%). None of these differences were

statistically significant.

The impact of treatment (Figs 6 and 7)

There was a statistically significant greater

%BIC in the test implants when compared to

the control implants in both week 2 (test

16.48 ! 6.13 vs. control 14.63 ! 8.52;

mean ! SD; P < 0.05) and week 4 animals

(test 37.78 ! 12 vs. control 26.4 ! 12.67;

mean ! SD; P < 0.05). The impact of implant

surface was independent of time with test

implants showing 9.89% (!3.90) greater %

BIC than control implants.

Greater %WB (11.17% !6.82) and %LB

(11.06% !3.67) were seen in the test implants

when compared to the control implants inde-

pendent of time. This was only statistically

significant for %LB (P < 0.05). A statistically

significant reduction of 16.78% (!6.19) in %

ST was noted in test implants when compared

to control implants (P < 0.05). Very little

(a)

(b)

Fig. 4. Magnified histological images illustrating peri-implant tissue of (a) test (SLActive!) implant after 2 weeks of

healing. (b) Further magnification of highlighted area (star) showing, new bone mostly as woven bone, directly

formed onto the implant surface in addition to residual ABBM particles. ABBM, anorganic bovine bone mineral par-

ticles; ERB, existing regenerated bone; LB, lamellar bone; NB, new bone; WB, woven bone. Toluidine blue staining.

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 5 | Clin. Oral Impl. Res. 0, 2016 / 1–9

Alayan et al "SLA! and SLActive! implants in maxillary sinus augmentation

difference was noted in %RG (<1%) between

test and control implants.

Discussion

This histological study was designed to gain

information on the impact of a hydrophilic

microrough surface (SLActive!) on early heal-

ing around implants placed into grafted max-

illary sinuses. Sheep is a well-established

animal model for maxillary sinus augmenta-

tion using an extra-oral approach (Haas et al.

1998, 2002; Aral et al. 2008; Saffarzadeh et al.

2009; Gutwald et al. 2010; Alayan et al.

2016). The characteristics of the Schneiderian

membrane in sheep are anatomically compa-

rable with humans (Estaca et al. 2008), and it

does not have the disadvantage of an

excessively thick cortical bone layer such as

that found in pigs (Estaca et al. 2008).

The design of the study attempted to

closely mimic routine clinical protocols,

whereby a staged implant placement was

undertaken following a 12 week period of

graft consolidation, which has been shown to

result in mature tissue in this model (Alayan

et al. 2016). The composite graft of anorganic

bovine bone and autogenous bone was cho-

sen as the graft of choice as this combination

has been utilized in multiple studies of max-

illary sinus floor augmentation (Maiorana

et al. 2000; Hallman et al. 2001a, 2001b,

2002, 2005, 2009; Hatano et al. 2004; Canniz-

zaro et al. 2009; Kim et al. 2009; Avila et al.

2010; Galindo-Moreno et al. 2010; de Vicente

et al. 2010; Alayan et al. 2016). Recent stud-

ies indicate that the combination of

autogenous bone and Bio-Oss (ABBM) has the

advantages of reduced graft resorption and

increased bone-to-implant contact formation

(Jensen et al. 2012b, 2013). A recent study

utilizing an identical sinus augmentation

procedure (Alayan et al. 2016) showed ade-

quate new bone formation using this

composite grafting material. In addition,

improved histological parameters were re-

ported in the AB-ABBM sites compared to

collagen-stabilized ABBM alone, especially

when assessing regions furthest away from

the resident bone.

In this animal model, healing time (4 vs.

2 weeks) led to significant improvements in

%BIC, %WB, and %LB in both test and con-

trol implants. %BIC around SLA! implants

increased from 14.63 ! 8.52 at week 2 to

26.4 ! 12.67 at week 4 (mean ! SD) and

SLActive! implants significantly increased

from 16.48 ! 6.13 at week 2 to 37.78 ! 12 at

week 4 (mean ! SD). This finding is consis-

tent with a systematic review which con-

cluded that BIC of implants placed in

augmented sinuses improved over time (Jen-

sen et al. 2012a). Studies included in this

review assessed BIC in both simultaneous

and staged implant placement after maxillary

sinus grafting using ABBM (Bio-Oss) via the

lateral window approach. The only other

study comparing SLA! and SLActive!

implants in augmented maxillary sinuses

(Philipp et al. 2014) reported similar %BIC

that also improved with time. %BIC for

SLA! implants increased from 21.5 ! 16.2 at

week 12 to 28.7 ! 13.3 at 26 weeks

(mean ! SD) and for SLActive! implants

from 20.1 ! 9.2 at week 12 to 34.1 ! 13.7 at

26 weeks (mean ! SD), but this was only sig-

nificant around SLActive! implants.

The use of a modified hydrophilic implant

surface (SLActive!) also led to increased %

BIC compared to the SLA! surface at both 2

and 4 weeks. This is consistent with other

studies which have shown improved out-

comes with SLActive! compared with SLA!

in terms of BIC in pristine bone (Buser et al.

2004; Lang et al. 2011) and bone healing in

augmented defects (Schwarz et al. 2008; Lai

et al. 2009). However, Philipp et al. (2014)

did not observe a statistically significant dif-

ference in %BIC between the two surfaces in

grafted sinuses at either 12 or 26 weeks.

They did, however, observe that SLActive!

implants placed into a grafted maxillary sinus

provided the best BIC values after 26 weeks

of healing. The differences between our

results and those from the above-mentioned

study may be due to the different implant

placement protocols (simultaneous vs. staged

(a)

(b)

Fig. 5. Magnified histological images representing samples from a (a) test (SLActive!) implant after 4 weeks of heal-

ing. (b) New bone is seen mostly as lamellar bone directly formed onto the implant surface. ABBM, anorganic

bovine bone mineral particles; ERB, existing regenerated bone; LB, lamellar bone; NB, new bone; WB, woven bone.

Toluidine blue staining.

6 | Clin. Oral Impl. Res. 0, 2016 / 1–9 © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Alayan et al "SLA! and SLActive! implants in maxillary sinus augmentation

implant placement), the different observation

times (12 and 26 weeks vs. 2 and 4 weeks) or

due to differences in bone graft composition

(hydroxyapatite and beta-tricalcium phos-

phate vs. ABBM + AB). Indeed, a number of

studies have also shown equivalence when

comparing SLActive! vs. SLA! surface at

later time points, after early observations

indicated an initial advantage to SLActive!

surfaces (Buser et al. 2004; Bosshardt et al.

2011; Lang et al. 2011). In review of the

available literature, it was concluded that a

stronger bone response was indeed present

during early healing around SLActive!

implants, which was not evident at later

stages (Wennerberg et al. 2011).

SLActive! implants also positively influ-

enced the bone healing in the vicinity of the

implants, as measured by the amounts of

woven and lamellar bone in the interthread

regions. Greater %WB and %LB were seen in

the test implants when compared to the con-

trol implants independent of time. This was

only statistically significant for %LB and

reflects the greater maturity of the new bone

around the SLActive! implants due to the

accelerated bone formation. Indeed,

transcriptome analysis from human peri-

implant human samples showed that com-

pared with the SLA! surface, SLActive!

exerts a pro-osteogenic and pro-angiogenic

influence on gene expression as early as

7 days postimplantation (Donos et al. 2011).

The outcomes of this pilot study, however,

must be interpreted with caution due to its

low power and higher probability of type-1

errors. These results should be validated in

larger studies with an increased number of

animals. Indeed, there appears to be a lack of

published studies that assess the influence of

surface modification in augmented sites gen-

erally. The small sample size was partly com-

pensated by the placement of a test and

control implant in every augmented sinus,

thus reducing the confounding influence of

variability in the healing potential at the

sinus level.

In conclusion, both time and the use of

hydrophilic implant surface had a positive

impact on %BIC around implants placed into

augmented maxillary sinuses. Hydrophilic

implant surfaces were also associated with a

more mature bone–implant interface. These

findings suggest that implant surface charac-

teristics influence the early wound healing

process at the implant–tissue interface lead-

ing to improved histomorphometric out-

comes.

Acknowledgements: Jamil Alayan is

the recipient of a scholarship from the

National Health & Medical Research

Council of Australia (ID: APP1056633). Bio-

Oss!, Bio-Oss Collagen! & Bio-Gide! were

kindly provided by Geistlich AG, Wollhusen,

Switzerland. We would like to thank Dr

Dimitrios Vagenas (Biostatistician, Institute

for Health and Biomedical Innovation,

Queensland University of Technology,

Kelvin Grove, Australia) for his assistance

and contribution to the statistical analysis in

this project.

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101

PART 2 – CLINICAL & RADIOGRAPHIC ASSESSMENT OF

COLLAGEN STABILIZED ABBM IN MSA

This section of the thesis includes the following chapter:

Chapter 5: A prospective controlled trial comparing xenograft/autogenous bone

and collagen-stabilized xenograft for maxillary sinus augmentation –

Complications, patient reported outcomes and volumetric analysis.

Chapter 6: A prospective controlled trial comparing xenograft/autogenous bone

and collagen-stabilized xenograft for maxillary sinus augmentation – Implant

supported restorations outcomes.

102

Chapter 5: A prospective controlled trial comparing xenograft/autogenous

bone and collagen-stabilized xenograft for maxillary sinus augmentation –

Complications, patient reported outcomes and volumetric analysis.

103

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED

PAPER

This chapter includes a co-authored paper. The bibliographic details of the

co-authored paper, including all authors, are:

Alayan J, Ivanovski S. (2018) A prospective controlled trial comparing

xenograft/autogenous bone and collagen-stabilized xenograft for maxillary sinus

augmentation – Complications, patient reported outcomes and volumetric analysis.

Clinical Oral Implants Research 29: 248 – 262. doi: 10.1111/clr.13107.

My contribution to the paper involved:

Design of the project, the provision of the data, the preliminary analysis and

categorization of the data into a usable format, the provision of direction on the

scope and structure of the analysis, the interpretation of results and drafting of the

article.

04th May 2018

Jamil Alayan

(Countersigned) 16th May 2018

Corresponding author of paper: Jamil Alayan

(Countersigned) ___________________________ 16th May 2018

Supervisor: Stephen Hamlet

Clin Oral Impl Res. 2017;1–15. wileyonlinelibrary.com/journal/clr  | 1© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Accepted: 29 October 2017

DOI: 10.1111/clr.13107

O R I G I N A L A R T I C L E

A prospective controlled trial comparing xenograft/autogenous bone and collagen- stabilized xenograft for maxillary sinus augmentation—Complications, patient- reported outcomes and volumetric analysis

Jamil Alayan  | Saso Ivanovski

School of Dentistry and Oral Health, Centre for Medicine and Oral Health, Griffith University, Southport, QLD, Australia

CorrespondenceJamil Alayan, School of Dentistry and Oral Health, Centre for Medicine and Oral Health, Gold Coast Campus Griffith University, Southport, QLD, Australia.Email: jamil.alayan@griffithuni.edu.au

AbstractObjective: Compare maxillary sinus augmentation (MSA) using two different materi-

als—anorganic bovine bone mineral (ABBM) + autogenous bone (AB) (control group)

vs. collagen- stabilized ABBM (test group) in terms of complications, patient- reported

outcome measures (PROMs) and volumetric analysis.

Materials and Methods: Sixty patients underwent sinus augmentation (30 control + 30

test group). Intra- and postoperative complications were recorded. PROMs measured

the impact of grafting on daily activities, pain and morbidity. CT scans were used to

measure graft volume, ridge height, material selection and degree of contact of graft-

to- surrounding sinus walls. Dental implant placement parameters were also

recorded.

Results: All complications were minor and did not prevent completion of the augmen-

tation or subsequent implant placement. Schneiderian membrane perforation was the

most frequently encountered complication. Both treatment groups reported moderate

limitation in the 1st 48 hr post- surgery but little or none by day 3 or 4. Jaw opening,

chewing and bruising were significantly higher in the control group. The impact on

work and social life was moderate initially but reduced to little or none by the 2nd day.

Mild to moderate pain and interference to daily activities were reported for the first

3 days requiring the use of NSAIDs only. A mean graft volume of 1.46 cm3 (±0.77) was

calculated in the control group and 1.27 cm3 (±0.65) in the test group. Extent of con-

tact between graft and surrounding sinus walls had a significant impact on bone vol-

ume. Shorter (8 mm) implants were utilized more frequently in the test group, which

was also more likely to require additional vertical augmentation, but this was not sta-

tistically significant.

Conclusion: MSA using a lateral wall approach is safe and associated with mild to mod-

erate pain and restrictions to daily activities for 48–72 hr. Patients’ reports of morbid-

ity were greater with autogenous bone harvesting. Collagen- stabilized ABBM provides

comparable bone volume to AB + ABBM that is sufficient for placement of implants of

adequate size with no need for further vertical augmentation. Engaging the surround-

ing sinus walls had a significant positive impact on graft volume.

2  |     ALAYAN ANd IVANOVSKI

1  | INTRODUCTION

Implant placement in the posterior maxilla is often prevented by

insufficient bone volume as a result of bone resorption and sinus

pneumatization. Maxillary sinus floor elevation is the technique most

frequently used to overcome this limitation and is considered a safe

and effective procedure with high implant survival rates (Chiapasco,

Casentini & Zaniboni, 2009; Del Fabbro, Testori, Francetti &

Weinstein, 2004). Nevertheless, clinically this surgical technique is

still being refined, with a large degree of variation in many aspects

of the procedure.

To date, a variety of bone substitute materials have demonstrated

clinically acceptable outcomes (Cordaro et al., 2008; Froum, Wallace,

Elian, Cho & Tarnow, 2006; Klijn, Meijer, Bronkhorst & Jansen, 2010;

Nkenke & Stelzle, 2009). Despite the established clinical efficacy of

the technique, the search continues for a bone substitute with optimal

properties including rapid osteogenesis as well as long- term volumet-

ric stability and space maintenance.

The composite graft of anorganic bovine bone mineral (ABBM)

and autogenous bone (AB) is well documented in multiple studies

of maxillary sinus floor augmentation (Avila et al., 2010; Cannizzaro,

Felice, Leone, Viola & Esposito, 2009; Galindo- Moreno et al., 2010;

Hallman, Cederlund, Lindskog, Lundgren & Sennerby, 2001; Hallman,

Mordenfeld & Strandkvist, 2005; Hallman, Sennerby & Lundgren,

2002; Hatano, Shimizu & Ooya, 2004; Kim, Yun, Kim & Lim, 2009;

Maiorana, Redemagni, Rabagliati & Salina, 2000; de Vicente,

Hernandez- Vallejo, Brana- Abascal & Pena, 2010). The ABBM/AB

combination is the most clinically and histologically studied mate-

rial for sinus augmentation (Jensen, Schou, Stavropoulos, Terheyden

& Holmstrup, 2012a,b; Jensen, Schou, Svendsen, et al., 2012) and

provides the advantages of AB whilst limiting its undesirable effects.

AB results in superior de novo bone formation (Schmitt et al., 2013),

but its harvesting is associated with postoperative patient morbidity

(Clavero & Lundgren, 2003; Cricchio & Lundgren, 2003), prolonged

surgical time and is only present in limited quantities. AB grafts can

also exhibit unpredictable resorption (Clavero & Lundgren, 2003;

Davis, Martinoff & Kaminishi, 1984; Sbordone et al., 2013; Wiltfang

et al., 2005), which is not a desirable characteristic in sinus graft-

ing. Indeed, sinuses augmented using AB as the sole grafting mate-

rial have been shown to undergo significant re- pneumatization and

loss of augmented volume (Arasawa et al., 2012; Johansson, Grepe,

Wannfors, Aberg & Hirsch, 2001; Johansson, Grepe, Wannfors &

Hirsch, 2001). The addition of a slower resorbing highly osteocon-

ductive material such as ABBM (Bio- Oss®, Gesitlich, AG, Wollhusen

Switzerland) provides superior space maintenance for de novo

bone formation (Berglundh & Lindhe, 1997; Haas, Baron, Donath,

Zechner & Watzek, 2002; Jensen et al., 1996; Orsini et al., 2005)

and volumetric stability (Galindo- Moreno et al., 2010; Jensen et al.,

2012a,b; Jensen, Schou, Svendsen, et al., 2012).

The supplementation of ABBM to AB grafts has also led to a re-

duction in the amount of autogenous bone needed, which in turn

eliminated the need for extra- oral harvesting thereby reducing pa-

tient morbidity, surgical time and expense (Scheerlinck et al., 2013).

Whilst the symphysis and retromolar regions of the mandible are rel-

atively safe sites to harvest particulate autograft, the need to create

a second surgical site remains. As such, collecting the bone removed

from the buccal wall of the maxilla during preparation of the buc-

cal antrostomy allows for the use of autogenous cortical particles

as part of a composite graft without the need for a second surgi-

cal site. Whilst further reductions in patient morbidity and operating

time would be expected using this technique, previous studies have

only emphasized histological/histomorphometric outcomes (Galindo-

Moreno et al., 2007, 2010, 2011). In general, there is an absence

of patient- reported outcome measures (PROMs) for maxillary sinus

augmentation, yet factors such as pain, bruising, oedema and their

impact on daily activities are an important consideration for patients

considering this treatment option.

ABBM is also available in a block form that is stabilized in 10% por-

cine type 1 collagen matrix (ABBM- C) (Bio- Oss Collagen®, Gesitlich,

AG, Wollhusen Switzerland). The contained nature of the block may

be advantageous in maxillary sinus augmentation when the common

surgical complication of Schneiderian membrane perforation occurs

(Chiapasco et al., 2009). The impact of this altered formulation of

ABBM has been investigated in terms of bone regeneration in max-

illary sinus augmentation (Alayan, Vaquette, Farah & Ivanovski, 2016;

Alayan, Vaquette, Saifzadeh, Hutmacher & Ivanovski, 2016), but there

are no studies reporting on the clinical outcomes of maxillary sinus

grafts using this material.

The primary aim of this controlled clinical trial was to determine

whether the test group (ABBM- C), used in defined clinical scenarios

(i.e., reduced local autogenous bone availability and/or sinus membrane

perforation), performs equivalently to the control group (ABBM+AB)

in terms of the prevalence of surgical complications, patient- reported

and volumetric outcomes.

2  | MATERIAL AND METHODS

2.1 | Ethics and consent

The University of Queensland School of Dentistry Dental Sciences

Research Ethics Committee provided ethical approval for this equiva-

lence non- randomized controlled clinical trial (Project Number: 1010).

The project complies with the provisions contained in the National

Statement on Ethical Conduct in Research Involving Humans and

K E Y W O R D S

anorganic bovine bone, Bio-Oss, Bio-Oss Collagen, bone substitute, clinical trial, guided bone

regeneration, patient-centred outcomes, Sinus floor elevation, volumetric outcomes

     |  3ALAYAN ANd IVANOVSKI

complies with the regulations governing experimentation on humans.

The STROBE (Strengthening the Reporting of Observational Studies

in Epidemiology) guidelines were adhered to in the preparation of this

manuscript.

2.2 | Inclusion criteria

To be considered eligible for the study, the following inclusion criteria

were applied:

1. Males and females, over 21 years of age at the time of

surgery

2. Must present with a bone deficiency in the maxillary sinus region,

which necessitates sinus floor augmentation to facilitate the place-

ment of one or more dental implants.

3. If bilateral sinus augmentation was required, only one sinus was

randomly selected and used for the purposes of this study.

4. Residual alveolar bone height of the edentulous maxilla below the

floorofthemaxillarysinus≤4.5mmand≥1mm(measuredatthemid-point of the edentulous space where both the coronal and sag-

ittal planes intersect).

5. Residualalveolarbonewidthof≥6mmasmeasuredonaCTscan.6. Generally fit and healthy and able to undergo oral surgical proce-

dures under local and general anaesthesia.

7. Patients with a history of chronic periodontitis were treated first

and subsequently enrolled in a supportive maintenance programme

prior to recruitment into the study.

8. Teeth at the surgical site which required removal were extracted a

minimum of 12 weeks prior to sinus floor elevation.

2.3 | Exclusion criteria

Patients were considered ineligible if any of the following criteria

were met:

1. Any systemic medical condition that could interfere with the

surgical procedure

2. Current pregnancy at the time of recruitment

3. Physical handicaps that would interfere with the ability to perform

adequate oral hygiene

4. Alcoholism or chronic drug abuse

5. Patients who smoke more than 10 cigarettes per day

6. Medications which interferes with bone formation

7. Mucosal diseases such as lichen planus

8. History of local radiation therapy

9. Severe bruxism or clenching habits

10. Signs and symptoms consistent with acute maxillary sinusitis

11. Significant soft tissue thickening or sinus opacification in the sinus

of interest.

12. Previous GBR (bone graft) or dental implant treatment in the poste-

rior segments of the maxilla

13. Benign or malignant tumours of the maxillary sinus

14. Pathological conditions that severely impact mucociliary clearance

including; chronic sinusitis, primary ciliary dyskinesia or cystic

fibrosis.

2.4 | Participant allocation and study design

To perform a comparison of bone volume between groups in this equiv-

alence study, a power analysis (PASS v. 11.0) indicated that we would

need 30 individuals in each group (total study population = 60 patients) to

achieve a power of 0.83 with a probability of 0.05 for type 1(alpha) errors,

assuming a standard deviation of 0.70 (Gultekin et al., 2016), a true dif-

ference of zero and a zone of clinical equivalence of ±0.55 cm3 (Figure 1).

Patients were prospectively recruited from the School of Dentistry

at the University of Queensland and from a specialist private prac-

tice from July 2010 to December 2015. Those that met the inclusion

F IGURE  1 Flow chart of treatment sequence and data collection, including the number of patients involved in each step. MSFE, maxillary sinus floor elevation; control (ABBM + AB), anorganic bovine bone mineral + autogenous bone; test (ABBM- C), collagen- stabilized anorganic bovine bone mineral; PCO, patient- centred outcomes

4  |     ALAYAN ANd IVANOVSKI

criteria then underwent the informed consent process. All patients

were referred specifically for replacement of teeth with implant-

supported restorations. Recruitment continued until 30 patients were

allocated to the control and test groups.

Allocation of each patient to either the test (ABBM- C) or the control

group (ABBM + AB) was based on two intra- operative parameters, the

presence of Schneiderian membrane perforation and availability of ade-

quate autogenous bone in the vicinity of the surgical site. ABBM- C was

selected if sinus membrane perforations occurred or if there was insuf-

ficient autogenous bone available for harvesting from in the site of the

buccal antrostomy. ABBM + AB was alternatively used if the Schneiderian

membrane remained intact and if there was sufficient autogenous bone

that could be harvested from the buccal wall of the maxilla.

Five–six months later, all participants underwent surgical implant

placement whereby they received a single implant in the augmented

site. The same clinician (JA) performed all of the surgical procedures.

Whilst clinician blinding was not possible at the time of the augmen-

tation surgery or implant placement, it was possible during the data

collection and analysis stages of this project.

2.5 | Primary and secondary outcomes

The overall objective of this study was to assess the success of the

maxillary sinus augmentation procedure.

Primary endpoint:

1. Augmented bone volume measured 5–6 months after maxillary

sinus augmentation and prior to implant placement.

Secondary endpoints include the following:

1. Intra-operative complications

2. Postoperative surgical complications and morbidity

3. PROMs such as pain and interference with daily life, in addition to

quality of life measures during the first week after maxillary sinus

augmentation

4. Need for further vertical augmentation at implant placement

5. Placement of implants with adequate dimensions

6. Complications during implant insertion, such as lack of primary

stability

2.6 | Bone graft materials and dental implants

Patients were assigned to one of the following groups:

1. Control group—small diameter particles (0.25 – 1.0 mm) of depro-

teinized bovine bone mineral (Bio-Oss®, Geistlich AG, Wollhusen,

Switzerland) + autogenous bone (ABBM+AB) in a 1:1 ratio, as

determined using a standardized 2 cc bone collecting chamber.

2. Test group—250 mg blocks of deproteinized bovine bone mineral +

collagen (ABBM-C) (Bio-Oss Collagen®, Geistlich AG, Wollhusen,

Switzerland).

All patients received a single Straumann (Institut Straumann AG,

Basel, Switzerland) tissue level dental implant 5 months post- grafting.

The implant had a standard microroughened sandblasted and acid-

etched (SLA) surface. The dimensions of the implants were chosen

according to the specific site characteristics.

2.7 | Pre- surgical procedures

All participants started systemic antibiotic therapy (500 mg amoxycil-

lin or 300 mg clindamycin) and 0.2% chlorhexidine mouth rinse 24 hr

prior to grafting surgery. A single dose of 8 mg dexamethasone was

also given preoperatively. All patients were assessed for periodontal

disease at the initial assessment appointment by specialist periodon-

tists (JA & SI) and according to the criteria of Armitage (Armitage,

1999). Those diagnosed were treated and deemed to be stable prior

to sinus augmentation surgery. Stability was defined as having a full

mouth bleeding score of ≤20% and ≤4sites of residual periodontalprobingdepth≥5mm(Lang&Tonetti,2003).

2.8 | Maxillary sinus floor elevation and grafting

All patients were treated by the same operator (JA) with the same

surgical technique consisting of sinus floor elevation via a lateral

wall (modified Caldwell- Luc) approach. After elevation of a full buc-

cal mucoperiosteal flap, a full antrostomy was created in the lat-

eral wall of the maxilla using a bone scraper (Safescraper Twist,

META, C.G.M Sp.A, Reggio Emilia, Italy). A piezoelectric surgical unit

(Implant Center, Satelec, Acteon, France) was subsequently used to

finalize the osteotomy and gain access to the maxillary sinus. The

size, shape and location of the antrostomy were variable and tai-

lored to the site and patient’s characteristics. The only consistent

feature of the antrostomy was the superior osteotomy, which was

located 10 mm apical to the alveolar bony crest (Figure 2). The full

buccal mucoperiosteal flap was limited in its vertical extension but

provided sufficient access to create this superior osteotomy. The

sinus membrane was then dissected and elevated in all directions

to create an intrasinus space that received one of the two grafting

materials. The membrane was elevated superiorly to allow for graft-

ing material packing to at least 10 mm apical to the alveolar bone

crest. This was achieved using standard sinus membrane elevator

spoons. Sufficient material was placed until the desired 10 mm of

vertical height was achieved. The material was carefully packed to

ensure maximum contact with resident bony walls, with care taken

to avoid over- packing.

The autogenous bone chips harvested during preparation of the

buccal antrostomy were collected using a bone scraper with a stan-

dardized bone collecting chamber (2 cc) and used for the autogenous

bone component needed for the control group. No other autogenous

bone harvesting was performed nor was the mucoperiosteal flap ex-

tended any further to widen the antrostomy zone for the purposes

of further autogenous bone harvesting. Any patient with insufficient

autogenous bone in the local area to achieve a 1:1 ratio with ABBM

was allocated to the test group. The reconstructed sites were covered

with a porcine collagen membrane (Bio- Gide, Geistlich AG, Wolhusen,

Switzerland). The mucoperiosteal flaps were then sutured using a

     |  5ALAYAN ANd IVANOVSKI

non- resorbable thermoplastic polyether- ester monofilament suture

(Dyloc 5/0 and 6/0 Dynek, Adelaide, Australia) to achieve primary

closure.

A postoperative regimen of amoxycillin 500 mg t.d.s for 7 days

(or if allergic 150 mg clindamycin q.i.d for 6 days) was prescribed, as

was paracetamol 500 mg, ibuprofen 200 mg and 0.2% chlorhexidine

mouth rinse b.d for 7 days. Narcotic analgesics were not prescribed

unless requested by the patient in cases of recalcitrant pain.

2.9 | Clinical follow- up and evaluation

Intra- operative complications and adverse events relating to the

maxillary sinus augmentation procedure were recorded and included

Schneiderian membrane perforation and intra- operative bleeding re-

quiring intervention. All patients were reviewed at 7 and 14 days post-

operatively. At the 7- day review, the following signs were recorded as

either present or absent: wound dehiscence, wound infection (sup-

puration and oedema), extra- oral bruising, oedema and other signs of

morbidity including altered sensation and nasal bleeding. Maxillary

sinusitis was also recorded and defined according to the clinical prac-

tice guidelines of the American Academy of Otolaryngology—Head

and Neck Surgery Foundation 2007 which defines acute maxillary

sinusitis as up to 4 weeks of purulent discharge accompanied by nasal

obstruction, facial pain/pressure or both as reported by the patient or

clinically observed (Rosenfeld et al., 2007).

Postoperative symptoms and the degree of interference with daily

activities were assessed in all patients that underwent maxillary sinus

augmentation under local anaesthesia. A condition- specific health-

related quality of life (HRQL) instrument was used which contained a

questionnaire and a VAS (Shugars, Benson, White, Simpson & Bader,

1996). The instrument collected data for 6 days postoperatively. The

self- administered questionnaire assessed limitations on daily activities

(jaw opening, chewing, talking, sleeping, work and social life) and post-

operative signs (swelling, bruising, bleeding) that cover oral and gen-

eral function, pain and other symptoms (Hashem, Claffey & O’Connell,

2006). A five- point Likert- type scale was used as the response for-

mat (lots, quite a bit, some, little or none). The VAS evaluated average

pain, and interference with daily activities. It consisted of a 10- cm line

with clearly defined endpoints. One end of the line was labelled “no

pain” and the other end of the line was labelled “most intense pain

imaginable.” A similar VAS was used to record interference with daily

activities. Patients were provided with six copies of the data- collecting

instrument and instructed to complete one instrument each evening

for the first 6 days postoperatively. The instrument was coded and

returned to a staff member but was anonymous to the operator (JA).

2.10 | Radiological/volumetric assessment

Volumetric imaging was performed using either multislice CT or cone

beam CT depending on the imaging centre the patient attended. All

scans were obtained at a small number of specialist imaging centres

under the supervision of an oral and maxillofacial radiologist. Pre-

maxillary sinus augmentation scanning was carried out on all patients

to assess site anatomy and to identify patients with significant sino-

nasal pathology. Subsequent scanning was performed no earlier than

5 months after maxillary sinus augmentation and prior to implant

placement. Patients attended the same centre for both scans to en-

sure scans were obtained on the same machines using the same scan

protocol thereby ensuring compatibility (Table 1). All CT scans were

taken with no gantry tilt. Scans were performed with the occlusal

plane approximately parallel with the primary X- ray beam during

acquisition. The occlusal plane was used as the reference plane for

subsequent image reconstruction.

F IGURE  2 A clinical photograph highlighting the placement of the superior osteotomy 10 mm form the bone crest accompanied by similar extent of membrane elevation

TABLE  1 Summary of CT scan protocols

Company Location Modality Scanner make/model kV Exposure (mAs)

Slice thickness (mm)

Image resolution (mm)

1 1 CT GE Lightspeed VCT 120 43.8 0.625 0.336

1 2, 3 CBCT iCAT/Imaging Science International

120 20 0.25 0.25

1 3 CT Toshiba Aquilion 16 120 40 0.5 0.3

2 1 CBCT Kodak Trophy K9500 90 108 0.2–0.3 0.2–0.3

2 2, 3 CT GE Brightspeed 120 168 0.625 0.336

3 1 CBCT Carestream Health/CS9300 85 60.25 0.25 0.25

6  |     ALAYAN ANd IVANOVSKI

All data sets were uploaded into the software and allocated an

identification number. The same operator, an experienced specialist

periodontist, performed all data set reconstruction and measurements

(JA). The protocol was discussed and confirmed with an experienced

oral and maxillofacial radiologist (RD).

DICOM data were then imported into a 3D dental implant plan-

ning software (Simplant Pro 17.0 Dentsply Implants NV, Research

Campus 10 3500 Hasslet Belgium). Only those data sets that in-

cluded the full volume of the maxillary sinus were included in the

study (i.e., from occlusal plane to the superior orbital rim). In addi-

tion, data sets were eliminated from the study if significant soft tis-

sue thickening or sinus opacification was noted that prevented the

accurate volumetric measurement of the maxillary sinus. A mask was

initially created to highlight the air- filled cavities, with subsequent

use of a segmentation tool to manually highlight the volume of the

maxillary sinus cavity proper. This allowed the software to calculate

the sinus volume in cubic centimetres. These measurements were

performed on both the pre- graft (Figure 3a) and post- graft CT data

sets (Figure 3b), and the difference between these measurements

was taken as the graft volume.

Two further radiographic parameters were measured using the

same software and included residual ridge height and contact of graft-

to- surrounding sinus walls (Simplant Pro 17.0 Dentsply Implants NV,

Research Campus 10 3500 Hasslet Belgium). Residual ridge height

was measured using a linear measurement tool at the mid- point of the

edentulous space where both the coronal and sagittal planes intersect.

To determine the degree of graft- to- surrounding sinus wall contact,

linear measurements were performed in regions where the graft con-

tacted resident walls at three predetermined coronal and three sag-

ittal sections through the grafted region. These three sections were

located by dividing the graft into three equal segments in both coronal

and sagittal planes (Figure 4a). Specifically, axial slices were assessed

to identify the slice which displays the maximum volume of graft in

the mesio- distal and transverse dimension. The mid- sagittal and the

mid- coronal bisecting lines were then identified on this axial slice. The

division of the graft into equal thirds was made at this axial slice level.

The division of the graft into equal thirds coronally was carried out

using this mid- sagittal slice, and similarly, the division into equal thirds

sagittally was performed using this mid- coronal slice. An average of

these contact measurements was obtained and used in the analysis

(Figure 4b).

2.11 | Dental implant placement

Dental implant placement was performed 5.5–6 months later. All

participants began systemic antibiotic therapy (1,000 mg amoxycillin

or 300 mg of clindamycin) 60 min prior to surgery. A 0.2% chlorhex-

idine mouth rinse was utilized for 60 s immediately prior to surgery.

Osteotomy preparation was completed using a standard surgical tech-

nique. The implant was placed with the border of the rough surface

approximating the alveolar crest leaving the machined neck portion

in the trans- mucosal area. A healing abutment was secured, and mu-

coperiosteal flaps were sutured using a non- resorbable thermoplastic

polyether- ester monofilament suture (Dyloc 5/0, Dynek, Adelaide,

Australia) to allow for a trans- mucosal healing modality. A small

amount of crestal gingiva was excised to allow for tight flap adapta-

tion, if necessary.

A postoperative regimen of amoxycillin 500 mg three times daily

for 7 days was prescribed, together with paracetamol 500 mg, ibupro-

fen 200 mg and 0.2% chlorhexidine mouth rinse twice daily for 7 days.

F IGURE  3 Volumetric assessment using digital reconstruction. (a) Pre- sinus augmentation CT – maxillary sinus volume (cubic centimetres) calculated by reconstructing the axial, sagittal and coronal segmentation. (b) Measurements repeated on the post- augmentation CT and the difference between these measurements taken as the graft volume

(a) (b)

     |  7ALAYAN ANd IVANOVSKI

Patients allergic to penicillin- based antibiotics were prescribed clinda-

mycin 150 mg four times daily for 7 days. Patients were reviewed 7

and 14 days later.

At implant placement, the following parameters were recorded;

implant dimension, the need for further vertical augmentation at

implant placement using osteotomes, and lack of primary stabil-

ity. Implant survival and success will be reported in a subsequent

study.

2.12 | Statistics

All data were analysed using IBM SPSS Statistics for Windows version

21.0 (IBM Corporation 2012©, Armonk, NY, U.S). Surgical outcomes

and complications were analysed using a comparison of proportion

calculator (MedCalc Software®). Generalized estimating equations

were used to analyse PROMs (VAS and survey) due to clustering of

data (VAS and survey were assessed on the same patients six times)

with the individual (subject) used as the clustering variable, and using

an ordinal logistic regression model for categorical and a linear model

for continuous outcomes. Treatment and time were used as explana-

tory variables along with treatment*time interaction. A backward

elimination procedure was used for arriving at the most parsimoni-

ous model. An independent t test was used to compare bone graft

volumetric data and a linear regression model to assess impact of

treatment (test vs. control), residual ridge height and extent of graft

contact with surrounding sinus walls on this parameter. Baseline pa-

rameters such as age and initial ridge height were compared using an

independent t test, and categorical parameters such as gender, smok-

ing and periodontal status were compared using a chi- squared test.

The null hypothesis was taken as non- equivalence in surgical

complications, in patient- centred outcomes and graft volume in aug-

mented maxillary sinuses using ABBM (Bio- Oss®) + AB or blocks of

ABBM- C (Bio- Oss Collagen®) alone. A p ≤ .05 was considered to rep-

resent statistically significant differences.

3  | RESULTS

3.1 | Study population

Sixty (n = 60) patients were enrolled in this study and underwent sinus

augmentation (Table 2). A total of thirty patients (n = 30) were allo-

cated to each group. There were no significant differences between

groups in terms of age, gender ratio or number of patients smoking.

Allsmokersreportedsmoking≤10cigarettesperday.Moderate-to-severe chronic periodontitis was diagnosed in four patients in the

control group and seven in the test group. All of these patients were

treated and were deemed stable (Lang & Tonetti, 2003) prior to place-

ment into a supportive maintenance programme and enrolment into

the study. All patients received maxillary sinus augmentation to allow

for the placement of a single implant in either a site bounded by natu-

ral teeth or in a distal extension situation. There were no significant

differences in the distribution of this arrangement between groups.

3.2 | Intra- operative complications

No major intra- operative complications were encountered during

the procedure or during the postoperative period (Table 3). All minor

complications were successfully managed and did not prevent com-

pletion of the augmentation procedure or subsequent implant place-

ment. Schneiderian membrane perforation was the most frequently

encountered complication with eight patients in total experiencing

sinus perforation and therefore allocated to the test group. These

perforations were small (≤3mm in diameter) to moderate (>3mm≤5mm) and repaired by creating a pouch using the placement ofa resorbable porcine collagen membrane (Bio- Gide, Geistlich AG,

Wolhusen, Switzerland) that was fixed using titanium tacs to the ex-

ternal surface of the lateral wall of the maxilla if needed (Proussaefs

& Lozada, 2003).

F IGURE  4 Representative CT scan showing method of determining degree of graft- to- sinus walls contact. (a) measurements were performed at three coronal (C1, C2, C3) and three sagittal (S1, S2, S3) sections through the grafted regions, located by dividing graft into three equal segments; (b) an average was taken of these linear measurements in regions where the graft contacted the surrounding sinus walls

(a)

(b)

8  |     ALAYAN ANd IVANOVSKI

3.3 | Postoperative surgical complications and morbidity

All 60 patients attended for post- surgical review (Table 3). Overall, no

major complications were encountered during the first 2 weeks post-

augmentation surgery. Minor complications were almost all seen at

the one- week review and were resolved by the second review without

specific intervention. Wound dehiscence (10% control and 3.3% test),

bruising (13.3% control and 6.7% test) and oedema (10% control and

3.3% test) were the most frequently recorded signs of morbidity albeit

at low frequencies. Fewer cases of dehiscence, bruising and oedema

were noted in the test group, but this was not statistically significant.

These bruising and oedema were also noted to be of lesser severity

in test patients compared to patients in the control group. This obser-

vation however was not objectively measured. Symptoms consistent

with postoperative sinusitis (nasal congestion and post- nasal drip and

facial pressure) were reported by a single patient in the control group

and resolved spontaneously within 48 hr after sinus grafting. A single

patient in the control group reported an isolated minor incident of

bleeding from the nose after the first 24 hr.

3.4 | Patient- reported outcome measures (PROMs)

A total of 30 patients (15 control + 15 test) completed the question-

naire and VAS for this part of the analysis. All these patients under-

went maxillary sinus augmentation using local anaesthesia only.

3.4.1 | Questionnaire—limitation to daily activities (oral function)

Overall, a trend of gradual and daily decrease in the degree of limita-

tion was seen across all activities. The majority of patients in both

ParametersControl (ABBM+AB) (n = 30)

Test (ABBM- C) (n = 30)

Group comparison (p)

Age (Mean years ± SD) 59.57 (±10.57) 58.87 (±9.43) .93

Gender ratio (f/m) 2.0 (f = 20/m = 10) 3.29 (f = 23/m = 7) .57

Smoking (no. patients) 1 2 1.0

Periodontal disease (no. patients)

4 7 .51

Initial ridge height (mm) (Mean ± SD)

3.13 ± 0.73 3.04 ± 0.72 .62

Implant arrangement—bounded by natural teeth (no. patients)

12 11 .98

Implant arrangement—distal extension (no. patients)

18 19 .99

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM- C, collagen- stabilized anorganic bovine bone mineral; p, p- value for intergroup comparison after proportion or means testing. Mean ± SD for continuous variables and counts for categorical variables.

TABLE  2 Baseline parameters

ParametersControl (ABBM+AB) (n = 30) Number (%)

Test (ABBM- C) (n = 30) Number (%) 95% CI

Group comparison (p)

Perforations - 8 - -

Haemorrhage 0 (0) 0 (0) - -

Wound dehiscence

3 (10.0) 1 (3.33) −9.3,23.5 .30

Wound infection

0 (0) 0 (0) - -

Extra- oral bruising

4 (13.3) 2 (6.67) −11.5,24.9 .40

Extra- oral oedema

3 (10) 1 (3.33) −9.3,23.5 .30

Acute maxillary sinusitis

1 (3.3) 0 (0) −8.7,17.2 .32

Other 1 (3.3) 0 (0) −8.7,17.2 .32

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM- C, collagen- stabilized anorganic bovine bone mineral; 95% CI, 95% confidence interval; p, p- value for intergroup comparison after pro-portion testing.

TABLE  3 Surgical outcomes from augmented maxillary sinuses

     |  9ALAYAN ANd IVANOVSKI

treatment groups reported to experience some or quite a bit of limi-

tation to all activities during the first 48 hr post- surgery that reduced

to some or little by day 3. Jaw opening and chewing were affected

by the augmentation procedure to a similar degree and were the oral

functions affected most during the postoperative period. Time was a

significant predictor of all three activities (opening p < .01, chewing

p < .01, talking p < .001) with reductions seen daily. Treatment was a

significant predictor for both opening (p < .005) and chewing (p < .01)

with test group experiencing significantly less interference in both of

these activities. The majority of the control group patients reported

some or lots of interference to both activities on the 1st 24 hr post-

augmentation as opposed to little or some in the test group. The

median interference to opening and chewing was reduced to little

or no interference by the 3rd day in the test group but remained

at some interference in the control group. This delay in reduction

was especially obvious for chewing in the control group. Talking was

mostly restricted in the control group of patients in the first 24 hr and

quickly improved by the 2nd and 3rd days.

3.4.2 | Questionnaire—limitation to daily activities (general function)

The greatest limitation on the first day after surgery was reported to

be on work and social life, but limitation to all three activities (sleeping,

work, social life) was reduced to little or none after the 2nd day. Time

was a significant predictor with reductions seen in all three activi-

ties (sleeping p < .001, work p < .005, social life p < .001) on a daily

basis across all these activities. Treatment group was not a significant

predictor in any of these three activities.

3.4.3 | Questionnaire—postoperative morbidity

Oedema was reported to occur from little to quite a bit by the majority

of patients for the first 3 days post- surgery reducing to some by day 4

and then little or none by days 5 and 6. Time was a significant predictor

for swelling (p < .001) with the median reducing from some to little or

none by the third day in the test group and 4th day in the control group.

Treatment was not a significant predictor. Time was not a significant

predictor of bruising, but treatment group was a significant predictor

(p < .05). The median impact of bruising was none on the first day after

surgery but increased to little on the second day and then to some for

the remainder of the 6 days in the control group, whereas it remained

unchanged and reported to affect patients little or none for the test

group; hence, the time*group interaction was also significant (p < .05).

Whilst time (p < .001) and group (p < .05) were significant predictors for

bleeding, it was reported at a level of little or none in both groups at all

times.

3.4.4 | VAS—average pain and interference to daily activities

Treatment did not significantly affect perception of pain but time

(p < .001) was a predictor of average pain with similar significant re-

ductions reported in both treatment groups. The majority of patients

reported that the average daily pain was mild to moderate for the first

3 days, which was halved by day 4 to become mild pain. Similarly,

treatment did not significantly affect perception of interference with

daily activities but time (p < .001) was a predictor with significant re-

ductions reported in both treatment groups. The majority of patients

reported that the interference with daily activities was mild to mod-

erate for the first 3 days, and this was halved by day 3 to become

mild interference. None of the patients requested narcotic analgesics

(Figure 5).

3.5 | Radiographic/volumetric analysis

The mean graft volume in the control group was 1.46 ± 0.77 cm3 and

1.27 ± 0.65 cm3 in the test group, which was not statistically different

(Tables 4 and 5). The mean initial ridge heights were very similar be-

tween the groups (control = 3.13 ± 0.73 mm; test = 3.04 ± 0.72 mm),

as was the average graft contact to resident walls (con-

trol = 18.67 ± 3.94 mm; test = 17.24 ± 3.08 mm). Multivariable linear

regression analysis performed indicated that treatment group and

initial ridge height were not significant explanatory variables for bone

graft volume but that average contact between graft and surround-

ing sinus walls was a significant explanatory variable with increased

volumes are seen in cases of greater contact (p < .01).

F IGURE  5 Box and whisker plot of VAS for (a) average pain and (b) interference to daily activities for the 1st 6 days post- maxillary sinus augmentation. (c), control group (ABBM + AB); T, test group (ABBM- C). Outliers (o) were identified as responses that lie below Q1 - (1.5IQR) or above Q3 + (1.5IQR)

10  |     ALAYAN ANd IVANOVSKI

3.6 | Implant placement outcomes

Two 8- mm implants were placed in the control group and six 8- mm

implants in the test group with the remainder of patients having suf-

ficient bone height for the desired 10- mm implant (Table 6). This

difference did not reach statistical significance. Additional vertical

augmentation using an osteotome mediated sinus floor elevation was

needed in order to place an 8- mm implant in three patients in the test

group with no such cases occurring in the control group. This differ-

ence did not reach statistical significance.

A single implant from both control and test groups did not achieve

primary stability, and these were classed as “spinners.” Adequate pri-

mary stability was achieved for all remaining implants.

4  | DISCUSSION

The aim of this equivalence trial was to prospectively examine a con-

servative approach to maxillary sinus augmentation that utilizes a lim-

ited flap design and locally harvested autogenous bone in combination

with Bio- Oss® (ABBM) versus Bio- Oss Collagen® (ABBM- C) alone and

compare the rates of surgical complications, postoperative morbid-

ity, PROMs and bone graft volume. Both groups were comparable in

terms of age, gender ratio, and rates of periodontal disease and extent

of edentulism.

This patient population did not experience major intra- or post-

operative complications, consistent with previous reviews (Chiapasco

et al., 2009). In addition, the minor complications were manageable

and did not prevent completion of the augmentation procedure. The

most frequent complication was membrane perforation and occurred

in 13% of our patient population (eight of 60). Multiple reviews have

also reported this to be the most frequently reported surgical com-

plication (Chiapasco et al., 2009; Nkenke & Stelzle, 2009; Pjetursson,

Tan, Zwahlen & Lang, 2008). The mean occurrence rates ranged from

10.0% to 19.5%, but some of the included studies reported perfora-

tions in up to 58% of cases. Most of these perforations were small in

diameter(≤3mm)andwererepairedwitharesorbableporcinecolla-gen membrane using a previously described technique (Proussaefs &

Lozada, 2003) with the addition of titanium tacs for additional stability

of the membrane where needed. The role of the collagen in the test

grafting material (ABBM- C) is to cohesively bind the ABBM granules

to form a block. Its advantage in sinus augmentation procedures over

particulate material is evident when perforations of the sinus mem-

brane occur and there is a risk of particulate biomaterial migrating

into the maxillary sinus cavity proper and obstructing the osteome-

atal complex. Previous studies have cited particulate migration into

the sinus cavity proper as a source of postoperative sinusitis (Doud

Galli, Lebowitz, Giacchi, Glickman & Jacobs, 2001; Quiney, Brimble

& Hodge, 1990; Wiltfang et al., 2000) which may require endoscopic

removal and re- establishment of drainage (Doud Galli et al., 2001;

Timmenga, Raghoebar, van Weissenbruch & Vissink, 2001). Our group

has previously shown that the use of ABBM- C for sinus augmentation

had no significant impact on histomorphometric outcomes except for

its delayed maturity as indicated by the lower proportion of lamellar

bone when compared to the control group (Alayan, Vaquette, Farah

& Ivanovski 2016). The significance of sinus membrane perforations

on implant survival or graft success remains contentious with some

studies reporting a negative impact especially in large perforations

(Hernandez- Alfaro, Torradeflot & Marti, 2008; Proussaefs, Lozada,

Kim & Rohrer, 2004), whilst others showed no differences (Becker

et al., 2008).

In terms of PROMs, the majority of patients reported that average

daily pain was mild to moderate for the first 3 days and subsequently

reduced to mild levels using NSAIDs only. The level of average pain

was similar across treatment groups. The majority of patients in both

treatment groups reported a moderate amount of limitation to both

oral and general activities during the first 48 hr post- surgery that re-

duced to little or none by day 3 or 4. The extent of interference to daily

activities was largely similar across the treatment groups, as assessed

using a VAS. A VAS is a valid, easily administered and reproducible tool

to assess dental pain perception (Seymour, Charlton & Phillips, 1983;

Tan, Krishnaswamy, Ong & Lang, 2014). Regardless of the treatment

group, all parameters improved after 48–72 hr. To the best of our

knowledge, this is the first study to report on PROMs for maxillary

TABLE  4 Outcomes of bone graft volume comparing control and test group materials

ParametersControl (ABBM+AB) (Mean ± SD)

Test (ABBM- C) (Mean ± SD) Mean difference 95% CI p value

Bone graft volume (cm3)

1.46 (±077) 1.27 (±0.65) 0.185 −0.18,0.55 .23

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM- C, collagen- stabilized anorganic bovine bone mineral; SD, standard deviation; 95% CI, 95% confidence interval of the mean difference; p, p- value for intergroup comparison after applying a t test.

TABLE  5 Results of linear regression for graft volume—patient used as the unit of analysis

b (SE) 95% CI p value

Treatment group

control 0.064 (0.16) −0.26,0.39 .70

test reference – –

Initial ridge height

−0.09(0.11) −0.31,0.13 .43

Contact sinus walls

0.09 (0.23) 0.045, 0.14 <.001

b, regression estimate coefficient; SE, standard error; 95% CI, 95% confi-dence interval.

     |  11ALAYAN ANd IVANOVSKI

sinus augmentation and indicates that 48–72 hr of recovery from this

procedure is needed for patients to resume oral function and daily

activities without limitations or the need for analgesics.

Jaw opening and chewing were the oral functions affected most in

both groups and to a significantly greater extent in the control group,

as was bruising after the first day. Whilst greater prevalence of bruis-

ing and swelling was noted clinically in the control group at the post-

operative review, this did not reach statistical significance. Bruising

and oedema were also noted to be of lesser severity in test patients

compared to patients in the control group, but this observation was

not objectively measured. This perception of increased limitation in

oral functions may reflect the increased morbidity associated with

greater autogenous bone harvesting (Cricchio & Lundgren, 2003).

Patients in the control group typically possessed a thick buccal wall of

bone that provided sufficient volume of autogenous bone needed to

support the elevated Schneiderian membrane at the requisite height.

The test patients on the other hand exhibited a thin buccal wall of

bone and therefore experienced far less bone removal. The inclusion

of the eight patients in the test group due to sinus membrane perfora-

tion may have reduced the extent of this difference in perception and

clinical signs of morbidity. Work and social life were the most affected

general activities with moderate levels of restrictions reported for the

first 48 hr in both treatment groups. It is likely those symptoms of pain

and perception of bruising and oedema translated to this limitation in

social life and work, and is generally consistent with anecdotal reports

from patients of abstaining from work for 48 hr post- surgery.

A slightly increased mean graft volume was measured in the

control group (1.46 ± 0.77 cm3) when compared to the test group

(1.27 ± 0.65 cm3). Whilst the volume of bone graft delivered into

the subantral space was tailored to the individual situation, the area

grafted was controlled in similar ways in both groups and therefore

should have resulted in the delivery of similar initial volume of graft

material. All patients were grafted for the purposes of placing a sin-

gle dental implant, and both groups had a very similar number of

sites bounded by natural teeth and distal extension situations. This

would have limited the mesio- distal dimension to a similar degree

across groups. The superior osteotomy was located 10 mm apical

to the bony crest in all patients and therefore would have similarly

limited the apico- coronal dimension. Interestingly, a cadaveric study

showed that 10 mm of membrane elevation translated to a volume

of 1.81 ± 0.79 cm3 (Uchida, Goto, Katsuki & Akiyoshi, 1998; Uchida,

Goto, Katsuki & Soejima, 1998) which is very similar to what was mea-

sured in our study in both groups. The only dimension not controlled

was the width of the sinus in a bucco- palatal dimension. A previous

study has reported this dimension to be largely consistent and not

influenced greatly by age or gender but influenced by whether the

patient is dentate or edentulous (Uchida, Goto, Katsuki & Akiyoshi,

1998; Uchida, Goto, Katsuki & Soejima, 1998), which was not a dif-

ferentiating factor between the groups in our study. To the authors’

best knowledge, this is the first study to utilize this technique to mea-

sure maxillary sinus graft volume in the context of dental implants. It

is however a utilized technique reported in the orthodontic literature

to measure airway volume and maxillary sinus volume (Darsey, English,

Kau, Ellis & Akyalcin, 2012; Oz, Oz, El & Palomo, 2017; Pangrazio-

Kulbersh, Wine, Haughey, Pajtas & Kaczynski, 2012). By measuring

the volumetric change in the air- filled maxillary sinus cavity, it over-

comes a major disadvantage of traditional techniques such as manual

perimeter tracing of a region of interest (Johansson, Grepe, Wannfors,

Aberg, et al., 2001; Johansson, Grepe, Wannfors, & Hirsch, 2001;

Uchida, Goto, Katsuki & Akiyoshi, 1998; Uchida, Goto, Katsuki &

Soejima, 1998; Wanschitz, Figl, Wagner & Rolf, 2006) or regions grow-

ing segmentation thresholding (Park, Kim, Kim, Kim & Chang, 2010)

by eliminating the need to delineate sinus graft from resident bone

especially after graft consolidation has taken place.

Whilst the use of autogenous bone provides the unique benefits

of osteoinductivity and osteogenic potential, extensive remodelling

may occur when it is used as the sole material for sinus augmenta-

tion (Schlegel, Fichtner, Schultze- Mosgau & Wiltfang, 2003). This has

been demonstrated to lead to undesirable volumetric changes after

grafting in the first year (Schmitt, Moest, Lutz, Neukam & Schlegel,

2014; Schmitt, Karasholi, et al., 2014). Recent animal studies indi-

cate that the combination of autogenous bone and ABBM (Bio- Oss®)

has the advantages of reduced graft resorption yet increased bone-

to- implant contact formation (Jensen, Schou, Svendsen, et al., 2012;

Jensen et al., 2013). Human studies however showed no signifi-

cant differences with respect to new bone formation when directly

Evaluated parameters

Control (ABBM+AB) (n = 30) Number (%)

Test (ABBM- C) (n = 30) Number (%) 95% CI

Group comparison (p value)

Primary stability

29 (96.7) 29 (96.7) −14.2,14.2 1.0

Implant length ≥10mm

28 (93.3) 24 (80.0) −6.4,32.8 .13

Additional vertical augmenta-tion

0 (0) 3 (10.0) −4.0,26.53 .078

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM- C, collagen- stabilized anorganic bovine bone mineral; 95% CI, 95% confidence interval; p, p- value for intergroup comparison after pro-portion testing.

TABLE  6  Implant placement outcomes into augmented maxillary sinuses

12  |     ALAYAN ANd IVANOVSKI

comparing trephine samples from sinuses grafted with ABBM and

ABBM + AB (Schmitt, Karasholi, et al., 2014; Schmitt, Moest, et al.,

2014). A recent clinical study showed that percentage fraction of

new bone in samples obtained from maxillary sinuses grafted with

ABBM- C (Bio- Oss Collagen®) are comparable to ABBM + AB (Alayan,

Vaquette, Farah & Ivanovski 2016). To date however, there are no

volumetric studies of the test material (ABBM- C) performance in

sinus augmentation. Even though this study did not assess volumet-

ric changes over time, some volumetric reduction can be assumed to

have taken place during the 6 months of graft consolidation. A re-

view by (Shanbhag, Shanbhag & Stavropoulos, 2014) indicated that

weighted average for volumetric reduction from controlled studies

for composite grafts was 21.88% (±7.38) at 6 months post- grafting.

This volumetric reduction is influenced by a variety of factors includ-

ing the choice of material. Multivariable linear regression analysis in

our study indicated that the choice of material did not correlate with

graft volume 6 months after augmentation and suggests that these

two grating materials exhibit similar volumetric changes over the

short term. Volumetric comparison between the two groups must

be made cautiously as no direct intra- operative measurement of the

graft volume was performed and no immediate postoperative CT

scan was carried out.

Multivariable linear regression analysis indicated that there is a sig-

nificant and positive relationship between the extent of graft contact

with surrounding sinus walls and graft volume. No significant relation-

ship was seen for residual ridge height. This is consistent with other

studies using a range of graft materials, reporting that native bone

height does not have a significant effect on the mean graft height

(Block, Kent, Kallukaran, Thunthy & Weinberg, 1998; Zijderveld,

Schulten, Aartman & ten Bruggenkate, 2009). It has also been shown

that adequate grafted ridge height can be achieved in cases with com-

plete loss of an intact maxillary sinus floor (Cortes, Pinheiro, Cavalcanti,

Arita & Tamimi, 2015). Histological studies have shown a positive im-

pact of increasing number or area of bony walls during healing on graft

outcomes (Alayan, Vaquette, Saifzadeh, Hutmacher & Ivanovski 2016,

Busenlechner et al., 2009; Haas, Donath, Fodinger & Watzek, 1998)

and support the principle ascertained form other models of guided

bone regeneration that surrounding bony walls are an important de-

terminant for de novo bone formation and graft particle incorporation

(Berglundh & Lindhe, 1997; Carmagnola, Adriaens & Berglundh, 2003;

Klinge, Alberius, Isaksson & Jonsson, 1992; Schenk, Buser, Hardwick

& Dahlin, 1994).

This conservative approach to sinus grafting is a safe procedure

that is associated with mild to moderate levels of pain and restric-

tions on oral function and daily activities for 48–72 hr. The use of

both locally harvested autogenous bone combined with ABBM or

collagen- stabilized ABBM solely provided adequate bone volume

for placement of implants of adequate size (8–10 mm) in the major-

ity of cases with no need for further vertical augmentation. The im-

portance of engaging surrounding sinus walls seems to have a more

significant impact on bone graft volume than material selection or

residual ridge height.

ACKNOWLEDGEMENTS

A portion of the Bio- Oss®, Bio- Oss Collagen® and Bio- Gide® uti-

lized in this study were kindly provided by Geistlich AG, Wollhusen,

Switzerland. The authors are grateful to Dr. Dimitrios Vagenas

(Biostatisticians, Institute for Health and Biomedical Innovation,

Queensland University of Technology, Kelvin Grove, Australia) for

his assistance and contribution to the statistical analysis in this pro-

ject. The authors are also grateful to Dr. Raahib Dudhia (RD) (Oral

and Maxillofacial Radiologist, private practice) for his assistance and

contribution to the radiological component of this study.

ORCID

Jamil Alayan http://orcid.org/0000-0003-4191-4776

Saso Ivanovski http://orcid.org/0000-0001-5339-0936

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SUPPORTING INFORMATION

Additional Supporting Information may be found online in the sup-

porting information tab for this article.

How to cite this article: Alayan J, Ivanovski S. A prospective

controlled trial comparing xenograft/autogenous bone and

collagen- stabilized xenograft for maxillary sinus

augmentation—Complications, patient- reported outcomes and

volumetric analysis. Clin Oral Impl Res. 2017;00:1–15.

https://doi.org/10.1111/clr.13107

119

Chapter 6: A prospective controlled trial comparing xenograft/autogenous

bone and collagen-stabilized xenograft for maxillary sinus augmentation –

biological and technical implant outcomes.

120

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED

PAPER

This chapter includes a co-authored paper. The bibliographic details of the

co-authored paper, including all authors, are:

Alayan J, Ivanovski S. (2018) A prospective controlled trial comparing

xenograft/autogenous bone and collagen-stabilized xenograft for maxillary sinus

augmentation – biological and technical implant outcomes. Clinical Oral Implants

Research (submitted).

My contribution to the paper involved:

Design of the project, the provision of the data, the preliminary analysis and

categorization of the data into a usable format, the provision of direction on the

scope and structure of the analysis, the interpretation of results and drafting of the

article.

04th May 2018

Jamil Alayan

(Countersigned) 16th May 2018

Corresponding author of paper: Jamil Alayan

(Countersigned) ___________________________ 16th May 2018

Supervisor: Stephen Hamlet

119

A Prospective Controlled Trial comparing Xenograft /

Autogenous Bone and Collagen-stabilized Xenograft for

Maxillary Sinus Augmentation – Biological and Technical

Implant Outcomes

Jamil Alayan, Saso Ivanovski

Authors’ affiliations:

Jamil Alayan & Saso Ivanovski, School of Dentistry and Oral Health, Centre for

Medicine and Oral Health, Griffith University, Southport, Australia.

Corresponding author:

Jamil Alayan

School of Dentistry and Oral Health, Centre for Medicine and Oral Health

Gold Coast Campus Griffith University

Southport, QLD 4215

Australia

Tel: +61 75 678 0741

Fax: +61 75 678 0708

Email: jamil.alayan@griffithuni.edu.au

Keywords: Sinus floor elevation, guided bone regeneration, anorganic bovine bone,

Bio-Oss Collagen, Bio-Oss, bone substitute, clinical trial, dental implants.

Running title: Collagen-Stabilized Xenograft in MSA - Implant Outcomes

120

Abstract

Objective: Report on biological and technical outcomes of implant supported

restorations placed into previously augmented maxillary sinuses (MSA) using

anorganic bovine bone (ABBM) + autogenous bone (AB) (control group) or collagen

stabilized ABBM (test group) after 1 year of function.

Materials & Methods: A single implant was placed 6 months (T0) after MSA in 27

control and 26 test patients. Fixed restorations were delivered 12 weeks later (T1 -

baseline) and reviewed 12 months after function (T2). Outcomes measured included

implant survival, marginal bone levels (DIB) and their changes, periodontal

parameters and incidence of biological and technical complications.

Results: A survival rate of 100% was reported. No significant inter-group differences

were noted for all parameters. Radiographic assessment 12 months after loading

revealed a mean (± SD) marginal bone level (DIB) of 0.60mm (0.33) and 0.58mm

(0.40) from the implant shoulder in the control and test group respectively. After 12

months of loading mean DIB loss of 0.32mm (± 0.24) and 0.35mm (±0.23) were

noted in the control and test group respectively. Mucositis (≥1 site BOP) was

diagnosed in 62.9% of control & 69.23% of test patients. Peri-implantitis was not

diagnosed in any patient. Screw retention and single crowns predominated. Technical

complications mostly comprised of ceramic veneer chipping and was noted in 7.4% of

control and 11.54% of test patients.

Conclusion: Based on a short observation period, implant reconstruction of the

partially edentulous posterior maxilla after MSA using ABBM + autogenous bone or

collagen stabilized ABBM led stable marginal bone levels, high implant survival and

low rates of technical complications.

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Introduction Maxillary sinus floor augmentation using the lateral wall approach (MSA) is well

established as a surgical technique for treating severe reduction of bone height due to

sinus pneumatization, in order to allow successful implant rehabilitation of the

posterior maxilla with high implant survival rates (Del Fabbro, et al. 2004, Chiapasco,

et al. 2009). When using a conservative approach and locally harvested AB, this

technique is also a safe procedure that is associated with mild to moderate pain and

restriction of daily activities for 2-3 days (Alayan & Ivanovski 2018).

The composite graft of anorganic bovine bone mineral (ABBM) and autogenous bone

(AB) is well documented in multiple studies of MSA (Maiorana, et al. 2000, Hallman,

et al. 2001, Hatano, et al. 2004, Galindo-Moreno, et al. 2007, Cannizzaro, et al. 2009,

Kim, et al. 2009, Avila, et al. 2010, de Vicente, et al. 2010, Galindo-Moreno, et al.

2010, Galindo-Moreno, et al. 2011). ABBM is also available in a block form that is

stabilized in 10% porcine type-1 collagen matrix (ABBM-C). The contained nature of

this material has been shown to have clinical utility when perforations of the sinus

membrane occur during MSA, as it eliminates the risk of particulate biomaterial

migrating into the maxillary sinus cavity proper and obstructing the osteomeatal

complex (Alayan & Ivanovski 2018). This altered formulation of ABBM has been

shown to have comparable histomorphometric and volumetric parameters when

compared to the composite graft (ABBM+AB) in MSA (Alayan, et al. 2016, Alayan,

et al. 2016, Alayan & Ivanovski 2018).

Improved implant survival rates have been shown when using rough implant surfaces

with a median survival rate of 100% (88-100%) when compared to 89% (61.2 –

100%) using machined implant surfaces (Jensen & Terheyden 2009). This is likely to

be due to histologically documented effects (Abrahamsson, et al. 2004) of improved

bone-to-implant contact (Buser, et al. 1998, Cochran, et al. 1998). Implant surface has

also been shown to affect the local tissue composition around implants placed into

augmented maxillary sinuses such as the area fraction of woven and lamellar bone in

a pre-clinical model (Alayan, et al. 2017). The SLA surface is a moderately rough

surface which is widely utilized clinically and documented in the literature in various

clinical scenarios (Bornstein, et al. 2005, Roccuzzo, et al. 2008, Buser, et al. 2012,

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Thoma, et al. 2014). A 100% survival rate has been reported in a clinical study using

one piece SLA implants in MSA at 12 and 60 months after implant placement

(Bornstein, et al. 2008).

Whilst survival data is available, there is a paucity of studies with well defined

success criteria reporting on implants placed into sinuses grafted with the composite

material (ABBM+AB 1:1) (Galindo-Moreno, et al. 2015) especially in terms of

clinical outcomes of the implant supported restorations and their technical

complications. Furthermore, there are no studies reporting on outcomes of implants

placed into augmented maxillary sinuses using ABBM-C.

As such, the aim of this controlled clinical trial was to test whether implant supported

restorations have similar biological and technical outcomes when placed into

previously augmented maxillary sinuses using stabilized ABBM (ABBM-C), as used

in defined clinical scenarios (i.e. reduced local autogenous bone availability and/or

sinus membrane perforation), when compared to those placed into a composite graft

of ABBM and autogenous bone (ABBM+AB).

Materials & Methods Ethics and Consent

The University of Queensland School of Dentistry Dental Sciences Research Ethics

Committee provided ethical approval for this equivalence non-randomized controlled

clinical trial (Project Number: 1010). The project complies with the provisions

contained in the National Statement on Ethical Conduct in Research Involving

Humans and complies with the regulations governing experimentation on humans.

The CONSORT (Consolidated Standards of Reporting Trials) checklist was used in

the preparation of this manuscript.

Study Settings & Participants Patients were prospectively recruited from the School of Dentistry at the University of

Queensland and from a specialist private practice (Brisbane, Australia) between July

2010 to December 2015. Those that met the inclusion criteria then underwent the

informed consent process. All patients were referred specifically for replacement of

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teeth with implant-supported restorations. Recruitment continued until 30 patients

were allocated into the control and test groups.

Patients were considered eligible for the study if they fulfilled the following criteria:

• Individuals were at least 18 years of age, medically healthy or with mild

controlled systemic disease, able to undergo oral surgical procedures under local

or general anaesthesia (ASA I and II; American Society of Anesthesiologists,

Schaumburg, Illinois, USA).

• Must present with a bone deficiency in the maxillary sinus region, which

necessitates sinus floor augmentation

• If bilateral sinus augmentation was required, only one sinus was randomly

selected and used for the purposes of this study.

• Residual alveolar bone height of the edentulous maxilla below the floor of the

maxillary sinus ≤4.5 mm and ≥ 1 mm (measured at the mid-point of the

edentulous space where both the coronal and sagittal planes intersect) and residual

alveolar bone width of ≥6mm.

• Teeth at the surgical site which required removal were extracted a minimum of 12

weeks prior to sinus floor elevation

• Patients with a history of periodontal disease as assessed by specialist

Periodontists (JA & SI) and according to the criteria of Armitage (Armitage 1999)

were treated and deemed to be stable prior to sinus augmentation surgery.

Stability was defined as having a full mouth bleeding score of ≤20% and no sites

of residual periodontal probing depth ≥5mm (Lang & Tonetti 2003).

Patients were considered ineligible if any of the following factors were evident:

• Pregnancy at the time of recruitment

• Physical handicaps that would interfere with the ability to perform adequate oral

hygiene

• Alcoholism or chronic drug abuse

• Patients who smoke more than 10 cigarettes per day

• Medications which interferes with bone formation

• Mucosal diseases such as Lichen Planus

• History of local radiation therapy

• Severe bruxism or clenching habits

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• Signs and symptoms consistent with acute maxillary sinusitis

• Significant soft tissue thickening or sinus opacification in the sinus of interest.

• Previous GBR (bone graft) or dental implant treatment in the posterior segments

of the maxilla

• Benign or malignant tumours of the maxillary sinus

• Pathological conditions that severely impact mucociliary clearance including;

Chronic sinusitis, Primary Ciliary Dyskinesia or Cystic Fibrosis.

Sample size

The patients in this study were part of a larger study designed to have sufficient

statistical power to detect differences in maxillary sinus graft volume between the two

groups (Alayan & Ivanovski 2018). Nonetheless, an analysis was performed for this

equivalence study to ensure adequate power to perform a comparison on marginal

bone level changes (G*Power 3.1.9, Faul, F., Erdfelder, E., Lang, A.-G., & Bychnner,

A, Germany). This analysis indicated that 25 individuals in each group (total study

population = 50) would be needed to achieve a power of 0.8 with a probability of 0.05

for type 1 (alpha) errors, assuming a standard deviation of 0.89mm, a true difference

of zero and a zone of clinical equivalence of ± 0.74mm (Galindo-Moreno, et al.

2014).

Participant allocation and study design

Allocation of each patient to either the control or test group was based on two intra-

operative parameters; the presence of Schneiderian membrane perforation and

availability of adequate autogenous bone in the vicinity of the surgical site. ABBM-C

(test) was selected if sinus membrane perforations occurred or if there was

insufficient autogenous bone available for harvesting in the vicinity of the buccal

antrostomy. ABBM + AB (control) was alternatively used if the Schneiderian

membrane remained intact and if there was sufficient autogenous bone that could be

harvested from the buccal wall of the maxilla.

The same clinician (JA) performed all of the surgical procedures. While clinician

blinding was not possible at the time of the augmentation surgery or implant

placement, it was possible during the measurement and analysis stages of this project.

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Bone graft materials and dental implants

• Control group – MSA with small diameter particles (0.25 – 1.0 mm) of

deproteinised bovine bone mineral (Bio-Oss®, Geistlich AG, Wollhusen,

Switzerland) + autogenous bone (ABBM+AB) in a 1:1 ratio, as determined by

using a standardized 2cc bone collecting chamber.

• Test group – MSA with 250mg blocks of deproteinised bovine bone mineral +

collagen (ABBM-C) (Bio-Oss Collagen®, Geistlich AG, Wollhusen, Switzerland).

All patients received a single (Straumann, Institut Straumann AG, Basel, Switzerland)

one piece dental implant. The implant had a standard micro-roughened sandblasted

and acid-etched (SLA) surface. The dimensions of the implants were chosen

according to the specific site characteristics.

Primary and Secondary Outcomes

Primary end point:

• Interproximal marginal bone levels (DIB) up to a period of 1 year after delivery

of prosthesis

Secondary end points:

• Peri-implant tissue health assessment (Periodontal probing depth, gingival

recession, bleeding on probing, suppuration, plaque index) • Implant supported restoration assessment (technical outcomes) • Implant and restoration survival

Clinical Interventions / Surgical Procedures

Maxillary sinus floor elevation and grafting

All patients received the same surgical technique consisting of sinus floor elevation

via a lateral wall (modified Caldwell-Luc) approach as previously described (Alayan

& Ivanovski 2018).

Dental implant placement

A single dental implant was placed 6 months later as previously described (Alayan &

Ivanovski 2018).

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Implant supported restorations

Abutment connection was carried out as per the manufacturer’s recommendations at

35Ncm, 3 months after implant placement by a small group of independent restorative

clinicians. No restrictions were made regarding the prosthesis design (single or multi-

unit) material type or type of retention. The baseline assessment was carried out after

insertion of restoration.

Follow Up Evaluations & Outcomes Measurements

Radiographic follow up & evaluation

Marginal bone levels were measured on intra-oral radiographs taken using a long cone

paralleling technique and a paralleling device. An exposure of 0.12s (70kV, 7mA,

Carestream CS2200) was used with a digital sensor (SOPIX2 SOPRO sensor, Acteon,

La Ciotat, France) with a pixel size of 20 x 20µm to acquire the radiographic images.

The digital images were imported into a free and open source code software program

(Horos v2.2.0 sponsored by Purview, Annapolis, MD USA www.horosproject.org).

The distance from the implant shoulder at the implant-abutment connection to the most

coronal bone-to-implant contact (DIB) was measured at the mesial and distal aspects

of each implant and expressed in millimeters (Roccuzzo, et al. 2001, Bornstein, et al.

2005). These linear measurements were calibrated to account for any distortion and

magnification by using the known inter-thread distance of the utilized implant

(1.25mm). The known distance of the smooth surface collar of the implant of 1.8mm

was subtracted to give the distance from the rough-smooth junction to DIB. All

radiographic measurements were recorded at placement (DIB0), at delivery of the

implant supported restoration (baseline) (DIB1) and then at 12 months post-placement

of the restoration (DIB2). The mean change per implant was then calculated between

T0 and T1 and then from T1 to T2 . All measurements were made by a single blinded

experienced periodontist (JA) after the radiographs were allocated an identification

number.

Clinical follow up & evaluation

The following parameters were assessed within 1 month after the placement of the

restoration (T1) (baseline) and then repeated 12 months post-placement of the

restoration (T2):

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• Assessment of peri-implant tissue – Periodontal probing depth (PPD) and measures

of inflammation were recorded. PPDs were measured at x6 sites per implant using

a manual periodontal probe (UNC 15, Hu Friedy, Chicago, U.S), with gentle force

and rounded off to the nearest millimeter. In addition, the same probe was used to

locate the mucosal margin (DIM) using the occlusal surface of the implant-

supported restoration as a fixed landmark. Recession was calculated as the change

in DIM between T1 and T2. A mean value was then calculated for each implant-

supported restoration. The presence or absence of sites with bleeding on probing

(BOP) and suppuration were assessed dichotomously (0, no bleeding / suppuration;

1, bleeding / suppuration) at six sites per implant supported restoration (Mombelli

& Lang 1994). Similarly, the presence or absence of plaque at the peri-implant

mucosal margin was recorded at six sites per implant supported restoration

dichotomously.

• Implant-supported restorations were assessed to determine if any of the following

features were evident or had taken place: implant fracture, loss of superstructure,

abutment fracture, framework or veneer fracture, abutment screw fracture,

abutment or abutment screw loosening, restoration screw loosening, loss of

retention (luting cement), veneer chipping and loss of access hole restoration

(Pjetursson, et al. 2012).

Definitions & Success Criteria

• % Survival – percentage of implants and restorations in-vivo and in function at the

time of assessment.

• Biological complications: included implants that exhibited signs of peri-implant

mucositis or peri-implantitis. Peri-implant mucositis was defined as any bleeding

on gentle probing without loss of supporting bone (Lang, et al. 2011). Peri-

implantitis was defined as bleeding on probing and / or suppuration with or

without concomitant deepening of peri-implant pockets, accompanied by marginal

bone level changes of greater than 1.0mm after baseline (Sanz, et al. 2012) (Lang,

et al. 2011). Furthermore, any treatment performed on the implant that was not

part of routine maintenance was by default classified as a complication.

• Technical complications (Pjetursson, et al. 2012): These were divided into (1)

major: such as implant fracture, loss of super-structure, (2) medium: such as

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abutment fracture, veneer or framework fracture and (3) minor: abutment and

screw loosening, loss of screw hole sealing, veneer chipping (requiring polishing)

and occlusal adjustments. In addition, any treatment performed on the restoration

that was not part of routine maintenance was by default classified as a

complication. If the implant was incorporated into an FPD, then any technical

complication affecting any part of the FPD was included in the analysis.

Statistics All data was analyzed using IBM SPSS Statistics for Windows version 25.0 (IBM

Corporation 2017©, Armonk, NY, U.S). Descriptive statistics such as mean (±

standard deviation) in addition to median (inter-quartile range) and range (minimum,

maximum) as well as absolute and relative frequencies were provided for baseline and

outcome parameters. Inter-group comparisons for continuous variables (DIB, PPD,

DIM) were performed using an independent t-test after being assessed for normality.

Homogeneity of variance was assessed using the Levene’s test. Mean difference,

confidence intervals and P values were adjusted if equal variance could not be

assumed. A paired t-test was used for intra-group time comparisons. Comparisons

using count data (BOP and PI) were assessed using a GEE with a Poisson regression

model. Proportions were analyzed using a Chi square or Fisher’s Exact test. A multi-

variable linear regression model was used to assess the impact of treatment group,

initial implant position (depth), residual ridge height, periodontal status, age and

gender on ABL1 and ABL2. The null hypothesis was taken as non-equivalence in

marginal alveolar bone loss, proportions of complications, and peri-implant

parameters around dental implants placed into augmented maxillary sinuses using

ABBM (Bio-Oss®) + AB or blocks of ABBM-C (Bio-Oss Collagen®) alone. A P ≤

0.05 was considered to represent statistically significant differences.

Results Study population & Survival (table 1)

Outcomes related to the maxillary sinus augmentation and implant placement stages

of treatment have been previously published (Alayan & Ivanovski 2018). Originally

sixty (n = 60) patients were enrolled into this study and underwent unilateral sinus

augmentation followed by single implant placement (n = 30 were allocated into each

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group) 6 months later. A total of 7 patients (3 control, 4 test group) were lost to follow

up as they did not return for their 1 year follow up assessment and were not

contactable. Analysis was performed on 53 patients (control = 27, test = 26). There

were no significant differences between groups in terms of age, gender, ratio or

number of smokers. Patients with chronic periodontitis (control = 3, test = 7 patients)

remained stable and continued to attend for supportive maintenance program (Lang &

Tonetti 2003) at the same private practice (Brisbane, Australia). There were no

significant inter-group differences in the number of weeks between insertion of

restoration and when baseline assessment actually took place (control = 9.29 ± 3.8

weeks) (test = 10.81 ± 2.7). Similarly, there were no significant inter-group

differences in the number of weeks between baseline assessment and the 12 month

assessment (control = 58.07 ± 19.61 weeks) (test = 59.73 ± 13.25). All 53 patients

presented with functional implant supported restorations resulting in a 100% survival

rate for both groups. Ten millimeter long implants were used in 92% of control

patients and 88% of test patients. The dimensions of all implants are shown in table 2.

Radiographic Outcomes

Marginal bone level values are shown table 3. No significant inter-group differences

(control vs. test) for DIB0 (marginal bone level at implant placement), at DIB1

(marginal bone level at delivery of the implant supported restoration) (baseline) or

DIB2 (marginal bone level after 12 months of function) were reported (P>0.05) (table

4). Intra-group comparisons indicated that these dimensions significantly increased at

baseline when compared to implant placement (DIB1 > DIB0) and at the 1 year

follow up when compared to implant placement (DIB2 > DIB1) in both the control

and test groups (P < 0.0001) (table 4).

When considering the change of marginal bone levels over time, there were no

significant inter-group differences (control vs. test) (P>0.05) (table 4). Intra-group

comparisons indicated a significantly greater ABL1 in both the control (P = 0.001)

and test groups (P = 0.001) when compared to ABL2. Multi-variable linear

regression analysis (table 5a) indicated that initial implant position (depth) was a

significant explanatory variable (P<0.0001) for ABL1 but that treatment group,

periodontal status, gender, age and residual ridge height were not significant

130

explanatory variables (P>0.05). None of these variables were significant explanatory

variables for ABL2 (P>0.05) (table 5b). Table 6 displays the frequency (no. of

implants / patients) distribution of marginal bone level changes (ABL) between T0-to-

T1 and between T1-to-T2 for both groups.

Biological Outcomes

All peri-implant parameters (PI, BOP, PPD, DIM) were similar for both groups and at

both time points (table 7). No suppuration was recorded in any site at either time point

for both groups. PI and BOP were low in both groups and at both T1 and T2.

Treatment group (control vs. test) was not a significant predictor for PI (P = 0.56) or

BOP (P = 0.63). Whilst both parameters increased at T2 (vs. T1) in both groups, this

did not reach a level of statistical significance PI (P = 0.07) or BOP (P = 0.09). There

were no significant inter-group differences in PPD at T1 (P = 0.45) (mean difference

0.13, 95%CI -0.49, 0.22) or at T2 (P = 0.81) (mean difference -0.05 & 95%CI -0.35,

0.45). Intra-group comparisons of PPD indicated no significant changes between T1

and T2 in both control (P = 0.39) (mean difference 0.1, 95%CI -0.13, 0.33) and test

groups (P = 0.49) (mean difference -0.85, 95%CI -0.33, 0.16). There were no

significant inter-group differences of DIM at T1 (P = 0.17) (mean difference -0.7,

95%CI -1.69, 0.30) or at T2 (P = 0.13) (mean difference -0.84, 95%CI -1.94, 0.26).

Intra-group comparisons of DIM indicated no significant changes between T1 and T2

in both the control (P = 0.51) (mean difference 0.38, 95%CI -1.51, 0.76) and test

groups (P = 0.28) (mean difference 0.52, 95%CI -1.47, 0.43). Using DIM

measurements to calculate gingival recession, there was minimal gingival recession at

T2 in both control (mean 0.40 ± 0.36) and test groups (mean 0.51± 0.61) with no

significant difference between treatment groups (P = 0.45) (mean difference 0.11,

95%CI -0.39, 0.17). Similar proportion of peri-implant mucositis was noted in both

groups with 62.9% (17/27 patients) of control implants and in 69.23% (18/26

patients) of test implants. There was no statistically significant association between

treatment group and proportion of peri-implant mucositis (P = 0.63). Peri-implantitis

was not diagnosed in any of the patients in this study.

Technical Outcomes

131

All patients were successfully restored without delay 3 months after implant

placement using metal-ceramic restorations. Table 8 summarizes type of restoration

utilized and the method of retention. The majority of patients were restored with a

single crown restoration (SC) in both treatment groups (67% control group and 77%

test group). Fewer number of implants were incorporated as part of a fixed partial

denture (FPD) (33% control group and 23% test group). The majority of FPDs were 3

units without cantilever extension with the distal implant placed into the augmented

maxillary sinus. A small of number of FPDs had a short mesial cantilevered

extension. Screw retention was the predominant type of retention utilized in both

treatment groups (78% control group and 77% test group). There was no statistically

significant association between type of restoration (P = 0.41) or type of retention

utilized (P = 0.94) and treatment group. Technical complications were noted in 7.4%

of patients in the control group (2/27 patients) and 11.54% in the test group (3/26

patients). A full description of these complications is shown in table 9. There was no

statistically significant association between proportion of complications and treatment

group (P = 0.67). All of these minor complications were successfully repaired by the

restoring clinicians.

Discussion

The aim of this equivalence trial was to report on the biological and technical

outcomes of implant supported restorations placed into augmented maxillary sinuses

using a conservative approach and locally harvested autogenous bone in combination

with Bio-Oss® (ABBM) versus Bio-Oss Collagen® (ABBM-C) alone. Both groups

were comparable in terms of age, gender ratio, rates of periodontal disease and

implant dimensions. This study revealed a 100% implant survival during the first year

of function for both groups, which is consistent with those reported in a number of

systematic reviews (Pjetursson, et al. 2008, Chiapasco, et al. 2009, Jensen &

Terheyden 2009, Nkenke & Stelzle 2009, Rickert, et al. 2012).

All patients in the present study were successfully restored without delay 3 months

after implant placement, suggesting that this is a sufficient time interval for

osseointegration to occur when using a moderately rough (SLA) implant surface using

both grafting materials. Systematic reviews report a mean loading time between 5 to

132

6.5 months (Chiapasco, et al. 2009, Jensen & Terheyden 2009) irrespective of the

biomaterial used or implant surface. The absence of autogenous bone from the

ABBM-C group did not lead to delays in restoration or decrease in survival rates even

though it has been shown to exhibit delayed maturity as indicated by a lower

proportion of lamellar bone (Alayan, et al. 2016). .

The one-piece implant system utilized in this study is designed to be placed with the

rough-smooth junction at the bone crest, leaving the implant collar in the trans-

mucosal region. Furthermore, the marginal bone remodeling process begins

immediately following placement of these implants and is influenced by the location

of the rough-smooth junction (Hammerle, et al. 1996, Hermann, et al. 2000, Hermann,

et al. 2001, Hermann, et al. 2001). These characteristics were also observed in this

study with greater initial marginal alveolar bone loss taking place with increasing sub-

crestal location of the rough-smooth junction. Initial implant position (depth)

however did not correlate with marginal bone level changes after 12 months of

functional load. This is in contrast to a previous clinical study (Hammerle, et al. 1996)

which reported greater marginal bone loss beyond initial remodeling in one piece

implants placed with a rough-smooth junction 1mm sub-crestal. Variations in in the

clinical model and the contrasting implant surfaces (TPS vs. SLA) may explain this

discrepancy. The vast majority of implants in the present study were placed with a

rough-smooth junction ranging from at the crest to 1.5mm below the bone crest.

Withstanding the short observation period, this suggests that the risk of excessive

marginal bone loss after initial remodeling due to mild sub-crestal placement of the

rough-smooth junction, is no different from crestal positioning in a group of healthy

compliant patients.

Both treatment groups exhibited stable and similar marginal bone level changes at the

1 year follow up. Significantly less change was seen in marginal bone levels during

functional load in both groups when compared to initial remodeling. This change may

represent bone loss due to exposure to bacterial load (Lang, et al. 2011) and

masticatory forces (Marcelis, et al. 2012) rather than physiological adaptation to

implant placement and biologic width formation. The marginal bone level changes

during the functional loading period in our study compare closely with a recent study

133

(Thoma, et al. 2014) that reported a mean marginal loss after 1 year of function of

0.25mm (±0.35) using the identical dental implant and radiographic protocol. This

study however placed implants in a variety of clinical scenarios with only a small

fraction of implants placed into MSA. Greater changes in marginal bone levels after

12 months of functional load were seen in the present study however than those

reported by Schincaglia et al. (Schincaglia, et al. 2015), who compared long implants

and MSA with short implants without MSA. They reported a mean loss of 0.16mm

(±0.62) in the grafted group. This variation between studies may have been caused by

differences in placement protocol and implant surface / design. Interestingly, whilst

the mean change in marginal bone level is smaller than the present study, the

variations (SD) are greater with 4 patients exhibiting marginal bone loss ranging from

1.0-2.5mm when compared to none in the present study.

Both treatment groups predominantly exhibited signs consistent with peri-implant

health and represent a compliant patient population who attend for maintenance and

performed adequate plaque control. Similar findings were reported in a recent study

on clinical parameters for implants placed into augmented maxillary sinuses after 12

months of functional load (Schincaglia, et al. 2015). They reported a mean PPD of 2.3

(±1.4)mm with 38% of the implants in the augmented sinus group exhibiting BOP.

The present study however diagnosed peri-implant mucositis in the majority of

patients in both treatment groups, when defined as any site of BOP without loss of

supporting bone. This was mostly due to the presence of one or two sites of BOP out

of 6 sites probed. The success criteria utilized in this study are based on the consensus

reports published after the 8th European Workshop on Periodontology (Pjetursson, et

al. 2012, Sanz, et al. 2012). It has been noted that variations in case definitions for

peri-implant disease (Derks & Tomasi 2015) leads to significant variations in reported

success rates (Karoussis, et al. 2003). Whilst the absence of BOP has a high negative

predictive value for disease progression (Jepsen, et al. 1996), the positive predictive

value of BOP becomes a useful parameter only once it reaches a frequency of ≥50%

at a site level (Luterbacher, et al. 2000). The case definition for mucositis also relies

on gentle probing (Lang, et al. 2011) and therefore probing force cannot be

discounted as a source of error in this study especially in the presence of low plaque

scores. Peri-implantitis was not diagnosed in any of the patients in this study.

134

However, recent meta-regression analysis demonstrated a positive association

between implant time in function and the prevalence of peri-implantitis (Derks &

Tomasi 2015). As such, our short observation period may be the main reason for the

absence of peri-implantitis and longer follow up will be required to more accurately

assess this outcome measure.

Fixed single crowns (SC) and multi-unit (FPD) implant restorations are well

documented in the literature (Pjetursson, et al. 2007, Jung, et al. 2008, Jung, et al.

2012, Pjetursson, et al. 2012). To the best of our knowledge this is the first

prospective study reporting on well defined technical outcomes for implant supported

restorations in MSA in the partially dentate patient. After one year of service, 100%

of the implant supported restorations were functional. The majority of patients were

restored with a SC restoration in both treatment groups. Screw retention was the

predominant type of retention utilized in both treatment groups and for both types of

restorations. There was no statistically significant association between type of

restoration or type of retention utilized and treatment group.

In the present study, the great majority of patients (90.56%) presented with

complication free implant supported restorations. Minor technical complications

comprising mostly of ceramic veneer fractures were seen in few patients. This is

consistent with a systematic review indicating that the most common technical

complication of implant supported restorations was veneer fracture (Pjetursson, et al.

2012). In the present study there was no statistically significant association between

proportion of complications and treatment group. From the total of 5 incidents of

reported complications, 4 affected screw retained restorations. This may be due to an

over representation of screw retained restorations in the present study but is also

consistent with a systematic review reporting higher technical complications for

screw retained restorations (Sailer, et al. 2012). Whilst the great majority of FPDs

were without cantilever extension a few 2-unit FPDs with cantilevered extension were

successfully utilized in this study. Whilst there are no comparative studies for this

treatment modality in maxillary sinus augmentation it appears to be consistent with

previous studies and systematic reviews (Aglietta, et al. 2009, Romeo & Storelli

2012, Kim, et al. 2014) on cantilevered restorations.

135

In conclusion, considering the short observation period, implant reconstruction of the

partially edentulous posterior maxilla after MSA using ABBM + autogenous bone or

collagen stabilized ABBM led to similar and stable marginal bone levels, high

implant survival, low rates of technical complications and no peri-implantitis. Whilst

high rates of peri-implant mucositis were similarly seen in both groups, its

significance remains unknown and is mostly related to definition threshold.

136

Acknowledgements The authors are grateful to Dr Dimitrios Vagenas (Biostatisticians, Institute for Health

and Biomedical Innovation, Queensland University of Technology, Kelvin Grove,

Australia) for his assistance and contribution to the statistical analysis in this project.

No external funding was received for this project.

137

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Table 1. Baseline Parameters

Parameters

Control

(ABBM+AB)

(n = 27)

Test

(ABBM-C)

(n = 26)

Group

comparison

(P)

Age (Mean years ±SD) 60.26 (±11.0) 61.58 (± 6.89) 0.61

Gender ratio (f/m) 2.0 (f=18/m=9) 2.71 (f= 19/m=7) 0.97

Smoking

(no. patients) 1 2 0.95

Periodontal disease

(no. patients) 3 7 0.89

Initial ridge height

(mm) (Mean ± SD) 3.11 ± 0.76 3.06 ± 0.70 0.81

Time (weeks)

restoration insertion to

baseline assessment

9.29 (3.8) 10.81 (2.7) 0.10

Time (weeks) baseline

assessment to 1 year

assessment

58.07 (19.61) 59.73 (13.25) 0.72

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM-C, collagen

stabilized anorganic bovine bone mineral; 95% CI, 95% confidence interval; P, P-value for

inter-group comparison after proportion testing or independent t-test.

Table 2. Implant dimensions – frequency table (no. of implants). Implants used were one piece with an SLA surface.

Implant

Dimensions

Control

(ABBM+AB)

(n = 27)

Test

(ABBM-C)

(n = 26)

RN 4.1 x 10mm 2 5

RN 4.8 x 10mm 1 0

WN 4.8 x 8mm 2 3

WN 4.8 x 10mm 22 18

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM-C, collagen

stabilized anorganic bovine bone mineral. RN, regular neck; WN, wide neck

Table 3. Marginal bone level (DIB) and changes at various time points (ABL).

Control (ABBM+AB) (n = 27) Test (ABBM-C) (n = 26)

Time Mean (± SD)

Median (± IQR)

Range (min, max)

Mean (± SD)

Median (± IQR)

Range (min, max)

DIB0 -0.38 (0.43)

-0.32 (0.68) -1.62, 0.20 -0.54

(0.45) -0.55 (0.75) -1.63, 0.14

DIB1 0.28 (0.31)

0.29 (0.42) -0.30, 1.12 0.24

(0.37) 0.24

(0.43) -0.70, 0.91

DIB2 0.60 (0.33)

0.58 (0.56) 0.08, 1.37 0.58

(0.40) 0.54

(0.55) -0.24, 1.57

ABL1 -0.66 (0.40)

-0.59 (0.5) -1.74, -0.02 -0.77

(0.48) -0.80 (0.62) -1.94, 0.21

ABL2 -0.32 (0.24)

-0.26 (0.38) -0.88, -0.02 -0.35

(0.23) -0.32 (0.29) -0.97, -0.01

DIB, distance from the implant shoulder at the implant-abutment connection to the

most coronal bone-to-implant contact; DIB0, mean DIB at implant placement (T0);

DIB1, mean DIB at loading of prosthesis (T1 baseline); DIB 2, mean DIB at 12

months after function (T2); ABBM, anorganic bovine bone mineral; AB, autogenous

bone; ABBM-C, collagen stabilized anorganic bovine bone mineral; ABL1, change in

DIB from T0 to T1; ABL2, change in DIB from T1 to T12; SD, standard deviation;

IQR, inter-quartile range. Negative ABL values denote alveolar bone loss.

Table 4. Group comparisons - marginal bone levels (DIB) and changes at various time points.

Inter-group comparison: Control (ABBM+AB) vs. Test (ABBM-C)

Parameter Mean Difference 95% CI P Value

DIB0 0.16 -0.08, 0.40 0.19

DIB1 0.05 -0.14, 0.23 0.61

DIB2 0.02 -0.18, 0.22 0.84

DIB0 - DIB1 -0.11 -0.35, 0.13 0.38

DIB1- DIB2 -0.03 -0.16, 0.10 0.68

Intra-group comparison

Parameter Mean Difference 95% CI P Value

Control

(DIB0 vs. DIB1) -0.66 -0.82, -0.50 <0.0001

Control

(DIB1 vs. DIB2) -0.32 -0.42, -0.22 <0.0001

Test

(DIB0 vs. DIB1) -0.77 -0.97, -0.57 <0.0001

Test

(DIB1 vs. DIB2) -0.35 -0.44, -0.25 <0.0001

Control

(ABL1 vs. ABL2) 0.34 0.15, 0.54 0.001

Test

(ABL1 vs. ABL2) 0.42 0.20, 0.64 0.001

DIB, distance from the implant shoulder at the implant-abutment connection to the

most coronal bone-to-implant contact; DIB0, mean DIB at implant placement (T0);

DIB1, mean DIB at loading of prosthesis (T1 baseline); DIB 2, mean DIB at 12

months after function (T2); ABL1, DIB change from (T0) to (T1); ABL2, DIB change

from (T1) to (T2); ABBM, anorganic bovine bone mineral; AB, autogenous bone;

ABBM-C, collagen stabilized anorganic bovine bone mineral; 95% CI, 95%

confidence interval of the mean difference; P, P-value for group comparison after

applying a t-test.

Table 5a. Results of multi-variable linear regression model for ABL1.

ABL1, marginal bone level changes from implant (T0) placement to baseline (T1); b,

regression estimate coefficient; SE, standard error; 95% CI, 95% confidence interval.

b (SE) 95% CI P value

Treatment group

control -0.02 (0.09) -0.19, 0.14 0.76

test reference - -

Periodontal status

healthy 0.07 (0.12) -0.16, 0.30 0.54

chronic disease reference - -

Gender

male 0.03 (0.09) -0.14, 0.21 0.71

female reference - -

Initial position -0.66 (0.10) -0.86, -0.47 <0.0001

Residual ridge height 0.10 (0.06) -0.02, 0.22 0.09

Age 0.0 (0.0) -0.01, 0.01 0.96

Table 5b. Results of multi-variable linear regression model for ABL2.

ABL2, marginal bone level changes from baseline (T1) to 12 months after function

(T2); b, regression estimate coefficient; SE, standard error; 95% CI, 95% confidence

interval.

b (SE) 95% CI P value

Treatment group

control -0.02 (0.07) -0.15, 0.11 0.72

test reference - -

Periodontal status

healthy 0.07 (0.09) -0.11, 0.24 0.46

chronic disease reference - -

Gender

male -0.11 (0.07) -0.24, 0.03 0.12

female reference - -

Initial position -0.03 (0.08) -0.19, 0.12 0.66

Residual ridge height -0.01 (0.05) -0.09, 0.09 0.95

Age 0.0 (0.0) -0.01, 0.01 0.85

Table 6. Frequency distribution (no. of implants / patients) of marginal bone level changes (ABL) across time.

ABL1, marginal bone level change from T0 to T1; ABL2, marginal bone level change from T1 to T12. T0,

implant placement; T1, insertion restoration (baseline); T2, 12 months after function; ABBM, anorganic bovine

bone mineral; AB, autogenous bone; ABBM-C, collagen stabilized anorganic bovine bone mineral.

ABL1 (T0-T1) ABL2 (T1-T2) ABL Interval

(mm) Control

(ABBM+AB) Test (ABBM-C) Control (ABBM+AB) Test (ABBM-C)

-2.5 < ABL ≤ -2.0 0 0 0 0

-2.0 < ABL ≤ -1.5 1 2 0 0

-1.5 < ABL ≤ -1.0 4 6 0 0

-1.0 < ABL ≤ -0.5 11 11 6 4

-0.5 < ABL ≤ 0 11 6 21 22

0 < ABL ≤ 0.5 0 1 0 0

0.5 < ABL ≤ 1.0 0 0 0 0

Total 27 26 27 26

Table 7. Clinical parameters of implants placed into augmented sinuses at baseline (T1) and after 12 months of function (T2).

Control (ABBM+AB) (n = 27) Test (ABBM-C) (n = 26)

Parameter Time Mean (± SD)

Median (± IQR)

Range (min - max) Parameter Time Mean

(± SD) Median (± IQR)

Range (min - max)

PI (no. sites)

T1 0.22 (0.51)

0.0 (0) 0.0 – 2.0

PI (no. sites)

T1 0.19 (0.49)

0.0 (0) 0.0 – 2.0

T2 0.41 (0.57)

0.0 (1.0) 0.0 – 2.0 T2 0.31

(0.55) 0.0

(1.0) 0.0 – 2.0

BOP (no. sites)

T1 0.96 (1.0)

1.0 (1.0) 0.0 - 3.0

BOP (no. sites)

T1 0.88 (1.03)

1.0 (1.25) 0.0 – 3.0

T2 1.03 (0.94)

1.0 (2.0) 0.0 - 3.0 T2 1.31

(1.16) 1.0

(2.0) 0.0 – 4.0

PPD (mm)

T1 2.34 (0.50)

2.33 (0.67) 1.33 - 3.33

PPD (mm)

T1 2.46 (0.76)

2.33 (1.22) 0.83 – 3.83

T2 2.50 (0.66)

2.33 (0.83) 1.50 – 4.17 T2 2.43

(0.83) 2.33

(1.13) 1.17 – 4.50

DIM (mm)

T1 8.10 (2.0)

7.67 (3.84) 5.17, 12.17

DIM (mm)

T1 8.78 (1.54)

8.84 (2.13) 6.17 – 12.0

T2 8.35 (2.09)

8.0 (3.43) 4.83, 12.33 T2 9.31

(1.94) 8.84

(1.71) 6.33 – 14.33

T1, baseline; T2, 12 months after function; ABBM, anorganic bovine bone mineral; AB,

autogenous bone; ABBM-C, collagen stabilized anorganic bovine bone mineral; PI, plaque index;

BOP, bleeding on probing; PPD, probing pocket depth; DIM, gingival marginal position; SD,

standard deviation; IQR, inter-quartile range.

Table 8. Technical Outcomes – type of prosthesis and retention

Control group (ABBM+AB)

(n = 27)

Type of prosthesis Test group (ABBM-C)

(n = 26)

Type of prosthesis

SC FPD Totals SC FPD Totals

Ret

entio

n Screw 15 6 21/27 (0.78)

Ret

entio

n Screw 16 4 20/26 (0.77)

Cement 3 3 6/27 (0.22) Cement 4 2 6/26

(0.23)

Totals 18/27 (0.67)

9/27 (0.33) Totals 20/26

(0.77) 6/26

(0.23)

ABBM, anorganic bovine bone mineral; AB, autogenous bone; ABBM-C, collagen

stabilized anorganic bovine bone mineral; SC, single crown, FPD, fixed partial denture.

Table 9. Technical Complications.

Patient Complication Category Treatment group Prosthesis Retention

1 Veneer fracture Minor Control SC Screw

2 Veneer fracture Minor Control FPD Screw

3 Restoration screw loosening Minor Test SC Screw

4 Veneer fracture Minor Test SC Screw

5 Veneer fracture Minor Test FPD (cantilever) Cement

Control group (ABBM + AB); Test group (ABBM-C); (ABBM, anorganic bovine bone mineral; AB,

autogenous bone; ABBM-C, collagen stabilized anorganic bovine bone mineral; SC, single crown,

FPD, fixed partial denture.

CONSORT 2010 Flow Diagram

Assessed for eligibility (n=67)

Excluded (n= 7) ¨ Not meeting inclusion criteria (n=3) ¨ Declined to participate (n=4) ¨ Other reasons (n=0)

Analysed (n=27) ¨ Excluded from analysis (n=0)

Lost to follow-up (not contactable = 2, relocated = 1) (n= 3)

Discontinued intervention (n=0)

Allocated to CONTROL (ABBM+AB) (n= 30) ¨ Received allocated intervention (n= 30) ¨ Did not receive allocated intervention (n=0)

Lost to follow-up (not contactable = 3, relocated =1) (n=4)

Discontinued intervention (n=0)

Allocated to TEST (ABBM-C) (n= 30) ¨ Received allocated intervention (n= 30) ¨ Did not receive allocated intervention (n=0)

Analysed (n=26) ¨ Excluded from analysis (n=0)

Allocation

Analysis

Follow-Up

Allocation (n= 60)

Enrollment

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CHAPTER 7 – DISCUSSION & FUTURE DIRECTIONS

157

DISCUSSION

The rehabilitation of partial and fully edentulous patients with dental implants is

considered to be a predictable therapeutic modality with favourable long term

functional outcomes (Buser, et al. 2012, Jung, et al. 2012, Pjetursson, et al. 2012).

Reduced alveolar bone height and bone density however are common limitations

when considering implant rehabilitation of the posterior maxilla (Jemt & Lekholm

1995). Maxillary sinus floor elevation (MSA) using the lateral window approach

(Boyne & James 1980) is the most commonly utilized procedure to overcome

maxillary sinus pneumatisation, resulting in predictable survival of implant

supported restorations (Bornstein, et al. 2008, Del Fabbro, et al. 2008, Pjetursson,

et al. 2008, Nkenke & Stelzle 2009, Corbella, et al. 2015, Antonoglou, et al. 2018).

Histomrphometric assessment of collagen stabilized ABBM in MSA

The significant limitations of autogenous bone (AB) in MSA has driven intense

research into various bone substitutes. Anorganic bovine bone mineral (ABBM)

(Bio-Oss®) is a very well documented xenograft in MSA when used alone or as a

composite graft with AB (ABBM + AB) (Galindo-Moreno, et al. 2011, Schmitt, et

al. 2015). More recently, collagen stabilized ABBM using 10% porcine type-1

collagen matrix has also become available for use (ABBM-C) (Bio-Oss

Collagen®). This formulation may have clinical utility in its use as a sole

biomaterial in MSA, especially in situations of Schneiderian membrane perforation.

There are however, no published studies on this material in MSA.

As such, the first part of this thesis was to test the suitability of this biomaterial in

a pre-clinical model. Collagen stabilized ABBM exhibited very similar

histomorphometric parameters to the composite graft of ABBM + AB in the

proximal zones to resident bone. The presence of AB however, seemed

advantageous in regions distant from resident sinus walls. This is consistent with

previous pre-clinical models reporting a gradient of graft consolidation

(Busenlechner, et al. 2009) which is dependent on the osteogenic response of the

host bone and the osteogenic profile of the bone substitute (Busenlechner, et al.

2008). This is also in line with early clinical observations of bone formation directly

from the sinus walls (Boyne & James 1980) with little evidence of new bone in

contact with the Schneiderian membrane (Boyne 1993, Scala, et al. 2010). New

158

bone was rarely observed in contact with the Schneiderian membrane in this study.

Whilst there are reports of successful MSA and simultaneous dental implants

placement without the use of adjunctive grafting material (Lundgren, et al. 2004),

bone formation does not appear to be extensive and is only present in areas proximal

to native bone walls of the maxillary sinus (Lundgren, et al. 2004, Palma, et al.

2006). Furthermore, the Schneiderian membrane has been shown to have

osteogenic potential (Gruber, et al. 2004, Srouji, et al. 2009), but this does not seem

to translate into actual bone formation in pre-clinical models (Haas, et al. 2002,

Scala, et al. 2010). It has also been shown that whilst new bone is formed after

membrane elevation only, this is unstable leading to resorption in areas where the

membrane collapses due to instability of the blood clot and a lack of mechanical

support (Xu, et al. 2005).

Whilst previous clinical comparative studies have failed to show an advantage of

adding AB to ABBM (Galindo-Moreno, et al. 2011, Corbella, et al. 2016), its use

in this pre-clinical experiment was associated with significant improvements in new

bone formation and degree of particle osseointegration, but only in the zone distant

to the resident sinus bone walls. When the above experiment was repeated in a

clinical model however, comparable histomorphometric outcomes were observed

for the two materials, with no signs of different gradients of consolidation. This

may be due to variety of reasons, such as differences between animal and human

models or the varying ability to detect a gradient in whole sinus samples compared

with biopsy harvested samples. Whilst there are no studies comparing these two

different sampling techniques, one could postulate that results would vary

depending on the location and depth of the biopsy (Margolin, et al. 1998, Artzi, et

al. 2005, Avila, et al. 2010). Another influencing factor may be the proximity of the

lateral walls of the sinus to the harvesting site (Suarez-Lopez Del Amo, et al. 2015).

Further pre-clinical comparisons between the standard particulate formulation of

ABBM alone and the ABBM+AB composite graft is required to definitively assess

the impact of adding AB to this model. There are currently no pre-clinical

histomorphometric comparative studies using ABBM+AB and ABBM alone.

159

The impact of hydrophilic dental implant surface on early osseointegration in MSA

Whilst grafting material influences the histological make-up of the regenerated

bone, other factors can also play a role including the dental implant surface. This

project assessed the impact of using a chemically modified hydrophilic implant

surface in MSA (SLActive®). Implant surface topography and chemistry have been

shown to affect osseointegration (Gittens, et al. 2012). They play a critical role in

the predictability of the bone response to implant placement (Junker, et al. 2009)

and are a major determinant of the rate and extent of dental implant osseointegration

(Wennerberg, et al. 2011). To date however, very few studies have compared

different implant surfaces in MSA and none at the early stages of osseointegration.

Results from this experiment showed that both time and the use of a hydrophilic

implant surface had a positive impact on BIC. Hydrophilic implant surfaces also

had a positive impact on the surrounding tissue composition. This finding is in

agreement with a number of studies using this same hydrophilic implant surface

(SLActive®, Struamann, Basel, Switzerland), indicating enhanced bone apposition

during the early stages of osseointegration compared to the standard SLA surface

(Lang, et al. 2011). The early advantage however, was not evident when assessing

BIC in a pre-clinical MSA model at later time points (Philipp, et al. 2013). Early

loading of modified hydrophilic implants may remain as an advantage, similar to

what has already been observed in non-grafted clinical models (Morton, et al.

2010).

Volumetric / radiographic assessment of collagen stabilized ABBM in MSA

Studies have shown higher resorption rates resulting in loss of graft volume when

using AB alone in MSA (Johansson, et al. 2001, Arasawa, et al. 2012), and an

increase in volumetric stability following the incorporation of ABBM, which was

positively correlated with increased proportions of ABBM in the ABBM + AB

composite grafts (Jensen, et al. 2012). The volumetric stability of collagen

stabilized ABBM (ABBM-C) was assessed in this study, to further determine its

suitability for use in MSA. A prospective clinical trial compared ABBM-C and

ABBM + AB with allocation based on defined clinical presentations (sinus

membrane perforation and/or insufficient local AB bone). ABBM-C exhibited

comparable bone volume to AB + ABBM that was sufficient for placement of

implants of adequate length, with little or no need for further vertical augmentation.

160

Longer follow up will be needed to determine if further changes in graft volume

takes place in both treatment groups over the long term.

Interestingly, further analysis indicated that engaging the surrounding sinus walls

had a significant positive impact on graft volume but not the residual ridge height.

Whilst previous studies have indicated that adequate graft height can be achieved

even in the presence of a sinus floor fenestration (Cortes, et al. 2015), this is the

first study to report on the association between engaging adjacent walls and graft

volume. Conceptually this finding is consistent with the overall impression that the

resident sinus walls are key defect parameters that influence not only histological

outcomes but also volumetric stability. Histological studies have shown a positive

impact of increasing both the number and total area of engaged bony on graft

healing outcomes (Haas, et al. 1998, Busenlechner, et al. 2009, Alayan, et al. 2017).

This is consistent with the principle, ascertained form other models of guided bone

regeneration, that surrounding bony walls are an important determinant for de novo

bone formation and graft particle incorporation (Klinge, et al. 1992, Schenk, et al.

1994, Carmagnola, et al. 2003).

Surgical complications & PROMs associated with MSA

Most of the clinical outcomes data for MSA is derived from medium to low level

evidence (clinical case series, retrospective analyses). Patients undergoing this

procedure also expect to be counselled about their expectations regarding pain and

the impact on their daily life in the post-operative period. Such information is not

currently available. There are few prospective controlled clinical trials applying

well defined success criteria to implant supported restorations placed in sites of

MSA and there are none on the use of collagen stabilized ABBM grafting material

in MSA. As such, the second part of this thesis explores the clinical, and patient

centred outcomes of MSA using these two biomaterials, followed by assessment of

the survival and success of the implant supported restorations placed into these

sites.

This patient population did not experience major intra- or post-operative

complications, consistent with previous reviews (Chiapasco, et al. 2009). In

addition, the minor complications were manageable and did not prevent completion

161

of the augmentation procedure. The most frequent complication was membrane

perforation, which occurred in 13% of the patient population. Multiple reviews

have also reported this to be the most frequently reported surgical complication

(Pjetursson, et al. 2008, Chiapasco, et al. 2009, Nkenke & Stelzle 2009). Whilst the

reported incidence varies greatly between studies, our rate of perforation is on the

lower end of the range and similar to more recent publications (Becker, et al. 2008,

Cortes, et al. 2015, Holtzclaw 2015). Most of these perforations were small in

diameter (≤ 3mm) and were repaired with a resorbable porcine collagen membrane

using a previously described technique (Proussaefs & Lozada 2003). The role of

the collagen in the test grafting material is to cohesively bind the ABBM granules

to form an intact 3-dimensional block. Its advantage in sinus augmentation

procedures over particulate material is evident when perforations of the sinus

membrane occur and there is a risk of endo-antral displacement of loose particles

and obstruction of the OMC (Quiney, et al. 1990, Wiltfang, et al. 2000, Doud Galli,

et al. 2001). The significance of sinus membrane perforations on implant survival

or graft success remains contentious with some studies reporting a negative impact,

especially in large perforations (Proussaefs, et al. 2004, Hernandez-Alfaro, et al.

2008), while others showed no differences (Becker, et al. 2008). Further follow-up

of our patient population did not reveal any consequences in outcomes on implant

loading, survival or biological / technical outcomes.

In terms of patient reported outcome measures (PROMs), the majority of patients

reported that average daily pain was mild to moderate for the first 3 days and

subsequently reduced to mild levels using non-steroidal anti-inflammatory drugs

(NSAIDs) only. The reported level of pain was similar across treatment groups. The

majority of patients in both treatment groups reported a moderate amount of

limitation to both oral and general activities during the first 48hrs post-surgery that

significantly further reduced by day 3 or 4. To the best of my knowledge, this is the

first study to report on PROMs for maxillary sinus augmentation and indicates that

48-72 hours of recovery from this procedure is needed for patients to resume oral

function and daily activities without limitations or the need for analgesics.

Whilst greater prevalence of bruising and swelling were noted clinically in the

control group at the post-operative review, this did not reach statistical significance.

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Bruising and oedema was also noted to be of reduced severity in test patients

compared to patients in the control group, but this observation was not objectively

measured. This perception of increased limitation in oral functions may reflect the

increased morbidity associated with greater autogenous bone harvesting (Cricchio

& Lundgren 2003). Patients in the control group typically possessed a thick buccal

wall of bone that provided sufficient volume of autogenous bone needed to support

the elevated Schneiderian membrane at the requisite height. The test patients on the

other hand exhibited a thin buccal wall of bone and therefore experienced far less

bone removal. It is likely those symptoms of pain and perception of bruising and

oedema translated to limitation in social life and work and is generally consistent

with anecdotal reports from patients of abstaining from work for 48 hours post-

surgery.

It’s important to note however, the sample size calculation for this part of the project

was performed for graft volumetric analysis and not for PROMs. Further studies

specifically designed at investigating PROMs after MSA would be needed before

firm conclusions can be made.

Biological & technical outcomes of dental implants placed into MSA sites

The same patient population was followed to assess implant supported restorations

after 12 months of function using well defined success criteria. Both groups

revealed 100% implant survival rates, which is consistent with high survival rates

reported in other MSA studies using micro-rough implant surfaces (Pjetursson, et

al. 2008, Chiapasco, et al. 2009, Jensen & Terheyden 2009, Nkenke & Stelzle 2009,

Rickert, et al. 2012). Whilst many factors may contribute to implant survival,

implant surface and graft composition are two important considerations. A median

survival rate of 100% (88-100%) is reported for rough implant surfaces when

compared to 89% (61.2 – 100%) using machined implant surfaces (Jensen &

Terheyden 2009). A100% survival rate was reported in a clinical study using one-

piece SLA implants in MSA at 60 months after implant placement (Bornstein, et al.

2008). This may be due to the documented favourable histological outcomes

(contact osteogenesis) with moderately rough implant surfaces (Abrahamsson, et

al. 2004), resulting in improved bone-to-implant contact (BIC) and stability

compared to minimally rough surfaces (Buser, et al. 1998, Cochran, et al. 1998).

163

Furthermore, from a graft material perspective, the 5-year implant survival rate of

implants placed in MSA sites grafted using composite graft AB+ABBM (1:1) was

reported to be 92.86% (Schmitt, et al. 2015) in a small number of patients.

It has been suggested that many factors influence osseointegration and the ability

of implants in augmented maxillary sinuses to withstand functional load (Jensen, et

al. 1998). This multitude of variables may explain the seemingly arbitrary loading

interval reported in studies. All patients in the present project were successfully

restored without delay 3 months after implant placement, suggesting that this is a

sufficient time interval for osseointegration to occur when using a moderately rough

(SLA) implant surface in either of the grafting materials. Studies using the same

composite graft (Galindo-Moreno, et al. 2015) or implants surface (Bornstein, et al.

2008) have utilized a loading time of 6 months and 10 weeks respectively.

Systematic reviews report a mean loading time between 5 to 6.5 months

(Chiapasco, et al. 2009, Jensen & Terheyden 2009).

We could speculate, based on our pre-clinical histological findings for the modified

hydrophilic implant surfaces, that these implants may be successfully loaded earlier

than the 12 week protocol we used for the SLA implants. This obviously needs to

be further investigated in an appropriately designed clinical trial.

The four most frequently used parameters for assessing success are related to the

implant level, peri-implant soft tissue, prosthesis and patient’s subjective

assessment (Papaspyridakos, et al. 2012). Similarly, based on consensus reports

published at the 8th European Workshop on Periodontology, outcome measures

should capture patient reported outcomes (McGrath, et al. 2012), peri-implant

health (Derks & Tomasi 2015) and restoration outcomes (Pjetursson, et al. 2012).

As such, for implant health we reported on marginal bone levels, tissue

inflammation (BOP / suppuration) and periodontal probing depth. For implant

restorations, we reported on longevity of the restorations and technical

complications. One limitation of this project was that PROMs were reported for the

MSA procedure only but not for the subsequent implant supported restorations.

164

Furthermore, we reported each success criteria outcome separately instead of

reporting the implant-prosthetic complex as a whole (Papaspyridakos, et al. 2012).

Generally, there is a paucity of studies reporting on implant outcomes using well

defined success criteria when placed into a composite graft (ABBM+AB 1:1)

(Galindo-Moreno, et al. 2015), and no studies on implants inserted in collagen

stabilized ABBM grafts. It has been shown however, that micro-rough implants

placed into MSA sites can exhibit high success rates with stable marginal bone

levels and healthy peri-implant parameters (Bornstein, et al. 2008). In this thesis,

both groups had very similar outcome measures across all categories. A mean

alveolar bone loss of 0.66mm in the control group and 0.77mm in the test group

was noted initially after implant placement (biologic width formation), that was

significantly associated with depth of placement and location of the rough-smooth

junction. This is consistent with studies indicating that marginal bone level changes

for one-piece rough surface implants are associated with remodelling immediately

following implant placement and are influenced by the location of the rough-

smooth junction (Hammerle, et al. 1996, Hermann, et al. 2000, Hermann, et al.

2001, Hermann, et al. 2001).

Significantly lower marginal bone loss was seen during the interval between

baseline and 12 months of function. Continued change in marginal bone levels after

this initial remodelling period has been observed in many studies. In this thesis, a

mean loss of 0.32mm and 0.35mm were noted in the control and test groups

respectively. This change may represent bone loss due to exposure to bacterial load

(Lang, et al. 2011) and masticatory forces (Marcelis, et al. 2012), rather than a

physiological adaptation to implant placement. The bone loss at 12 months in our

patient population approximates that seen in a comparable study with a mean

marginal bone loss of 0.25mm (Bornstein, et al. 2008), but was double that of

another study reporting a mean loss of 0.16mm (±0.62) (Schincaglia, et al. 2015)

during the same interval. These variations between studies may have been caused

by differences in the placement protocol and implant surface / design. The authors

have attributed the small amount of marginal bone loss to the platform switching

feature of the implants utilized in their study.

165

Both treatment groups predominantly exhibited signs consistent with peri-implant

health. At the 12 months follow up, they were characterized by a median of 1 site

of BOP, a mean PPD <3mm and negligible gingival recession. Both treatment

groups represent a compliant patient population who attend for maintenance and

performed adequate plaque control. This is similar to a recent study that reported

on clinical parameters for implants placed into augmented maxillary sinuses after

12 months of functional load (Schincaglia, et al. 2015). They reported a mean PPD

of 2.3mm (±1.4). They also reported that 38% of the implants in the augmented

sinus group exhibited BOP. This is lower than the present study, with mucositis

diagnosed in approximately 2/3 of patients in both treatment groups (62.9% control

patients and 69.23% of test patients), when defined as any site of bleeding on gentle

probing without loss of supporting bone (Pjetursson, et al. 2012, Sanz, et al. 2012).

This was mostly due to the presence of one or two sites of BOP out of 6 sites probed.

Indeed, 96% of control and 85% of test patients had BOP at fewer than 3/6 (i.e. 0,

1 or 2) sites at the 12 months follow up. Whilst the absence of BOP has a high

negative predictive value for disease progression (Luterbacher, et al. 2000, Coli, et

al. 2017), the significance of a single recording of positive BOP in determining

disease progression is not determined and its dichotomous nature limits its

correlation to disease status and severity (Sanz-Sanchez, et al. 2018). Furthermore,

excessive probing force may induce false positive readings (Gerber, et al. 2009).

A high prevalence of peri-implant mucositis was reported by a recent systematic

review estimated the weighted mean prevalence to be 43% (95% CI: 32, 54%)

(Derks & Tomasi 2015). The case definitions for peri-implant disease vary

significantly in terms of threshold (Derks & Tomasi 2015) leading to significant

variations in reported success rates (Karoussis, et al. 2003). The success criteria

utilized in this study are based on the consensus reports published at the 8th

European Workshop on Periodontology (Pjetursson, et al. 2012, Sanz, et al. 2012).

When an alternative, previously proposed definition of mucositis was used (≥1 site

with PPD ≥ 5mm and BOP without loss of supporting bone) (Mombelli & Lang

1994), mucositis was diagnosed in 11.1% in the control group and 15.4% in the test

group. There was no statistically significant association between treatment groups

and proportion of peri-implant mucositis. It is possible that our sample size was

insufficiently powered to detect this difference. Peri-implantitis was not diagnosed

166

in any of the patients in this study. However, it is noteworthy that a recent meta-

regression analysis demonstrated a positive association between implant time in

function and the prevalence of peri-implantitis (Derks & Tomasi 2015). As such,

our short observation period may be the main reason for the absence of peri-

implantitis and longer follow up will be required to more accurately assess this

outcome measure.

To the best of our knowledge there are no prospective studies reporting on well

defined technical outcomes for implant supported restorations placed in MSA sites

in the partially dentate patient. Fixed single (SC) and multi-unit (FPD) implant

restorations are well documented in the literature (Pjetursson, et al. 2007, Jung, et

al. 2008, Jung, et al. 2012, Pjetursson, et al. 2012) with a reported 5 year survival

of 96.3% and 95.4% respectively (Jung, et al. 2012, Pjetursson, et al. 2012). The

majority of the restorations in this study were screw retained SCs or short span

FPDs with a small number of cement retained restorations. Both forms of retention

have their advantages and disadvantages (Michalakis, et al. 2003, Chee & Jivraj

2006). Overall, the 5 year survival rates for fixed implant supported reconstructions

is 96.03% (CI: 93.85 – 97.43%) and 95.55% (CI: 92.96 – 97.19%) for screw

retained and cement retained reconstructions respectively (Wittneben, et al. 2014).

When assessed according to prosthesis type, the estimated 5-year survival rate of

screw retained SC and FPD reconstructions is similar to that of cemented

reconstructions (Sailer, et al. 2012, Wittneben, et al. 2014, Millen, et al. 2015). The

short observation period of this project and the 100% survival rates prevent any

comments or conclusions on restoration survival.

The most common technical complication of implant supported restorations was

reported to be veneer fracture (Pjetursson, et al. 2012), which was consistent with

the findings of this thesis. The overall rate of technical complications of fixed

implant reconstructions has been reported to be significantly greater for cement

retained when compared to screw retained fixed restorations (Wittneben, et al.

2014, Millen, et al. 2015). When the data is analysed according to the type of

prosthesis however, screw retained SC restorations exhibit greater rates of technical

complications due to their significantly greater rates of abutment screw loosening

(Millen, et al. 2015). Whilst our results seem to be consistent with these reports,

167

they are however influenced by the significantly greater number of SCs and screw

form of retention, in addition to the very short observation period of this project.

This prevents any meaningful analysis or conclusion.

FUTURE DIRECTIONS

• There are few studies reporting on long term outcomes for implant supported

restorations placed into MSA using well defined success criteria as well as

volumetric graft stability. A long term follow-up of the present population

would therefore be a useful contribution to the field.

• It would be interesting and novel to assess the histomorphometric and

volumetric outcomes in a controlled clinical MSA model that is analyzed and

stratified according to extent of engagement of maxillary sinus walls.

• The impact of implant surface characteristics on the loading protocols of

implants placed in MSA sites has been underexplored. This thesis showed that

chemical modification can result in accelerated osseointegration as measured

by improved early bone-implant contact. However, the clinical implications of

these findings are not clear and it would be interesting to explore whether using

the chemically modified SLActive implants would facilitate the use of shorter

loading protocols.

• Tissue engineering is a rapidly developing multi-disciplinary field. Continued

development in this area may lead to the production of a bone substitute that is

not only biocompatible and highly osteoconductive but also osteoinductive and

is gradually resorbed and replaced by host tissue. This is likely to involve the

use of 3-dimensional bioresorbable alloplastic scaffolds with or without the

addition of growth factors and cells. This may lead to further reductions in

treatment time and possibly morbidity.

168

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APPENDIX A

SCHOOL OF DENTISTRY

DENTAL SCIENCES RESEARCH ETHICS COMMITTEE

APPROVAL FORM

Chief Investigator: Jamil Alayan Project Title: Randomised clinical trial comparing DBBM vs. BCP with and without autogenous bone deficient maxillary Sinuses for the purpose of dental implant placement. Supervisor: A/Prof CS Farah Co-Investigator(s): N/A Department(s): Dentistry Project Number: 1010 Duration of Approval: 3 years

Comments/Conditions/Restrictions: This project complies with the provisions contained in the National Statement on Ethical Conduct in Research Involving Humans and complies with the regulations governing experimentation on humans. A/Prof Camile Farah Chairperson, Research Committee Date:___________________ Signature: _______________________________________

University Animal Research Ethics CommitteeANIMAL ETHICS APPROVAL CERTIFICATE

QDPI Scientific Use Registration Number 0052

Date of Issue:4/10/11 (supersedes all previously issued certificates)

UAECs must advise investigators and teachers of their decisions in writing as promptly as possible and activities must not commence until written approval has been obtained from the UAEC. This Approval Certificate serves as your written notice.

Within this Approval Certificate are: * Activity Details * Animal Details * Conditions of Approval (Specific and Standard)

The following standard conditions are particularly important: (a) prompt notification to the UAEC of any adverse or unexpected effects that impact on animal wellbeing; (b) submission to the UAEC for consideration, any changes to the project as detailed in the proposal application.

Further information regarding your ongoing obligations regarding animal based research can be found via the Research Ethics website (http://www.research.qut.edu.au/ethics/) or by contacting the Research Ethics Coordinator on 07 3138 2091 or ethicscontact@qut.edu.au.

If any details within this Approval Certificate are incorrect please advise Research Ethics within 10 days of receipt of this certificate.

Activity Details

Dear Prof Dietmar Hutmacher

1100001051Approval Number:

Bone regeneration in a sinus model: a comparison between several bone grafting materials

Activity Title:

The aims of this project are to compare the histological appearance, histomorphometric parameters, immunohistochemical features and expressed gene profiles of biopsies harvested from the maxillary sinus cavity in a sheep model (ovis aries)after sinus ?oor augmentation with either deproteinised bovine bone mineral (DBBM; Bio-Oss, Geistlich AG, Wolhusen, Switzerland) or Bio-Oss Collagen (DBBM-C; Bio-Oss Collagen, Geistlich AG, Wolhusen, Switzerland) and to assess the impact of adding autogenous bone on these parameters by means of a prospective randomized clinical trial.

Activity Summary:

Approved Until: 04-Oct-2014 (subject to annual reports to the UAEC)Date Approved: 04-Oct-2011

Location(s): MERF

Chief Investigator: Prof Dietmar Hutmacher

Other Staff/Students:Investigator Name Type RoleAdj/Prof Saso Ivanovski Internal Ethics - Alternative

InvestigatorMr Cedryck Vaquette Internal Associate InvestigatorA/Prof Yin Xiao Internal Associate InvestigatorMr Stephen Hamlet External Associate InvestigatorMr Jamil Alayal External Associate InvestigatorDr Siamak Saifzadeh Internal Ethics - VeterinarianDr Eugene Huang External Associate Investigator

Investigator Details

Animal Details

RM Report No. E802 Version 3.1 Page 1 of 3

University Animal Research Ethics CommitteeANIMAL ETHICS APPROVAL CERTIFICATE

QDPI Scientific Use Registration Number 0052

Date of Issue:4/10/11 (supersedes all previously issued certificates)

Sheep: Domestic Mammals Ovis aries --- 3-4yrs maleFurther Details:Animal Type:No. Used to Date:Total No. Approved: 045

Monitoring of Animals: During procedures, the depth of general anaesthesia will be monitored throughout the operation using the following parameters: physiological parameters (HR, RR), reflexes (palpebral, corneal and web pinch reflexes) and muscle tone (jaw tension), the oxygen saturation of the sheep using pulse oximetry, end tidal CO2 level. After procedures - The oral surgical wounds will be checked on a daily basis for any evidence of post-operative complications, such as failure in suture integrity (wound dehiscence), bleeding, inflammation, and infection.

Housing of Animals: The sheep will be housed in a group of 10/yard. Group of 3/pen for overnight fasting. Environmental enrichment: grass, pellet, trees, and rocks.

Disposal of Animals: As per MERF protocols

Conditions of Approval

Specific Conditions of ApprovalNo special conditions placed on approval by the UAEC. Standard conditions apply.

Standard Conditions of ApprovalUnder its Terms of Reference the UAEC monitors the conduct of approved activities until their completion so as toensure continued compliance with relevant requirements, and may withdraw approval;

Investigators and teachers have personal responsibility for all matters related to the welfare of the animals they use and must act in accordance with all requirements of the Code. This responsibility begins when an animal is allocated to a project and ends with its fate at the completion of a project;

Work must be conducted in accordance with university policy, NHMRC / AVCC guidelines and regulations, and in accordance with the provisions of any legislation which is relevant to your project;

Immediately advise the UAEC, via the Research Ethics Coordinator, of any unforseen events that might affect the continued ethical acceptability of the project;

Immediately advise the UAEC, via the Research Ethics Coordinator, if any complaints are made, or expressions of concern raised in relation to the project;

Report on the progress of the approved project at least annually, or at intervals determined by the Committee. The Committee may also choose to conduct a random audit of your research;

Ensure that adequate records are kept of the acquisition, breeding, health, care, use, transportation, housing, handling and disposal of the animals. The Committee may wish to see these records at the end of the experimentation;

It is the responsibility of all investigators to ensure that all personnel involved, or connected with the project, including research assistant and students, are adequately trained in all handling and safety procedures;

Investigators and teachers must make arrangements so that they, or other responsible persons, can be contacted in the event of emergencies.

Modifying your Ethical ClearanceRequests for variations must be made via submission of a Variation Request Form (available online at http://www.research.qut.edu.au/ethics/forms/ani/var/var.jsp) to the Research Ethics Coordinator. Minor changes will be assessed on a case by case basis.

It generally takes 7-14 days to process and notify the Chief Investigator of the outcome of a request for a variation.

Major changes, depending upon the nature of your request, may require submission of a new application.

Audits

RM Report No. E802 Version 3.1 Page 2 of 3

University Animal Research Ethics CommitteeANIMAL ETHICS APPROVAL CERTIFICATE

QDPI Scientific Use Registration Number 0052

Date of Issue:4/10/11 (supersedes all previously issued certificates)

All active ethical clearances are subject to random audit by the UAEC. You will be contacted by the Research Ethics Unit if this is to occur.

End of Document

RM Report No. E802 Version 3.1 Page 3 of 3

ANIMAL ETHICS COMMITTEE

23 April 2013 Dear Professor Saso Ivanovski, I write further to the additional information provided in relation to the conditional approval granted to your application for ethical clearance for your project "Bone regeneration in a sinus model: a comparison between several bone grafting materials." (GU Ref No: DOH/02/13/AEC) detailed below. Is this submission only for the approval of Dr Alayan to work on the project? Yes Where will the animal work take place? All procedures involving the animals will take place at QUT’s Medical Engineering Research Facility, Chermside, Brisbane. A Griffith University staff member is listed on the original AEC application to QUT. Why was an application not submitted to the Griffith University AEC in 2011? Since all of the animal work was done at QUT, I was not aware that we need Griffith University ethical approval. I have close collaborators at QUT and hold an adjunct appointment there. This application has now been put through the Griffith Ethics Committe, because Dr Alayan, a PhD student who will be doing the sheep surgery, has obtained an NHMRC scholarship, and the Office of Research requires ethics approval through Griffith in order to formally accept the scholarship. The additional information provided in relation to the conditional approval granted to your application for ethical clearance has satisfied all the conditions and you are authorised to immediately commence this research on this basis. Regards Dr Amanda Fernie Animal Ethics Committee Secretary Office for Research Bray Centre, Nathan Campus Griffith University ph: 3735 6618 fax: 3735 7994 email: A.Fernie@griffith.edu.au Researchers are reminded that the Griffith University Code for the Responsible Conduct of Research provides guidance to researchers in areas such as conflict of interest, authorship, storage of data, & the training of research students.

2

You can find further information, resources and a link to the University's Code by visiting http://policies.griffith.edu.au/pdf/Code%20for%20the%20Responsible%20Conduct%20of%20Research.pdf PRIVILEGED, PRIVATE AND CONFIDENTIAL This email and any files transmitted with it are intended solely for the use of the addressee(s) and may contain information which is confidential or privileged. If you receive this email and you are not the addressee(s) [or responsible for delivery of the email to the addressee(s)], please disregard the contents of the email, delete the email and notify the author immediately.

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