Development of a New Experimental Model for Investigating Osseointegration of Titanium Mini-Screws

Placed Intraorally

by

Charles Tremblay

A thesis submitted in conformity with the requirements for the degree of Master of Science

Faculty of Dentistry University of Toronto

© Copyright by Charles Tremblay 2020

ii

Development of a New Experimental Model for Investigating Osseointegration of Titanium Mini-Screws Placed Intraorally

Charles Tremblay

Master of Science

Faculty of Dentistry University of Toronto

2020

Abstract

A novel in-vivo pilot study was developed using the rabbit mandibular diastema to evaluate the

effect of implant micro- and nano-surface topography on the rate of osseointegration. Six rabbits

received one titanium mini-screw from both topographies in each diastema. Two rabbits were

euthanized at 6, 22 and 85d post-implantation. Harvested mandibles were measured and

processed for reverse torque, microCT, SEM and histological analyses. At 22 days, the nano-

surface screws had higher mean torque values but both topographies reached nearly identical

mean values after 85 days. Localized areas of crestal bone resorption were seen around all screw

heads at 22 days. Sub-periosteal bone formation lateral to the screw heads led to complete

coverage of the screws at 85 days. The epithelium of the overlying mucosa was parakeratinized

stratified squamous. This intra-oral animal model is suitable to study osseointegration.

iii

Acknowledgments

This thesis results from a collaboration of many people and I wish to express my respect. I begin

with my co-principal investigators, Dr. Vanessa Mendes and Dr. John E. Davies. Vanessa, your

work ethic, dedication toward your students and friendly personality makes you a role model for

far more than academics. Your guidance as a clinical instructor, research supervisor and now

program director helped me in the direction of professionalism and humanism. You taught me

evidence-based periodontics and research fundamentals as a program mentor. JED, it was a

privilege and honor to be part of your Bone Interface Group. Your vision of the project

stimulated me throughout the study. I am grateful for the patience and guidance you have given

to this work. Your knowledge and generous support made this experience truly enjoyable.

I would like to express my profound gratitude to my advisory committee members, Dr. Sean Peel

and Dr. Michael Goldberg. Your availability and support were fundamental in the development

of this document. Thank you also for your valuable interest and scientific contribution to this

project.

I am grateful for the opportunity I had to work along the members of the Bone Interface Group.

Zhen-Mei Liu, Sophie Yang, thank you so much for your help and time in the laboratory. I could

always count on you two. Robert Liddell, your participation in the reverse torque component and

power analysis was key. I wish to express my kindest thanks to the Department of Comparative

Medicine (DCM) support staff for their assistance in the animal care. Special thanks to Dr. Kate

Banks, Dr. Chereen Collymore, Rainerio De Guzman, Arin Dunning and Jean Kontogiannis. I

am also thankful to Professor Giovanni Grasselli at the Department of Civil and Mineral

Engineering for allowing me to use their equipment. In addition, I would like to thank Nancy

Valiquette from the Faculty of Dentistry for providing unremarkable histology sections.

I would sincerely like to thank the funding aid from Zimmer Biomet 3i.

Regards to Dr. Jim Lai, my clinical instructors, fellow residents and assistants from the graduate

periodontology department for your friendly relationships.

To my fiancée, family and friends, thank you for your understanding. Much love!

iv

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Tables ................................................................................................................................ vii

List of Figures ................................................................................................................................ ix

List of Appendices ......................................................................................................................... xi

List of Abbreviations .................................................................................................................... xii

1 Introduction ...................................................................................................................................1

1.1 Animal models .....................................................................................................................1

1.1.2 Large animal models ...................................................................................................3

1.1.3 Small animal models ...................................................................................................4

1.2 Mechanical disruption tests ...................................................................................................6

1.3 Endosseous osseointegration .................................................................................................8

1.4 Implant surface topography ..................................................................................................9

1.5 Rationale .............................................................................................................................11

1.6 Hypothesis ...........................................................................................................................12

1.7 Objectives ............................................................................................................................12

2 Materials and Methods ................................................................................................................13

2.1 Miniature implants and screws ...........................................................................................13

2.1.1 Materials ...................................................................................................................13

2.1.2 Methods of surface modification ..............................................................................13

2.2. Development of the surgical models ..................................................................................14

2.2.1. Rats ..........................................................................................................................14

2.2.2. Rabbits .....................................................................................................................15

v

2.3 In vivo implantation study ..................................................................................................17

2.3.1 Animal numbers ........................................................................................................17

2.3.2 Sample harvesting and measurements ......................................................................19

2.4 Reverse torque analysis .......................................................................................................19

2.4.1 Sample preparation ...................................................................................................19

2.4.2 Method ......................................................................................................................19

2.5 Imaging ...............................................................................................................................20

2.5.1 Micro computed tomography ....................................................................................20

2.5.2 Scanning electron microscopy ..................................................................................20

2.5.3 Histological analysis .................................................................................................21

3 Results .........................................................................................................................................22

3.1 Miniature implant and screw surface characterization ........................................................22

3.2 Rats ......................................................................................................................................23

3.2.1 Results .......................................................................................................................23

3.2.2 Discussion .................................................................................................................25

3.3 Rabbits ................................................................................................................................28

3.3.1 Cadaveric dissections and surgical simulations ........................................................28

3.3.2 In vivo implantation study ........................................................................................30

3.3.3 Dimension of mandibular diastema ..........................................................................31

3.3.4 Reverse torque analysis .............................................................................................31

3.3.5 Micro computed tomography and radiography .........................................................32

3.3.6 Scanning electron microscopy analysis ....................................................................36

3.3.7 Light microscopy ......................................................................................................38

3.3.8 Power-analysis for future experiments .....................................................................40

4 Discussion ...................................................................................................................................42

4.1. Development of the model .................................................................................................42

vi

4.1.1 Position of the screws ...............................................................................................42

4.1.2 Reverse torque values ...............................................................................................43

4.1.3 Bone resorption and formation .................................................................................44

4.2. Effect of surface topography ..............................................................................................46

4.2.1 Reverse torque values ...............................................................................................46

4.2.2 Bone bonding and contact osteogenesis ...................................................................47

4.3 The rabbit mandibular diastema ..........................................................................................47

5 Conclusion ..................................................................................................................................49

References ......................................................................................................................................50

Appendix 1 .....................................................................................................................................63

Appendix 2 .....................................................................................................................................64

vii

List of Tables

Table 1: Chemical products used in the surface modification of the mini-screws ....................... 13

Table 2: Mandibular diastema dimensions (intact rabbits) ........................................................... 64

Table 3: Distance from mesial alveolar process of mandibular 1st premolar to have 3mm of bone

height above the lower incisor (intact rabbits) .............................................................................. 64

Table 4: Maximal bone height and distance from mesial alveolar process of mandibular 1st

premolar (intact rabbits) ................................................................................................................ 65

Table 5: Mandibular diastema dimensions (pilot study rabbits) ................................................... 65

Table 6: Distance from mesial alveolar process of mandibular 1st premolar to have 3mm of bone

height above the lower incisor (pilot study rabbits) ...................................................................... 66

Table 7: Maximal bone height and distance from mesial alveolar process of mandibular 1st

premolar (pilot study rabbits) ........................................................................................................ 66

Table 8: Mandibular diastema dimensions (all rabbits included) ................................................. 67

Table 9: Distance from mesial alveolar process of mandibular 1st premolar to have 3mm of bone

height above the lower incisor (all rabbits included) .................................................................... 68

Table 10: Maximal bone height and distance from mesial alveolar process of mandibular 1st

premolar (all rabbits included) ...................................................................................................... 69

Table 11: Screws’ position from placement to 85 days ................................................................ 70

Table 12: Reverse torque mean values ......................................................................................... 32

Table 13: Reverse torque values ................................................................................................... 70

Table 14: Relationship of mental foramen with posterior screw .................................................. 71

Table 15: Screw’s distance to lower incisor ................................................................................. 71

viii

Table 16: Values including outliers .............................................................................................. 40

Table 17: Values excluding outliers ............................................................................................. 41

Table 18: Chang (2009) ................................................................................................................ 41

ix

List of Figures

Figure 1. Mesial-distal span between two sites with a minimum height of 3mm ........................ 16

Figure 2. Maximal bone height above the incisor ......................................................................... 16

Figure 3. Dimensions of the rabbit mandibular diastema ............................................................. 16

Figure 4. Screw placements within the rabbit’s mandibular diastema ......................................... 17

Figure 5A. Miniature implant and screw surface characterization ............................................... 22

Figure 5B. Micrographs of screw surface characterization .......................................................... 23

Figure 6A. Axial view of the 1st and 2nd mandibular molar’s roots. ............................................. 25

Figure 6B. Alveolar bone dimensions of the 1st mandibular molar’s distal root .......................... 25

Figure 6C. Implant placement next to retained root tips .............................................................. 25

Figure 7A. A,B,C screws and osteotomy showing the variance in the mandibular height and

width of a 24 weeks old rabbit ...................................................................................................... 30

Figure 7B. Horizontal and vertical dimensions available to accommodate a 1.4mm x 3.0 mm

screw ............................................................................................................................................. 30

Figure 8. Reverse torque mean values at 6, 22 and 85 days for both surface topographies ......... 32

Figure 9A. Micro computed tomography: left coronal anterior and posterior ............................. 34

Figure 9B. Micro computed tomography: left sagittal .................................................................. 34

Figure 9C. Micro computed tomography: right coronal anterior and posterior ........................... 35

Figure 9D. Micro computed tomography: right sagittal ............................................................... 35

Figure 10. Subperiosteal bone formation and crestal bone resorption .......................................... 36

x

Figure 11. Screw surface topography before placement and after removal ................................. 37

Figure 12. Scanning electron microscopy of crestal bone resorption around osteotomies ........... 38

Figure 13. Low magnification image of the mandibular diastema. .............................................. 39

Figure 14. Histology of the mucosa overlying the mandibular diastema ..................................... 39

Figure 15. Suggested range for placement of two screws measuring 1.4 x 3.0 mm within the

mandibular diastema of rabbits 21 weeks and older ..................................................................... 43

Figure 16. Scanning electron microscopy of crestal bone resorption around osteotomies ........... 46

Figure 17. Partial thickness incision design and excision of muscles and salivary glands .......... 48

xi

List of Appendices

Appendix 1.

A. MicroCT images showing implant drift towards the shaft in the rat tibia after different

time points........................................................................................................................63

B. MicroCT images illustrate unicortical implant engagement at earlier time-points (5-28

days) transformed into bicortical contact with time (56 and 168 days)...........................63

Appendix 2.

Table 2-11, 13-15...........................................................................................................64-71

xii

List of Abbreviations

BIC: bone-to-implant contact

PDL: periodontal ligament

SEM: scanning electron microscopy

µCT: micro computed tomography

1

1 Introduction

Tooth loss can significantly impact the quality of life of an individual. Fortunately, the

introduction of osseointegrated titanium dental implants in the 1960s revolutionized tooth

replacement options.1 Initial concepts of osseointegration originated from vital microscopic

studies evaluating bone marrow vascularization of titanium chambers embedded in rabbit fibula.2

As bone grew, an inseparable incorporation of the chambers was discovered.3 Since then, animal

studies have contributed significantly to the evolution of implant dentistry.2,4,5 Indeed, the

development and improvement of dental implant therapy still largely depends on pre-clinical

models. Currently, the success rate of this treatment modality is reported to be over 90% after 5-

10 years of implant placement.1,6 However, conditions like early implant placement or loading,

and placement in compromised sites or individuals, have been associated with lower success

rates.7-10

Pre-clinical studies allow investigations precluded in humans.5 Similarly, principles of humane

treatment, animal welfare and emphasis on replacing, reducing and refining the use of animals

now strongly encourages in-vitro modeling.11 These models can represent some in-vivo

phenomena, permit a mechanistic understanding of early peri-implant healing,12 and are suitable

to evaluate cytotoxicity, genotoxicity, cell proliferation and differentiation.13-15 As such, animal

use is often prevented. In addition, in-vitro conditions are generally easier to standardize and

quantify compared to in-vivo environments.14 However, biocompatibility, mechanical function

and tissue responses to new materials are generally better assessed with in-vivo studies.13

1.1 Animal models

Animal models are defined as living animals with inherited or induced pathological processes or

lesions capable of answering a research hypothesis.16 Dental implant research typically focuses

on mechanisms of osseointegration, peri-implant pathologies, and treatments.17 Accordingly, an

ideal animal model should have an anatomy, physiology, disease progression, and risk factors

2

similar to those of humans.18,19 In addition, the surgical site, technique and outcome should also

be comparable to those seen in human treatments.20 Unfortunately, no species entirely

corresponds to these criteria and inappropriate model selection can lead to irrelevant or

misleading information.13,20 Several factors must be taken into consideration when extrapolating

data from animal models.4,11,17,21,22 Inherited characteristics such as bone micro- and macro-

structure, cortical-cancellous distribution, metabolism and remodeling must be considered.11,18

The use of each species also involves unique ethical implications, cost, handling and housing

demands.23 Due to these disparities, findings from in-vivo studies are generally stronger when

correlations are made with comparable studies using different animals.4,24 Therefore, multiple

models are normally needed to fully evaluate a research question.

Both intra-oral and ectopic small and large animal models are utilized in implant research.13,22,25

A recent review of in-vivo research in implant dentistry reported that dogs and rabbits were the

most frequently used species.25 Dogs, non-human primates and mini-pigs were primarily

employed as intra-oral models whereas rabbits, rodents, goats and sheep typically served as

extra-oral, or ectopic, models.17,25 The most common intra-oral model involved the mandible of

dogs whereas the femur, tibia and calvaria of rats and rabbits were the most popular extra-

orally.4,25 In general, the long bones of larger animals (dog, non-human primate, goat, sheep)

tend to approximate the size, mineral composition and biology of human bone.19,23,26,27 When

mature, they provide a stable skeletal environment as they undergo continuous bone remodeling

but minimal growth.19 Some suggest they may be better suited for biomechanical evaluations in

orthopaedics.20 However, it is proposed that they would provide little advantage over smaller

animals for answering biological questions.20 Therefore, due to their lower cost and faster

healing time, smaller species are frequently used to test implants and associated materials in

preliminary trials.11,13,20 Comprehensive descriptions of the bony anatomy and physiology of the

most commonly used species in implant research are found elsewhere.5,13,18,23,28 Important

characteristics are presented below.

3

1.1.2 Large animal models

1.1.2.1 - Dogs

Pioneer studies on the discovery of osseointegration, were performed in dogs.2 Their docile

character and convenient size facilitates intra-oral access, use of human sized dental implants and

post-operative maintenance.5,29 The bone micro- and macro-structure of dogs are moderately

similar to human bone.5,29 A study comparing the bone composition and density of several

animals concluded that canine bone was the most similar to humans when compared to pigs,

cows, sheep, chickens, and rats.30 Yet, their remodeling cycles are generally 25% shorter and can

vary within and between dogs.5,23 Irrespective of their rich history in implant research, their

status as a companion animal has increased social concerns and ethical issues regarding their

use.4

1.1.2.2 - Non-human primates

Non-human primates are the most suitable species in terms of anatomy, morphology and

physiology when compared to humans.4,31 The structure of their periodontium closely resembles

the human masticatory apparatus which facilitates implant loading.4 However, strict animal care

regulations, ethical issues related to their evolutionary affiliation, cost, demanding handling and

maintenance as well as the risk for zoonotic infections limits their use.4,13,29

1.1.2.3 - Mini-pigs

Mini-pigs have many similarities in terms of bone anatomy, morphology, healing and

remodeling when compared to humans.22,28,29 Their lamellar bone structure and bone remodeling

is similar to humans.32 The bone mineral density and concentration approximates human bone

but includes a denser trabecular network.30,32 The bone regeneration rate of the adult mini-pig

mandible (1.2-1.5 mm/day) is comparable to that of humans (1.0-1.5 mm/day).33 However, their

rapid growth, difficult handling and temperament limit their practicality.19,28,34 In addition, the

sizes of their tibiae or femora do not allow the use of human sized dental implants.28

4

1.1.2.4 - Sheep and goats

The use of sheep and goats in orthopaedic research has increased due to the negative public

perception of using companion animals.5,13 Their long bone dimensions allow the insertion of

human dental implants.27,34 These animals are generally docile but can be frail and less resilient

when alone.19 The bone structure and density of sheep are affected by age, gender and

anatomical site.13,35 When immature, they predominantly have primary bone structure and a

lower density while mature sheep have secondary Haversian remodeling and a significantly

higher trabecular bone density.13,28,34 Nevertheless, the trabecular bone density of sheep is almost

twice that of humans.4,35 The bone composition and remodeling of goats is very similar to

humans but they lack homogenously distributed Haversian systems.13,28

1.1.3 Small animal models

The small size, cost, availability and docile character of rats and rabbits makes them attractive as

pre-clinical models.13,18,36 Despite having a different bone micro- and macro-structure, repair rate

and remodeling compared to humans, they remain two of the most commonly used animals in

basic research involving the skeleton.5,11,13,18,30 Rabbits were recently reported as the most

commonly used animals for biomechanical studies in implant dentistry.25 Unlike rats, rabbit long

bones allow sufficient volume to accommodate multiple human dental implants.13 Nevertheless,

the distinct physical and physiological properties of their bones must be recognized.19,20,31

The skeleton of rats consists of woven bone, compared to the lamellar secondary osteonal

structures of human bone,5,11 while rabbit bone comprises both woven and lamellar bone. Unlike

humans and rabbits, the long bones of rodents essentially lack intracortical remodeling and have

less trabecular bone than rabbits.19 In rabbits, the medullary spaces are rich in fat, whereas

human bone marrow mostly contains hematopoietic cells and less adipocytes.37 The bone

turnover of rabbits is faster than humans.11,34,38 As such, osseointegration is reached after

approximately six weeks in rabbits compared to 3-4 months in humans.39 Rabbits reach skeletal

maturity at around 6-7 months of age.37,40,41 In contrast, the growth plates of long bones in

rodents remain open throughout life but little endochondral elongation is observed after 20

weeks.19,42 Accordingly, due to the rapid or continuous growth of the appendicular skeleton of

these species, implants placed uni-cortically within the metaphysis in young animals may engage

5

both circumferential cortices of the diaphysis after long periods of implantation (Appendix

1).11,22 This modified relationship must be recognized when the outcome is a mechanical

measurement of osseointegration.43

Minimally invasive intra-oral implant placement protocols in the rat and rabbit are scarce. Intra-

oral models frequently include dental extractions, bone grafting procedures and extra-oral access,

which all involve substantial animal distress.11,44-49 Several anatomical factors limit the

practicability and suitability of the alveolar bone of rats and rabbits to study peri-implant

healing.13,37,50 In rats, the presence of long continuously growing incisors, thin root diameters and

small quantity of cancellous bone makes tooth extraction and implant placements very

difficult.18,19,51 In rabbits, extraction is associated with high risk of dental and bone fracture due

to the presence of long roots deeply embedded within the alveolar bone.18 The use of the alveolar

socket as a study model is also complicated by the continuous eruption of their teeth. Due to their

open apices, these teeth are subject to re-development and eruption if not fully extracted.37 The

small volume of alveolar bone available also limits the use of human dental implants in both

species.11,36

Interestingly, an anatomical analysis revealed that the rabbit mandibular diastema, the edentulous

space anterior to the 1st premolar, could offer 43-96 mm3 of bone volume available for

experimentation.37 Over 3 mm of bone height was measured over the lower incisor in rabbits at 4

months of age.52 A developmental analysis also illustrated that the mandible reached 90% of its

final length after 4 months.53 A study created bony defects (8mm (length) x 3mm (width) x 3mm

(height)) within the diastema to evaluate the spontaneous healing response of rabbits.54 At the

end of the 3rd week, woven and mature bone trabeculae had completely filled the cavity and

became mature bone 2 weeks later. This study exposed the rapid regeneration rate of this animal

model.55 Therefore, the accessibility, absence of teeth and minimal growth found after 4 months

of age could potentially justify the rabbit mandibular diastema as a valuable intra-oral model for

implant placement. Close attention will be given to this species in the upcoming sections.

6

1.2 Mechanical disruption tests

Various histological, biomechanical and radiographical applications have commonly been

utilized to evaluate the effect of implant surface topography on osseointegration.21,25 Histological

sections are considered the gold standard in quantifying new bone formation but are technically

demanding.21,56 The technique sensitive process of sample fixation, decalcification, dehydration,

embedding, staining and sectioning can lead to numerous compromising artifacts. Several

reports found correlations between histological bone-to-implant contact (BIC) and

biomechanical tests.57-60 Pushout, pullout and reverse torque tests apply mechanical forces and

measure the peak force required to disrupt implant integration.11,61 Such mechanical disruption

tests allow an assessment of the level of osseointegration.21 Reverse torque tests are clinically

relevant as cylindrical screw-threaded implants can be utilized.43 The architecture of the bony

tissue observed with micro computed tomography (µCT) has been positively correlated with its

mechanical properties.62 However, a lack of mineralization and metal-induced artifacts created

by beam hardening or scattering often limits the quantification of new peri-implant bone

formation with radiographic assessments.21 The animal model, study design and implant

selection can significantly impact the strength and rate of osseointegration evaluated by

mechanical disruption tests.

The implant recipient site can also influence the biomechanical test values. The bone

morphology and cortical-cancellous proportions are critical.61,63,64 These factors are directly

related to the selected animal species, breed, age and associated growth.61,63,65,66 Cortical-

cancellous bone proportions significantly affects the initial implant stability, BIC and reverse

torque values.24,67 Intuitively, one would expect correlations between reverse torque values and

BIC.65 However, in several rabbit studies, even when higher BIC was obtained in the presence of

cancellous bone, reverse torque values were primarily related to the amount of cortical bone

surrounding the implant and to the maturation of woven bone into lamellar bone.65,68 As such,

due to the greater elastic modulus of cortical bone, reverse torque values were mostly affected by

its presence, even if that meant lower BIC.65,67-69 In addition, bi-cortical engagements were

shown to offer higher reverse torque values than uni-cortical implant stabilization.24,66,67,69 In

long bones, as previously mentioned, the skeletal growth can affect the implant’s cortical

relationship after longer experimental periods. As rabbits reach skeletal maturity at 6-7 months

7

of age, studies employing older rabbits or shorter implants reduce the risk of this phenomenon

affecting outcome measures.37,40,41,69,70 Differences in the cortical bone thickness between

different rabbit breeds and individual variations could also potentially impact reverse torque

values.65,66

The designated testing time-points and the performance of the disruption test will also influence

the biomechanical data.61,64,65 A careful selection of specific euthanasia time points is important

to properly evaluate the rate of osseointegration.66,70,71 Implant primary stability or the insertion

torque depends on mechanical interlocking within cortical and cancellous bone.21 Cortical bone

resorption occurring after implant placement will diminish this frictional engagement and lead to

the stability dip, illustrated by Raghavendra et al (2005).72 This temporary state will soon be

replaced by the secondary stability provided by bone formation and remodeling.21 It was

demonstrated that the primary stability dip in rabbit long bones occurred between 0 and 7 days.73-

75 In addition, it was illustrated that it takes about 6 weeks for woven bone to be replaced by

mature lamellar bone in rabbits.76 Similarly, the osseous healing after placement of a titanium

chamber in the rabbit tibia was shown to be well advanced after 6 weeks.77 As such, Sennerby et

al (1997) did not identify any significant difference in reverse torque values after 6 weeks, 3 and

6 months of implant placement while Gotfredson et al (1995) found significant differences as his

tests were performed after 3 and 12 weeks in rabbits.65,69 One must also note that techniques used

to measure removal torque are prone to numerous potential measuring errors.65 For example, bias

due to misalignment between the implant and the torque device or bone coverage of the implants

leading to potential incomplete bone removal can affect reverse torque data.61,65

Reverse torque values can be affected by various implant characteristics such as the composition,

dimension, geometry and surface topography.70 The metal type and biocompatibility was shown

to influence the overall BIC and reverse torque.57,58 The implant length, diameter and geometry

also affects the total bone-implant surface area, which frequently correlates with reverse torque

values.67,69,70,78 The impact of implant surface topography on bone anchorage was demonstrated

in preliminary findings by Hann and Palich (1970).79 Subsequently, Wilke et al (1990) and Buser

et al (1991) compared the BIC and reverse torque values of implant surfaces modified either by

additive (spraying, coating) or subtractive (blasting, etching) techniques.59,80 Extensive data now

indicates that implants with topographically complex surfaces should offer a greater rate of bone

apposition along with a stronger mechanical anchorage compared to machined

8

implants.24,59,69,70,81-85 These improvements have allowed the introduction of different prosthetic

designs, loading and surgical protocols.86 Therefore, the effect of surface topography on the rate

of osseointegration has stimulated much interest from clinical, commercial and scientific points-

of-view.25 Nevertheless, understanding early peri-implant bone healing mechanisms is important

in order to realize the effect of surface topography on osseointegration.

1.3 Endosseous osseointegration

Successful endosseous integration largely depends on the early peri-implant healing

mechanisms. After implant placement, activated platelets from the blood clot are fundamental in

creating a gradient of cytokines and growth factors. This causes the recruitment of osteogenic

cells and undifferentiated mesenchymal progenitors from the neighbouring peri-vascular

connective tissue.12,87-89 To reach the implant surface, these cells apply a tractional force on the

fibrin network formed during clot formation.87,88 The recruitment and migration of these cells is

called osteoconduction.87,88 The strength of the fibrin attachment to the implant will influence the

ability of these cells to reach the surface and impact subsequent osteogenesis.87 In successful

cellular migration, the progenitor cells will gradually differentiate into osteogenic cells and

secrete non-collagenous proteins in the form of individual calcified globular accretions directly

on the implant surface.12,87 These globules will progressively fuse and form the cement line, a

continuous mineralized matrix rich in calcium, phosphorous, osteopontin and bone

sialoprotein.12 Shortly after, the differentiating osteogenic cells will synthesize collagen that will

assemble to form a fibrous extracellular collagenous matrix over the cement line. These cells will

eventually differentiate into mature polarized osteoblasts and the collagen matrix will mineralize

to produce de novo bone.88,90 The combination of osteoconduction and de novo bone formation

is called contact osteogenesis.87,88,91 The continued apposition of bone to the implant will result

from ongoing arrival of osteogenic cells at the implant surface.88

In contrast, the osteogenesis process does not always happen directly on the implant surface. As

progenitor cells migrate towards the implant, some may spontaneously begin their differentiation

before reaching the implant.87 A similar result occurs when the fibrin network detaches from the

implant surface during cell migration.87 In such scenarios, the ossification occurs on the surface

9

of the bone in proximity to the implant. Therefore, the implant will gradually be surrounded by

bone but will remain separated by a layer of extracellular matrix and osteoblasts that may

eventually perish due to a lack of vascularity.87 This healing pattern is called distance

osteogenesis.88,91 It is also typically visualized within the cortical bone compartment.88 The

minimal vascular architecture of compact bone causes the entrapment of blood and cell remnants

within the limited space formed by the implant threads and osteotomy walls. With the exception

of porous coated implants that provide a larger peri-implant blood clot volume, these conditions

will inhibit capillary anastomoses, limit colonization of osteogenic cells, cause blood stagnation

and eventually lead to bone necrosis.12,92 However, de novo bone formation may be detected

within cortical bone through remodeling osteons that impinge directly on the implant

surface.12,87,88 Nevertheless, the remainder of the transcortical portion will eventually be

surrounded by necrotic bone or connective tissue space.87 Consequently, cortical healing will

mostly rely on slow osteonal remodeling and re-arrangement due to osteoclastic invasion from

the underlying medullary space.12,87,88,93 Since trabecular bone contains bone marrow with

mesenchymal progenitor cells and an extensive vascular network essential for angiogenesis, it is

not surprising that its remodeling rate is much faster than cortical bone.87 Yet, using specific

implant surface topographies can still optimize the early peri-implant healing reactions in

trabecular bone.

1.4 Implant surface topography

Buser et al (1991) questioned the role of implant surface topography in cancellous and cortical

bone healing.59 In type III or IV bone (Lekholm & Zarb, 1985), where minimal cortical bone is

available to ensure early stabilization, optimizing contact osteogenesis is of great interest.90,94

Indeed, three scale-ranges of implant surface topography must be optimized.82 Macro topography

(ranges from mm-µm) influences the implant geometry, improves the initial implant stability and

allows mechanical interlocking with bone.95 The addition of coarse-micron (>10µm) and micron

(<10µm) features contributes to the long-term stability by increasing the BIC and by improving

the mechanical interlocking at the implant interface.59,82,95,96 This is particularly advantageous in

trabecular bone healing.61 The micro features mimics those created by single osteoclast

resorption pits whereas coarse-micron features resemble those left by osteoclast resorption tracts

10

in bone.82 The presence of nano-topography (ranges from 1-100nm) could also have biological

effects mediated by protein and molecular phenomena (see below).24,60

A number of reports have shown that complex implant surface topographies have significant

impacts on the early blood cell reactions previously described.73,88 Implant surfaces with micro-

or nano-topography offer more surface area, which allows greater fibrinogen adsorption, fibrin

attachment and strength compared to machined surfaces.24,73,97 This can up-regulate the

agglomeration of red blood cells along with platelet and neutrophil adhesion, number and degree

of activation.97-100 As such, a higher density of cytokines and growth factors is secreted, which

potentiates osteoconduction as more leukocytes, endothelial cells, and osteogenic cells are

attracted towards the implant.82,84,88 Surface topography modifies the pattern of

neovascularization, as new blood vessels tend to develop closer to topographically complex

surfaces.89 Similarly, the remodeling rate of the vascular network seems to be accelerated in

presence of such surfaces.89 Therefore, the possibility of de novo bone formation on the implant

surface and contact osteogenesis is increased which may accelerate the rate of

osseointegration.43,61 In contrast, it was shown that machined implants have slower rates of

osseointegration as they are associated with distance osteogenesis.61,69,88

The presence of contact osteogenesis and undercuts at the sub-micron level can allow

mechanical interlocking of the cement line with the implant surface.101 This mimics bone

deposition on the floor of Howship’s lacunae as the floor also exhibits a complex surface with

undercuts at the sub-micron level.82,101 The cement line will typically penetrate the underlying

resorbed bone or implant surface to a depth of approximately one micron and become bonded by

mineralizing inside the undercuts.82,102,103 This interdigitation on the implant surface creates an

interface called bone bonding.82,101 This phenomenon may affect mechanical disruption test

values as the force may cause a fracture of the bone surrounding the implant instead of totally

releasing the implant from the native bone.43,84,101,104 The beneficial effect and biological

relevance of moderately rough implant surface on bone anchorage has recently been illustrated

with mathematical parameters.43

11

1.5 Rationale

The Davies laboratory has employed different animal models using the rat appendicular skeleton

to investigate mechanisms of peri-implant healing described above and analyze various

osseointegration parameters.82-84 Mechanical disruption tests have been used to assess the

importance of implant surface topography at the functional bone/implant interface.61 Using such

tests, a mathematical model has been developed to reflect the osseointegration potential of

implants with different surface topographical designs.43 The resultant exponential recovery curve

allows an assessment of the force required to disrupt the interface over time and can be compared

to the secondary stability curve famously theorized by Raghavendra et al. (2005).72 During the

post-implantation period, as a result of osseointegration, the force to disrupt the interfacial bone

tends to approach a plateau. The timing at which bony homeostasis is reached and the associated

force value are both affected by the type of implant surface topography.43 However, when using

long experimental time points, endosseous implants, when placed in the appendicular skeleton of

young rodents, can easily be affected by the continuous growth of long bones (as discussed

above and illustrated in Appendix 1). This variation can significantly affect data obtained by

mechanical testing and introduce undesired variables to research experiments.43 Clearly to avoid

such problems would need a different anatomical model.

Since an intra-oral implant placement model would approximate a clinical scenario, and the

growth pattern of the mandible differs from that of long bones, authors have focused on this

anatomical site.11,105,106 As explained earlier, large animals such as non-human primates and dogs

have widely been used in dental implant research,5,30 but their high costs and ethical issues limit

their utility.29 Therefore, our interest has focused on exploring smaller animals to potentially

accommodate and test candidate implant materials over time. Being distant from the secondary

growth centers of the mandible,105,106 implants inserted within the alveolar bone should be at

minimal risks of drifting or variations in their cortical relationship caused by facial growth.107,108

In addition, the correlation of this anatomical position to the one from human implant dentistry

would increase the clinical relevance of this intra-oral animal model in dental implant research.

12

1.6 Hypothesis

The anatomy and growth of the rat mandibular alveolar bone and rabbit mandibular diastema

provide suitable sites for intra-oral placement of screw-shaped implants and testing of

osseointegration with time.

1.7 Objectives

Our objectives for the development of this study were two-fold:

1. To develop an effective intra-oral animal model aimed at investigating peri-implant

healing and the osseointegration potential of mini-threaded titanium implants with micro-

and nano-surface topographical designs.

2. To conduct a pilot study to test this animal model.

13

2 Materials and Methods

2.1 Miniature implants and screws

2.1.1 Materials

Micro-surfaced implants with a size of 2.0 x 4.0 mm (diameter x height) obtained from Zimmer

Biomet, previously employed by the Davies group, were available and tested during the

cadaveric surgical simulations. The micro or nano-surface topographies of these implants have

been described elsewhere.43 However, they were too large for intra-oral placement in rats, and a

smaller alternative implantable screw was sourced.

Thus, commercially pure grade 2 titanium mini-screws, 1.6 x 3.0 mm, were purchased

(ChinaTiScrew, Titanium screws M1.6x3 Small Pan Head 0# Phillips Driver Ti GR2 Polished 50

pcs) through Aliexpress.com. The chemical products used to create various topographical surface

designs are presented in Table 1.

Chemical product Formulation and molecular

weight (MW) g/mol Vendor

Laboratory detergent Decon 75, Fisher Sci. Cat.#: 36 099 513

Hydrofluoric acid HF MW: 20.0063

Sigma-Aldrich Cat.#: 695068

Nitric acid HNO3 MW: 63.01

Caledon Cat.#: 7525-1

Sodium Hydroxide NaOH MW: 39.997

Sigma-Aldrich Cat.#: 58045

Table 1: Chemical products used in the surface modification of the mini-screws

2.1.2 Methods of surface modification

The mini-screws were cleaned with diluted detergent (2% dilution using distilled-H2O) in an

ultrasonic machine for 20 minutes. They were then twice rinsed thoroughly with d-H2O and

placed back in the ultrasonic with d-H2O for 15 min before being kept overnight in d-H2O. The

next day, the mini-screws were further treated to create a micro-topography using a mixture of

14

HF and HNO3 (HF: 0.11 mol/l, 250 fold diluted from commercial HF; HNO3: 0.09 mol/l, 170

fold diluted from commercial HNO3) at room temperature for 15 minutes. The mini-screws were

subsequently rinsed three times with d-H2O and placed inside an oven at 50oC for 2 days. After

these modifications, some had further surface treatment to create a nano-topography. First, a 4M

NaOH (160g NaOH Pellets dissolved in 1000mL RO/DI H2O) solution was made and

subsequently heated to 60oC. A solution mixer with a stir bar was used and set at 200rpm before

submerging the micro-surfaced mini-screws into the heated solution for 50 minutes. After, the

mini-screws were again rinsed three times with d-H2O and placed inside an oven at 50oC for 2

days. Both the micro- and nano-surface screws were examined by scanning electron microscopy

(SEM) to evaluate the different topographies. The microscope (Quanta FEG 250 environmental

SEM with BF/DF STEM detectors, FEI, USA) was located at Centre for Nanostructure Imaging,

Department of Chemistry, University of Toronto. Images of the uncoated screws were taken at

10 kV with various magnifications (up to200k). Before the surgery, the screws were packed into

autoclave bags and sterilized by gamma-irradiation (25 kGy). After surface transformation, both

mini-screws had final dimensions of 1.4 x 3.0 mm.

2.2. Development of the surgical models

As our laboratory has been involved in multiple protocols using the rat’s appendicular skeleton,

the intra-oral cavity of this animal was first considered. Cadaveric dissections were conducted

for a better understanding of their anatomy, osseous and dental development. Multiple implants

were placed during the simulations. An identical approach was also employed with a larger

animal model, the rabbit.

2.2.1. Rats

Twelve Wistar rat cadaveric heads (animals were between 4-20 weeks-old at the time of

euthanasia) were dissected and subject to various dental extractions. Other cadaveric rats were

kept intact for anatomical measurements. The extractions were done using modified dental

instruments and a buccal-lingual odontectomy was made to facilitate the extraction of the

mandibular roots using a fine cylindrical carbide bur mounted in a high-speed hand piece. Mini

titanium dental implants were inserted on several specimens after tooth extraction. The samples

15

were examined by a µCT system (MicroCT40, Scanco Medical, Basserdorf, Switzerland). The

samples were stabilized by sponges to prevent any movement during the scanning and were

immersed in 10% neutral buffered formalin. The samples were scanned at medium resolution

(isotropic voxel size of 20 µm) with a 0.5mm aluminium filter and radiographic parameters of

70kVp, 114µA. Acquisition files were obtained in 3 planes at 500 projections per 180o rotation.

The scanned region extended from the mesial of the premolar to the distal of the third molar for a

length of approximately 6mm. The scan created 280-300 z-axial slices for analysis. These

allowed an evaluation of the individual tooth anatomy and position in the alveolar bone, as well

as the maxillary and mandibular anatomy and developmental pattern. Anatomical measurements

of intact mandibles were also taken with an imaging software (Image J, Java, USA) from the tip

of the buccal and lingual crest down to the superior portion of the mandibular canal to evaluate

the bone height and width available for immediate implant placement at the distal socket of the

1st mandibular molar.

2.2.2. Rabbits

Six New Zealand female white rabbits, named C1 to C6, aged between 21-24 weeks were

dissected. Extraction of the 1st mandibular premolar was attempted in one rabbit and implant or

screw insertions in the mandibular diastema (edentulous region between the incisor and 1st

premolar) were performed in three rabbits. These mandibles were subjected to µCT qualitative

analysis using a micro computed tomography system (MicroCT40, Scanco Medical, Basserdorf,

Switzerland), as described above for the rat samples but employing radiographic parameters of

55kVp and 72µA. Acquisition files were obtained in 3 planes at 500 projections per 180o

rotation. The scanned region extended from the incisor tip to the distal of the third molar for a

length of approximately 44mm. The scan created 2195 z-axial slices for analysis.

The samples were also scanned by plane film imaging (HF8015+dlp ultra light, Minxray inc.,

USA) at 50kV and 0.38 mAs/sec and were subsequently digitalized (CR 30-X, AGFA

Healthcare, Belgium). These images allowed an assessment of the screw’s position in relation to

the lower incisor, the bone height above this tooth and the mesial-distal span of bone available to

accommodate 3mm long screws (Figure 1 and 2). All measurements were taken using the same

imaging software (ImageJ, Java, USA). In addition, a caliper (Inoxyd, Helios-Preisser, Germany)

was used to evaluate the length of the mandibular diastema at all time points. Measurements

16

were taken on the crest of the alveolar process from the mesial aspect of the mandibular 1st

premolar (Figure 3, landmark A) to the distal aspect of the mandibular incisor (Figure 3,

landmark C). The distance from the mesial aspect of the mandibular 1st premolar to a vertical

line extending from the mesial portion of the mental foramen (Figure 3, landmark B) and to the

mesial portion of the mandibular incisor was also measured (Figure 3, landmark D). For

standardization, as it was recognized that the alveolar process mesial to the 1st mandibular

premolar and distal to the incisor had an oblique shape, measurements were always taken in the

middle of the process.

Figure 1. Mesial-distal span between two sites with a minimum height of 3mm

Figure 2. Maximal bone height above the incisor

Figure 3. A: mesial aspect of the mandibular 1st premolar; B: mesial extent of the mental foramen; C: distal aspect of the mandibular incisor; D: mesial portion of the mandibular incisor

17

2.3 In vivo implantation study

2.3.1 Animal numbers

A study was undertaken to investigate the osseointegration of screws placed in the rabbit’s

mandibular diastema. A total of 6, 12-weeks old male New Zealand white rabbits (Charles River

Laboratories), named D1 to D6, at the time of delivery to the animal care facility (Department of

Comparative Medicine (DCM), University of Toronto) were employed. Rabbits were kept 5

additional weeks to accommodate mandibular growth and acclimatization. A total of twenty-four

1.4 x 3.0 mm screws were inserted in which one micro- and nano-surface screw was positioned

in each mandibular diastema. The anteroposterior position of each screw was alternated per side

and per animal (Figure 4).

Figure 4. Screw placements within the rabbit’s mandibular diastema The surgical protocol was approved by the Local Animal Care Committee of the Faculty of

Medicine, University of Toronto. To minimize the risk of stress, dysbiosis and anorexia, animals

received pre-surgical habituation to a high fiber palatable diet e.g. pumpkin. Technicians from

the DCM did the surgical preparation of the animals with the assistance of the operator. On the

day of the surgery, the rabbits were anesthetized by an intramuscular injection of ketamine

35mg/kg and xylazine 5 mg/kg followed by inhalation of isoflurane at 1-3% for maintenance

using a v-gel supraglottic airway device. The level of anesthesia required for an operative

procedure was monitored by pulse oximetry, heart rate, respiratory rate and toe pinches during

the surgery. To avoid any pain or infection, 0.12 mg/kg of buprenorphine and 5mg/kg of

enrofloxacine were administered subcutaneously. To prevent hypothermia and maintain body

weight, a warm water circulation pad was installed on the surgery table, onto which a sterile

towel was placed prior to surgery. After this initial preparation, the rabbits were transferred to

the surgery table. Special care was taken to maintain sterility and ensure the well-being of the

18

animals during the entire procedure.

Once transferred to the surgery table, a sterile drape was placed over the rabbit and the oral

cavity was maintained in the open position using a rabbit tabletop mouth gag (IM3). A standard

toe-pinch test was made to ensure effective anaesthesia prior to the surgical procedure. Local

anaesthetic was then administered at both mental foramina with a gauge 30 needle using 0.5ml of

2% lidocaine, 1:100000 epinephrine. The peri-operative area was cleaned with a cotton gauze

soaked in 0.12% Chlorhexidine. A 1.5 cm crestal incision was made intra-orally on the

mandibular diastema (between the central incisor and first premolar), approximately 1.0 cm

anterior to the 1st mandibular premolar. A conservative full thickness flap was elevated on both

sides exposing the mandibular crest by blunt dissection of the attached keratinized tissue, oral

mucosa, underlying muscle and periosteum. Two unicortical drill holes were made

approximately 1-2 mm apart and within an imaginary plane perpendicular to the long axis of the

crest, in the exposed cortical bone through to the marrow cavity, using a cylindrical carbide

dental burr (twist drill 1.3mm diameter, Brasseler, USA). A final similar cylindrical carbide

dental burr (twist drill 1.38mm diameter, Brasseler, USA) was used with an endodontic bur

stopper to complete the osteotomy to a depth of 3mm. The osteotomy was drilled at a speed of

1400 rpm using a handpiece mounted on an electric motor (BIOMET 3i DU1000 motor, and WI-

75 LED- G dental hand piece). Saline irrigation with suctioning was maintained throughout the

drilling sequence to avoid overheating and to remove bony debris. The two osteotomies received

a 1.4 x 3.0mm (diameter x height), mini-screw, which was tightened using a modified precision

screwdriver (Mastercraft, USA). After crestal placement and final irrigation of the surgical area

to remove any bone debris, the flap was reapposed and four single interrupted sutures (Covidien

Polysorb 5.0) were used to obtain primary closure of the wound. The distance of the anterior and

posterior screw in relation to the mandibular 1st premolar’s mesial crest was measured in one

rabbit before suturing to evaluate the difference in their position 85 days later.

Following surgery, a dose of 0.2-0.3mg/kg of meloxicam was administered once a day

subcutaneously immediately post-operatively and subsequently once/day for up to three days by

the DCM technicians. The option to continue the antibiotic post-surgically depended on the

clinical appearance of the rabbits and as per recommendation of the veterinarian. The animals

were monitored daily for signs of infection and distress.

19

2.3.2 Sample harvesting and measurements

Animals were euthanized according to the DCM’s protocol using an embutramide, mebezonium

iodide and tetracaine hydrochloride injectable solution (0.3ml/kg of T-61) after reaching a

surgical plane of anesthesia with 1-3% of isoflurane. Two rabbits were euthanized at each of 6

days (D1,D2), 22 days (D4,D5) and 85 days (D3, D6) and bony samples containing the screws

were harvested. The mandibles were dissected, excised and cleaned of soft tissue. Immediately

after dissection, the samples from 22 and 85 days were imaged with plane radiographs

(HF8015+dlp ultra light, Minxray inc., USA) at 50kV and 0.38 mAs/sec and were subsequently

digitalized (CR 30-X, AGFA Healthcare, Belgium). The same digital and manual measurements

as the ones performed on the cadaveric rabbits identified as C1 to C6 (see Figures 1-3) were

made. In addition, the distances from the anterior and posterior screws to the mesial aspect of the

mandibular 1st premolar were measured in one rabbit euthanized at 85 days in order to evaluate if

there was any significant change in their original position relative to the growth of the mandible

3 months later. To maintain hydration, the samples were quickly placed into vials filled with a

sucrose solution until ready for reverse torque analysis.

2.4 Reverse torque analysis

2.4.1 Sample preparation

Once all measurements were taken, the mandibles were further trimmed with a cylindrical

diamond bur (Brasseler, Canada) mounted in a high-speed dental drill (KaVo Dental, Canada), to

approximately an inch in length from the anterior screw to the distal portion of the mental

foramen, leaving the mandibular diastema containing the screws. This section of bone was then

embedded into a dental composite (Pattern Resin LS, GC, USA), which maintained the screws

aligned vertically inside the torque-testing device (Torque Meter, Sper Scientific).

2.4.2 Method

A steadily increasing reverse torque motion was applied using the same modified precision

screwdriver (Mastercraft, USA) until dissociation of the bone-screw interface. The peak torque

20

required for rotation of the screw was recorded for all screws. Once completed, the sample was

removed from the torque-testing device and fixed in neutral formalin until µCT analysis.

2.5 Imaging

2.5.1 Micro computed tomography

All samples were subjected to µCT imaging for qualitative analysis of bone formation around the

screws. The apical portion of the samples was surrounded by the same dental composite (Pattern

Resin LS, GC, USA), allowing a firm stabilization inside a screw-capped centrifuge tube filled

with 10% formalin. The resin prevented any movement of the samples during scanning. The

samples at 6 and 22 days were scanned with an industrial high-resolution micro computed

tomography system (Phoenix v|tome|xs, GE measurement, Billerica, USA) while the samples

from 85 days were scanned with a different system (SkyScan1272, Bruker, Billerica, USA). The

samples harvested at 6 days and 22 days were scanned at high resolution (17µm), 150KeV,

100µA, while the samples from 85 days were also scanned at high resolution (16,4 µm), 100kV

and 100 µA. 1023 slices (samples from 6 days and 22 days) and 1086 slices (sample from 85

days) over 360 degrees. These images were reconstructed and visualized in a cross-sectional and

sagittal plane using the same imaging software (ImageJ, Java, USA). An image representative of

the central slice of the screw in a coronal and sagittal view was selected for evaluation. One non-

blinded examiner qualitatively analyzed the images. Observations were made concerning the

anatomical position of the screw (proximity to lower incisor and mental foramen), the presence

of crestal bone resorption or apposition as well as the thickness of the surrounding bone.

2.5.2 Scanning electron microscopy

One left mandible containing 2 mini-screws, D4 left, was subjected to SEM analysis to evaluate

the bone surrounding the osteotomies at 22 days. The samples were fixed in 10% formalin after

dissection and then rinsed with distilled water. Before dehydration, soft tissue surrounding the

osteotomies was carefully removed using forceps under a stereomicroscope after being immersed

in 6% w/v sodium hypochlorite for 10 minutes for soft tissue digestion. Once devoid of soft

tissue, the mandible was rinsed with PBS. Dehydration was done with graded ethanol

concentrations and the sample was critical point dried (BAL-TEC CPD 030) following a

21

standard dehydration protocol (50% 70%, 80%, 85%, 95%, 5 min x three times; 100%, 10 min x

3 times, and kept in 100% ethanol overnight in a fridge for critical point dry). The dried samples

were fixed on a stub using conductive adhesive (graphite) and gold sputtered (LEICA EM

ACE200) to obtain surface coating of 15 nm. Micrographs were obtained using an environmental

SEM microscope (Quanta FEG 250 with BF/DF STEM detectors, FEI, USA) at 10 kV with

various magnifications (up to 200k).

2.5.3 Histological analysis

One intact mandible of a 4 months old rabbit cadaver was dissected and processed for

histological analysis of the type of keratinization of the epithelium overlying the diastema. After

fixation, the two mandibular halves were placed in a glass beaker and washed with running tap

water for 30 minutes. The samples were then placed on a shaker in another sealed plastic

container containing double distilled-H2O for 5 minutes before replacing it by 12% formic acid

decalcifying solution (Immunocal, StatLab, McKinney, USA) and leaving it for 14 days. The

solution was changed every four days. Decalcification was confirmed by verifying the hardness

of the lower incisor with needle perforations. Samples were then dehydrated by immersion in

ascending concentrations of ethanol (70, 95, 100 v/v), which were each changed twice and left

for 1 hour. Then, the two mandibular halves were left for overnight infiltration in a solution of

toluene. This solution was changed the next morning for 1 additional hour, which was then

followed by 3 rounds of paraffin infiltration of at least 1 hour each in an oven at 60oC. The last

round of paraffin infiltration employed a vacuum. At the time of embedding, the samples were

oriented for sagittal and coronal sections of the mandible. They were sectioned at a thickness of

6µm and mounted onto microscope slides (1-2 samples per slides). Prior to staining, the samples

were un-waxed by three xylene infiltrations lasting 5 minutes each. Then the samples were re-

hydrated with decreasing concentrations of ethanol (100, 95, 70, then water v/v). The samples

were stained with hematoxylin and eosin. Qualitative histological analysis by light microscopy

was done with the Leitz Aristoplan microscope (Leica, Germany) and the Leica Wild M3Z Type

S microscope (Heerbrugg, Swizterland). Images were acquired by a digital camera (Zeiss

Axiocam ICc 5, Germany) coupled to the microscopes and transferred using an imaging software

(Zeiss Zen 2.3 lite, Germany), to be visualized, analyzed and saved.

22

3 Results

3.1 Miniature implant and screw surface characterization

Images and micrographs of the screws as received, after cleaning, after acid etching and

treatment with sodium hydroxide are seen in Figure 5A and 5B.

Titanium screws 1.6 mm X 3.0 mm: As received: After cleaning: After dual-acid etching:

Figure 5A. Miniature implant and screw surface characterization

23

Figure 5B. Micrographs of screw surface characterization

3.2 Rats

3.2.1 Results

Cadaveric dissections and µCT imaging provided an understanding of the rat mandibular molar

anatomy and development. The 1st molar has four roots while the 2nd and 3rd each have three.

From Figure 6A, it can be seen that the diameter ranges for the mesial and distal roots of the 1st

mandibular molar are 1.08-1.42mm while those for the buccal and lingual roots are 0.42-0.56

mm. For the 2nd mandibular molar, the diameter ranges for the mesio-buccal and mesio-lingual

roots are 0.50-0.95 mm while the distal root had a diameter of 1.52mm. From the 4th to the 14th

week of age, Yoneda noted that the 1st mandibular molar’s mesio-buccal root length increased by

1.5 mm and noted a continuous deposition of cementum, making the canal space narrower.109

The prominent lingual inclination of the molars also revealed a height discrepancy of about 1.5

mm between the lingual and buccal alveolar bone plate (Figure 6B).

Extractions of the 1st molar in 4 week-old rats often led to complete removal of the mesial and

distal roots while the smaller buccal and lingual roots commonly fractured in situ. In 10-20

week-old rats, complete extractions frequently occurred at the distal root while the remaining

24

three roots were typically partially removed. Extractions of the 2nd molar in 4-20 week-old rats

again often led to complete extractions of the distal root while one of the two mesial roots

fractured. Having very short roots, the 3rd molar was twice accidentally extracted during the

elevation of the 1st and 2nd molar. This accidental extraction once caused a puncture of the

inferior alveolar artery as it runs in a close proximity to this tooth.

As mentioned, the distal root of the 1st mandibular molar was typically the largest, longest and

the easiest to remove in its entirety. As such, this location was initially selected for implant

placement. Due to the root proximity between the 1st and 2nd mandibular molars (Figure 6A),

extraction of the 2nd mandibular molar was also performed on several occasions to avoid any root

contact and assure enough mesio-distal width. Yet, as previously demonstrated, the height of the

lingual alveolar process in younger rats was insufficient to obtain full coverage of our implants.

We have simulated the surgical procedure for immediate implant placement knowing that some

lingual threads would be exposed. With 4 week-old rats, the osteotomy either led to a perforation

of the buccal or lingual plate where the minimal available width was approximately 2.4mm. The

implant osteotomy angulation followed the external oblique ridge of the mandible for optimal

implant positioning. The lingual alveolar process was at a high risk of perforation if the

osteotomy relied on the inclination of the sockets or the crown angulation of the 3rd molar.

Implant placements were only possible in rats aged 10-20 weeks where adjacent root tips were

left remaining from partial extractions (Figure 6C).

25

Figure 6A. Axial view of the 1st and 2nd mandibular molar’s roots. Root diameters are identified in millimeters

Figure 6B. Alveolar bone dimensions (in millimeters) of the 1st mandibular molar’s distal root

Figure 6C. Implant placement next to retained root tips

3.2.2 Discussion

The rat superior incisors and maxillary molars have been the most commonly extracted teeth in

animal models reported in the literature.51 However, the continuous growth of the incisors and

the masticatory burden caused by their extraction must be considered. Also, the maxillary first

molar complex five-rooted anatomy as well as the fragility and close proximity of the alveolar

bone with the maxillary sinus increases the risk of severe tooth and osseous fractures. As such,

our interest has been directed towards the extraction of mandibular molars as a potential site for

implant placement. Dental extractions of these teeth were highly influenced by the radicular and

B L

4 weeks 20 weeksFigure 1A

2.367 2.8881.335

2.057

3.4062.980

B LAlveolar bone dimensions (in milimeters) of the 1st mandibular molar’s distal root.

Mesial root of 2nd molar

Mesial root of 1st molarBuccal and lingual roots of 1st molar

Figure 1C

26

osseous development as well as the surgical technique. Yoneda et al. (2017)109 demonstrated that

the mean mesial root length of the 1st mandibular molar increased from 1.7 mm to 2.6 mm

between the 4th and 8th week of age. The Wistar rat 1st mandibular molar radicular development

was completed at 8 weeks of age.109 In another study, tooth eruptions were shown to be

completed at around 35 days of age and were then followed by secondary cementum

deposition.110 This continuous deposition of cementum caused the mean root canal width to

decrease from 0.66 mm to 0.37 mm from the 4th to the 12th week of age.109 The increasing root

length and continuous cementum deposition renders complete extraction of all roots an

overwhelming task due to the high risk of crown or root fracture.51,111,112 As such, previous rat

models involving tooth extractions have mostly relied on 4-5 weeks old rats even if their young

age limits the maximal mouth opening, access and visualization.49,51,113-115 Our µCT analysis and

surgical simulations have illustrated similar results and conclusions. Interestingly, the distal root

increased resistance to fracture may be related to its straight shape and the fact it has the largest

diameter of all roots on molars. Therefore, the distal root socket of the 1st mandibular molar was

selected for our immediate implant placements in cadaveric simulations. The bone dimension

available and feasibility of implant insertion was dependent on the rat’s age where immediate

placements were only possible in older rats.

The extraction technique previously described by Zecchin et al. (2007)51 was followed in this

procedure, however, our surgical simulations resulted in further observations and adjustments to

this technique. Considering the high risk of fractures, the position of the buccolingual

odontectomy was related to the tooth radicular anatomy. For the 1st mandibular molar, the

odontectomy was positioned either between the mesial and middle roots or the distal and middle

roots. While an odontectomy in the middle of the crown would most likely lead to a partial

extraction of the buccal or lingual root, a section located too close to the marginal ridge would

lead to a fracture of the crown. As the 2nd and 3rd mandibular molars have two mesial roots and

one distal root, the odontectomy was located in the center of their crowns.

Considering the minimal bone dimension available for implant placements after extractions in 4

weeks old rats, the idea of a staged protocol was investigated. The amount of bone fill obtained

after extraction of mandibular molars in younger rats has been discussed by previous

studies.111,115 Kumasaka et al. (2015)115 evaluated the amount of bone fill and growth obtained

27

following the extraction of the 1st mandibular molar in 4 weeks old rats. The buccal and lingual

alveolar bone height measurements were taken from the tip of the crest down to the superior

portion of the inferior alveolar canal. Pre-operatively, the buccal and lingual crests had

approximate heights of 2.7mm and 1.5mm respectively. At the 3rd week post-extraction, these

buccal and lingual estimated levels decreased to 1.5mm and 0.9mm. No further dimensional

changes were observed after the 3rd week but the bone density was still increasing at the

termination of the study 6 weeks post-operatively. A similar approach was followed by

Guglielmotti et al. (1985)111 where this time, all three mandibular molars were removed in 4

week-old rats. Using the same measurement landmarks, a combined average of all three molars

buccal and lingual alveolar bone heights was obtained. As such, the pre-operative buccal and

lingual crests had approximate heights of 2.2mm and 1.5mm respectively. During the 60 days

post-extraction period, the average buccal bone reached a maximal height at the 14th day while

this simultaneously corresponded to the lowest lingual average level. The final crest position

ended at around 2.4mm on the buccal and 1.4mm on the lingual aspects. These slightly higher

and lower post-operative positions are in contrasts to the results mentioned above by Kumasaka

et al. (2015).115 This disparity is most likely related to the use of a single average to represent all

three extraction sites and the longer follow-up period. Unfortunately, the difference in width was

not calculated. Nevertheless, these two studies offered an idea on the amount of residual alveolar

bone available for implant placement if a staged protocol was considered.

Rats’ bone micro- and macro-structure, rapid and continuous growth rate and high healing

capacity reduce human biological relevance of the model.5,30 Also, their radicular and osseous

development need to be considered if dental extractions or implant placement are planned as

these can be associated with risks of fractures or insufficient bone quantity. Based on previous

literature and our µCT examinations, the maximal dimensions for complete implant submersion

should be 1.4 x 1.5mm (diameter x height) if an immediate implant protocol was used in a 4

week-old rat after extraction of the 1st mandibular molar.111,115 This dimension should be reduced

to 1.4 x 1.0 mm if a one month healing period was allowed.111,115 Previous staged protocols have

been described using longer implants after extraction of the 1st mandibular molar in older rats but

these did not obtain complete implant submersions or their figures showed partial root

extractions.48,49,116 Socket preservation and various bone grafting techniques have not been

investigated and could potentially be included in a staged protocol in order to use longer

28

implants. Otherwise, the feasibility of developing a shorter rough surface implant would need to

be investigated if the avenue of intra-alveolar implant placement in rat mandibles is to be further

considered.

In conclusion, numerous anatomical limitations surrounded the extractions and implant

placements in the rat’s mandible. First, the access and visibility of the mandibular molars was

very limited. The fine caliber of the roots also complicated most extractions as these often

fractured. Consequently, considerable ostectomy was necessary to remove the remaining roots.

In addition, regardless of the rat’s age, the height of the lingual plate was insufficient to

completely cover our mini-implants. Even if smaller implants were developed, the presence of

any retained roots in proximity could disturb implant insertion or healing. Thus, an alternative

had to be explored.

3.3 Rabbits

3.3.1 Cadaveric dissections and surgical simulations

The extraction of the 1st premolar lead to a horizontal radicular fracture which was difficult to

manage due to the presence of a long root, dense bone and thin buccal osseous plate. Due to the

high risk of root or osseous fractures associated with extractions, the dimension of the

mandibular diastema was investigated as a location for implant placement. From 21 to 24 weeks,

a negligible (≤1mm) difference in the mean mandibular length at each anatomical landmark was

noted (Table 2 - Appendix 2). On average, a minimal bone height of 3mm was observed at a

distance of 8.83mm from the mesial aspect of the mandibular 1st premolar. This minimal height

extended anteriorly for about 8.44mm, which represents a distance of 17.28mm from the

premolar (Table 3 - Appendix 2). However, one must note that this mesial-distal span varied up

to 2.70mm. The mean maximum height was very similar at both time points (3.63mm and

3.75mm) with a difference of only of 0.12mm (Table 4 - Appendix 2). The minimal mandibular

width of the alveolar crest overlying the lower incisor was constantly around 3mm throughout

the diastema.

29

The mental foramen was determined to be located in the distal third of the mandibular diastema,

anterior and above the apex of the incisor. Its anterior opening was located approximately 7-8

mm anterior to the mesial aspect of the 1st premolar. The region contained a circumferential

cortex surrounding cancellous bone except at the inter-mandibular suture. The height from the

superior portion of the incisor’s periodontal ligament (PDL) to the crest varied throughout the

length of the diastema due to the curvature of the lower incisor (Figure 7A). In order to

accommodate screws measuring 3mm in height, Figure 7B shows that the screws must be placed

in the 8mm area anterior to the mental foramen to avoid impeding on the lower incisor. As such,

in a 24 weeks old rabbit, two 1.4 X 3.0mm screws were placed safely placed. The rabbit’s

diastema was considered a feasible model, which warranted progressing to an in vivo study.

30

Figure 7A. A,B,C screws and osteotomy showing the variance in the mandibular height and width of a 24 weeks old rabbit

Figure 7B. Horizontal and vertical dimensions available to accommodate a 1.4mm x 3.0 mm screw. Note that the posterior screw is located just anterior to the mental foramen to maximise the height.

3.3.2 In vivo implantation study

A total of 6 male New Zealand rabbits received two bilateral screws under anesthesia induced by

intramuscular infiltration of ketamine and xylazine and inhalation of isoflurane and oxygen. The

surgical procedures and post-operative period were uneventful. All animals were ambulatory

after recovery from anesthesia and none of them suffered from medical disorders or adverse drug

reactions. All animals continued a normal diet after a high fiber palatable diet e.g. pumpkin

lasting 1 week. Hematuria was once signalled in two rabbits on the day following the surgery

3.000 3.459

7.750

Horizontal and vertical dimensions available to accommodate a 1.4 mm x 3 mm implant. Note that the posterior implant is located just anterior to the mental foramen to maximise height.

Figure 2B

31

and according to the veterinarian, this was most likely related to the physiological stress caused

by the intervention.

The animals were euthanized at the time-points previously established (6, 22 and 85 days). The

harvested mandibles were measured, trimmed and subjected to reverse torque analysis, µCT

qualitative analyses, and representative specimens were selected for scanning electron

microscopy, histological processing and light microscopy qualitative analysis.

3.3.3 Dimension of mandibular diastema

On average, based on the measurements obtained from the pilot study rabbits, a minimal bone

height of 3mm was observed within a mesial-distal span of approximately 11mm at 21 weeks

and 16mm at 30 weeks (Table 6 - Appendix 2). The mean maximum height was 3.76 at 18 weeks

and 6.09mm at 30 weeks (Table 7 - Appendix 2). As the width of bone was constantly

approximating 3mm at the crest of the diastema, it was not measured for every rabbit. Tables 8-

10 (Appendix 2) show combined data of intact mandibles (Table 2-4) and implanted mandibles

(Table 5-7). Furthermore, from the time of placement to 85 days later, the position of the two

screws that were measured after placement were located approximately 2.3mm more mesial

(Table 11 - Appendix 2).

3.3.4 Reverse torque analysis

The torque values of each screw type have increased with time (Tables 12-13 and Figure 8).

Micro-surfaced screws had no difference in reverse torque values between 6 and 22 days. At 6

days, the torque values of all screws ranged between 0.3-2.1Ncm. The screws with a micro-

topography had a slightly higher mean torque value by approximately 0.4Ncm. However, at 22

days, the mean torque value of the nano-surfaced screws was higher by 1.1Ncm. It is important

to note that the standard deviation was 1.86Ncm for the nano-surfaced screws. At 85 days, the

values of the micro and nano screws were of 8.25Ncm and 8.75Ncm respectively, which each

represents an increase of 535% and 265%. Both surface topographies reached almost identical

mean values. The standard deviation must again be considered as micro-screws had values

ranging from 5.8-12Ncm.

32

Table 12: Reverse torque mean values 6 days 22 days 85 days Micro 1.35Ncm 1.30Ncm 8.25Ncm Nano 0.93Ncm 2.40Ncm 8.75Ncm *See Table 13 - Appendix 2 for all values, standard deviations and confidence intervals

Figure 8. Reverse torque mean values at 6, 22 and 85 days for both surface topographies

3.3.5 Micro computed tomography and radiography

A representative image of the screws can be viewed in a coronal and sagittal plane (Figure 9 A-

D). Regardless of the screw’s position in relation to the mental foramen, the screw was always

completely surrounded by bone (Table 14 - Appendix 2). The screw would only impede on the

inferior alveolar canal or be located within the mental foramen if its position was to be far too

buccal. The position of the foramen in relation to the screw can be visualized in D1 and D4 left

posterior on a coronal view. The screw’s apex was always found to be above the periodontal

ligament of the lower incisor with the exception of one rabbit (Table 15 - Appendix 2). In D4,

one screw was found to be in contact with the lower incisor and two screws were within the PDL

space.

33

Bone growth around the screw threads was detected qualitatively and increased over time. At 6

days, the osteotomy wall could still be observed next to the implanted screws. At 22 days,

localized areas of crestal bone resorption were seen around all screw heads. Sub-periosteal bone

formation lateral to the screw heads led to partial or complete coverage of the screws at 85 days

(Figure 10). At 85 days, bone growth could be observed within almost all screw threads.

However, there are some localized areas of radiolucency next to the screw’s mesial and distal

surface. This phenomenon was not observed on the buccal and lingual. In addition, the screws

were almost entirely surrounded by dense cortical bone.

34

Figure 9A. Micro computed tomography: left coronal anterior and posterior

Figure 9B. Micro computed tomography: left sagittal

35

Figure 9C. Micro computed tomography: right coronal anterior and posterior

Figure 9D. Micro computed tomography: right sagittal

36

Figure 10. 22 days: Asterisk: subperiosteal bone formation. Arrow: crestal bone resorption. 85 days: partial or complete bone coverage

3.3.6 Scanning electron microscopy analysis

Layers of tissue deposition, most likely representing cement line globules, are covering the nano-

surface features at 22 days post-implantation (Figure 11A). However, the shear stress applied on

the screw’s surface during the reverse torque has disturbed the true architecture of the overlying

particles. Therefore, this impedes any detailed information of the implant interface. Regardless,

there is evidence that some elements have remained attached to the screw’s surface due to its

undercuts. It can be assumed that layers of collagen fibers are also most likely seen overlying

these particles. In contrast, on the micro-surface screw, the surface features can still be identified

and fewer particles are found compared to the nano-surface screw at the same time point (Figure

11B).

37

Figure 11. Screw surface topography before placement and after removal Wide areas of resorption are exposing the trabecular bone and marrow spaces around the

osteotomies of D4 left (Figure 12A). Osteocytes lacunae along with canaliculi are also visible

due to the resorption process (Figure 12B). A localized field of bone resorption is observed along

the osteotomies (Figure 12C). At higher magnification, one could assume that digested collagen

fibrils are left exposed at the bottom of the resorption pits (Figure 12D).

38

Figure 12. Bone resorption around osteotomies: A: wide zone of resorption around an osteotomy exposing trabecular bone; B (arrow in figure A): osteocyte lacuna and its canaliculi; C (asterisk in figure A): localized area of bone resorption; D (asterisk in figure A): most likely digested collagen fibrils.

3.3.7 Light microscopy

A cross-section of the mandibular diastema is seen in Figure 13. Higher magnification images

illustrate that the epithelium of the overlying mucosa is parakeratinized stratified squamous and

that the thickness of the keratin layer varies from the buccal to the lingual aspect (Figure 14A).

In addition, the soft tissue covering the alveolar bone over the mandibular incisor is largely

composed of salivary glands and muscle attachments (Figure 14B).

39

Figure 13. Low magnification image of the mandibular diastema.

Figure 14. Histology of the mucosa overlying the mandibular diastema; A: Parakeratinized stratified squamous epithelium with underlying connective tissue; B: Salivary tissue is seen overlying the buccinator muscle inserting on the alveolar ridge

40

3.3.8 Power-analysis for future experiments

The mean data and standard deviations from our pilot study were used for this power analysis

(Table 16). A sample size formula for two-sided two-sample t test was utilized (see below):

n = ((σ12+σ2

2)(Z1-α/2+ Z1-β)2) / (µ1-µ2)2

n = sample size; σ = standard deviation; µ = mean

For significance level (α) = 0.05, Z1-α/2 = 1.96

For power (1-β) of: 90% (β = 0.10), Z1-β = 1.282

80% (β = 0.20), Z1-β = 0.842

70% (β = 0.30), Z1-β = 0.530

A second analysis was performed eliminating the evident outliers from our data (Table 13 and

17). Outliers were defined as values outside the mean ± standard deviation when all values are

included. This elimination allowed a much more obvious difference between the torque values of

both groups and subsequently a lower needed sample size.

P-values at each time points were determined by the standard normal deviate using the means,

standard deviations and sample sizes from the reverse torque values of our pilot study (Table 16).

The table of the standard normal deviate was utilized to identify the p-value:

SND, z = µ1-µ2 / √ ((σ12/n1) + (σ2

2/n2))

n = sample size; σ = standard deviation; µ = mean

Table 16: Values including outliers Time points (days)

Topography Torque values: mean ± SD (Ncm)

Confidence interval 95% (Ncm)

Variance in the mean

p-value Expected sample size required to see similar difference with power of: *(α = 0.05):70% 80% 90%

6 Micro 1.35 ± 0.76 0.61-2.09 0.58 0.3 28 35 47Nano 0.93 ± 0.45 0.48-1.37 0.20

22 Micro 1.3 ± 0.94 0.38-2.22 0.88 0.3 23 29 38Nano 2.4 ± 1.86 0.57-4.23 3.47

85 Micro 8.25 ± 2.65 5.65-10.85 7.03 0.7 210 267 357Nano 8.75 ± 1.21 7.56-9.94 1.47

41

Table 17: Values excluding outliers Time points (days)

Topography Torque values: mean ± SD (Ncm)

Confidence interval 95% (Ncm)

Variance in the mean

Expected sample size required to see similar difference with power of: *(α = 0.05): 70% 80% 90%

6 Micro 1.70 ± 0.36 1.29-2.11 0.13 4 5 6 Nano 1.13 ± 0.21 0.90-1.37 0.04

22 Micro 1.2 ± 0.42 0.61-1.78 0.18 12 15 20 Nano 2.4 ± 1.56 0.24-4.56 2.42

85 Micro 7.00 ± 1.08 5.77-8.22 1.17 8 10 13 Nano 8.27 ± 0.9 7.26-9.28 0.80

Previous work from Chang used implants with an almost identical diameter but of a longer

length, 1.3mm x 8mm, to compare the reverse torque values of different implant topographies at

various time points in the tibia of rabbits (Table 18).96 This study had mean values and standard

deviations within a comparable range from the ones obtained in our pilot study. As their implants

were also inserted unicortically, their slightly larger reverse torque values may be related to their

longer implants lengths.

Table 18: Chang (2009)

Study design Reverse torque values: mean ± SD (Ncm) - 24 rabbits (tibia) -Implant size: 1.3mm x 8mm -3 implant surface topographies (machined, macro, micro) -3 implants per time points: 14, 28, 56, 84 days

Machined Macro sandblast (Al2O3),

alkaline-etch (NaOH)

Micro sandblast (Al2O3),

acid etch (HCl/H2SO4)

14 d: 4.42 ± 1.36

28 d: 3.76 ± 1.65 56 d: 4.47 ± 1.99 84 d: 6.20 ± 2.05

14 d: 5.14 ± 2.14 28 d: 4.15 ± 1.13 56 d: 7.25 ± 1.67 84 d: 9.48 ± 2.36

14 d: 3.62 ± 0.59 28 d: 4.64 ± 1.28 56 d: 8.16 ± 0.13 84 d: 8.04 ± 1.05

42

4 Discussion

4.1. Development of the model

To our knowledge, the rabbit mandibular diastema has not served as a placement site in implant

research. As such, limited information on this anatomical location was available prior to the

development of this pilot study. Several aspects of the model that affected the results will be

discussed below.

4.1.1 Position of the screws

Pre-operative radiographs of the rabbit mandibular diastema are impractical. Therefore, average

dimensions of the diastema are essential in guiding implant positioning. The rabbit mandibular

growth and dimensions have previously been documented.53 While its development is minimally

affected by gender, standard deviations related to its size are mostly due to individual

variability.53 To our knowledge, the development, and dimensions, of the mandibular diastema

have never been specifically examined. Our measurements allowed a general assessment of its

growth rate and dimensions from 18-30 weeks. Nonetheless, our small sample size limits the

generalization of our mean dimensions. Our preliminary intact cadaveric measurements

suggested that two 1.4 x 3.0mm screws could be safely placed within the diastema. However,

during the pilot study, one screw was placed in contact with the mandibular incisor. To prevent a

similar outcome, a mesial-distal range where 3mm of bone height is found above the lower

incisor was determined after combining our samples (Table 9, Appendix 2). An antero-posterior

span limited to 11-16mm from the mesial alveolar crest of the mandibular 1st premolar was

identified (Figure 15). This range applies to rabbits 21 weeks of age or older and confirms that

only two screws should be placed within the diastema. An increased sample size of 16 weeks old

rabbits would be necessary to solidify the data regarding the mesial distal range available for

screw placement and possibly allow a more favorable spread between the screws. The screw

length should not exceed 3mm when placed in rabbits up to 24 weeks of age (table 10, Appendix

2). The bone formed over the screw heads at 85 days prevented a proper estimate of the height

available in rabbits at 30 weeks. Based on the buccal position of the inferior alveolar canal, the

implants should be centered on the ridge, even if that corresponds with the position of the mental

43

foramen (Figure 9 A,C). In order to standardize the position of each osteotomy, a surgical

template could be fabricated to help guide the operator.

Figure 15. Suggested range for placement of two screws measuring 1.4 x 3.0 mm within the mandibular diastema of rabbits 21 weeks and older

4.1.2 Reverse torque values

Several factors related to each euthanasia time point may have affected our reverse torque

values. At the time of screw placement, one osteotomy (D2 right anterior) was over-prepared and

led to a lack of primary stability. Another osteotomy (D1 left posterior) was also overly enlarged

compared to the other osteotomies on the µCT images at 6 days. Interestingly, these two screws

had the lowest reverse torque values (0.3Ncm for both screws representing about 20-30% of their

respective means). Insertion torque standardization could have reduced some of the variances in

the values at 6 days. The crestal bone resorption most likely affected the mechanical values at 22

days. One mandibular half (D4 left) visually had more resorption and extensive cortical bone

loss. Consequently, its screws had much lower reverse torque values (0.3Ncm and 0.4Ncm

representing about 10-20% of their respective means). At 85 days, the unequal bone removal

around the submerged screw heads was another source of bias.65 For example, one screw head

(D3 right anterior) had its periphery fully surrounded by bone and the highest reverse torque

value. In contrast, the screw with the lowest value (D6 right posterior) had no bone surrounding

its head. Its proximity with the adjacent screw prevented any bone formation on the mesial and

44

the bone surrounding the distal was completely removed prior to reverse torqueing. A tissue

level implant or an implant design allowing the insertion of a healing abutment could prevent

this bony coverage.22,76

Other factors need to be recognized as potential modifiers of the reverse torque values. The

individual variance of each rabbit in regards of their bone density and cortical plate thickness

must be considered.65,68 The increased thickness of the cortical bone at 85 days is likely related

to the considerably higher values of the latest time point. In addition, as these screws were not

intended to be dental implants, their thread design and pitch could have affected the early

osseointegration process.117 Bias due to misalignment of the reverse torque screw-driver with the

screw head could also have influenced the data.61,65

4.1.3 Bone resorption and formation

Areas of bone resorption and formation were observed along the implanted screws at 22 and 85

days. The underprepared osteotomy, by 0.02mm, and the dense cortical bone likely contributed

to the high primary stability often perceived manually. Although not measured, the high insertion

resistance and the presence of a screw head could have generated detrimental pressure on the

alveolar ridge. High torque values have previously been associated with potential microfractures

in the cortical bone and compression necrosis.118,119 It was illustrated that under sizing the

osteotomy to achieve greater torque values can lead to osteocyte apoptosis.118 Additionally,

osteocyte apoptosis at the bone surface promotes more resorptive signals compared to signals

found in deeper tissues.119-121 Therefore, high insertion torques were previously correlated with

greater levels of peri-implant bone loss during early healing.122 As such, pressure necrosis could

be responsible for the crestal bone resorption observed at 22 days. Interestingly, the resorption

seemed to be accentuated on the lingual aspect of the diastema (Figure 16). The bone is

anatomically slightly higher on the lingual as it serves as an insertion point for the buccinator

muscle (Figure 14B). Even though this bony protuberance was flattened before making the

osteotomies, a discrepancy could have remained between the two heights. Therefore, the lingual

aspect of the ridge potentially accumulated more pressure compared to the buccal. Subsequently,

the bone covering the screw heads at 85 days was consistently higher on the lingual (Figure 10).

The adjacent muscle fibers that remained attached on the lingual protuberance next to the

surgical site could have created areas of tension near the screw heads. This could partly explain

45

why sub-periosteal bone formation was mostly found on the lingual aspect of the screw heads. In

retrospect, to prevent this resorption process, a drilling protocol offering an osteotomy

corresponding to the same screw diameter would be recommended due to the thick layer of

cortical bone. Bone tapping could also be considered before screw placement. Moreover, an

implant design devoid of any screw head would also likely help reduce the resorption observed

at 22 days.

At 85 days, localized radiolucent spaces were found on the mesial and distal surface of the

screws (Figure 9 B, D). These could represent areas of resorption created by altered mechanical

forces, a process referred to as stress shielding.123 Similar to orthopedic prostheses, the

implantation of the screw within the diastema has disrupted the distribution of the tension and

flexion applied on the bone during the rabbit masticatory motion. As such, the buccal and lingual

cortical plates, which were left undisturbed, concentrated most of the forces applied on the

diastema. As a result, increased cortical bone thickness and subperiosteal bone formation over

the screw heads may have been formed in reaction to the stress applied on the weakened

diastema. This relates to the flexure-drift hypothesis, where forces applied on bone surfaces

loaded in compression induce rapid osteogenesis as a short-term measure to reinforce the

weakened bone.76 In contrast, the bone surrounding the mesial and distal surface of the screws

would have borne lower physiologic loads, leading to these apparent areas of resorption. The

geometrical design and material characteristics of the implanted screw are known to influence

the resultant bone resorption.124 Again, our implanted screws were not designed to minimize

bone loss.

46

Figure 16. A: screws before reverse torque; B: screws and crestal bone resorption; C: osteotomies devoid of soft tissue; D: SEM of osteotomies

4.2. Effect of surface topography

4.2.1 Reverse torque values

The results of our pilot study support the concept that an appropriate surface roughness can

accelerate bone deposition at the implant interface.24 Varying cellular responses have been

identified based on the various surface treatments available to create nano-topography.125,126

Alterations using NaOH were previously shown to produce a reticulate sodium titanate layer,

which increases the surface roughness and facilitates hydroxyapatite deposition from tissue

fluid.125,126 Similar to our results, surface modification has been previously shown to provide

higher mean BIC and reverse torque values compared to machined surfaces in rabbit tibia.96

Likewise, another study showed that micro-topography (hydrofluoric and nitric acid treatment)

increased BIC and reverse torque values in rabbit tibia.60 In our pilot study, the mean torque

values of the nano-surface screws were higher than the micro-surface values at 22 days and both

surfaces reached similar mean values after 85 days. This corresponds to similar differences in the

rate of osseointegration and the maximal disruption force plateau of these two topographies

previously obtained in rat femora.43 It also correlates with mean reverse torque values and

associated rate of osseointegration of micro and nano-surface screws previously measured in

studies using the rabbit tibia.96,98 However, the mean reverse torque values of the present study

47

are much lower compared to other studies.67,69,70 The small screw dimension, uni-cortical

engagement, selected testing time points and younger rabbits used in this study are probable

contributing factors.43,96,98 Nonetheless, the limitations encountered during the development of

this model along with our small sample size have an effect on the significance of the mean

reverse torque values obtained for the micro and nano surface screws. Analysis or comparison of

this data in relation to the effect of implant surface topography on the early phases of

osseointegration should be made cautiously.

4.2.2 Bone bonding and contact osteogenesis

The micrographs of the micro- and nano-screws before, and 22 days after, placement revealed

their capacities to retain tissue particulate on their surfaces. After reverse torque testing, the

micro-topography was clearly visible while numerous particles, likely representing the cement

line and potential collagen fibers, were covering the nano-topography. This correlates to the bone

bonding capacities of implants with sub-micron topographies with undercuts.82,101 No

conclusions could be made as to which of these surfaces could lead to contact osteogenesis but

the tissue deposition on the nano-screws reveals that this topography could be beneficial. This

may also explain the higher mean reverse torque values observed with the nano-screws at 22

days.

4.3 The rabbit mandibular diastema

Rabbits are attractive from a scientific, economic, practical and ethical point of view.127 Their

mandible has proven appropriate for intramembranous bone research and this intra-oral location

increases the clinical dental relevance.128 The surgical restrictions associated with their limited

mouth opening are diminished by the accessibility of the mandibular diastema. This location

obviates any invasive extra-oral approach, tooth extraction or bone augmentation.127 However, it

is not suitable for implant dimensions used in humans or direct loading protocols.36,69 Despite

being very sensitive to any oral disturbance, the rabbits responded very well to the surgical

procedure.

This model was developed to avoid undesired variables related to the continuous growth of long

48

bones in rats (Appendix 1). The rabbit mandibular diastema has minimal growth after 4 months

of age and its fast bone turnover shortens the timeline needed to evaluate the rate of

osseointegration.53,60 As such, the implanted screws were fully osseointegrated after 85 days and

their initial position was relatively unchanged. However, the cortical bone thickness around the

implanted screws increased with time (Figure 9). This variation could have affected the reverse

torque data similar to the transition from a unicortical bone implant contact to a bicortical thread

engagement (Appendix 1).22,43,127 As mentioned, a different implant design could possibly

prevent this issue. However, either bone formation or resorption in reaction to the implantation

of the mini-screws, confirmed the suitability of this model to study biological mechanisms of

peri-implant healing.

The presence of muscle and salivary tissue within the mucosa overlying the mandibular diastema

is not representative of human peri-implant soft tissues. A surgical approach has been explored

where these anatomical structures would be excised from the implantation site before implant

placement (Figure 17). Histological sections would be required to examine the anatomy of the

resulting tissue. The presence of a fibrotic connective tissue could potentially serve in future

studies investigating the overlying soft tissue reactions to the transmucosal portion of an implant,

and also provide a foundation for a new model of peri-implantitis.

Figure 17. A: Partial thickness incision design; B: The superficial connective tissue layer is preserved *Muscles and salivary glands are excised to expose bone; C: Primary closure

*

CBA

49

5 Conclusion

Based on the findings from this study it was concluded that:

1) The minimal height of the rat’s mandibular alveolar bone limits the insertion of mini-titanium

screws with a dimension of 1.4 mm x 3.0 mm.

2) The rabbit mandibular diastema of 4 months old rabbits is a suitable intra-oral model to study

the osseointegration of mini-titanium screws with a dimension of 1.4 mm x 3.0 mm.

3) The minimal growth of the rabbit mandibular diastema after 4 months of age allows a stable

implant position after 85 days.

4) Despite the limitations of the study, screws with nano topography showed higher mean

reverse torque values at 22 days post-implantation and both surface topographies reached similar

values at 85 days. This data should be interpreted with caution due to the small sample size,

which reflected in the low statistical significance.

5) Due to the challenges and limitations encountered during the development of this pilot study,

future studies are needed to validate our observations.

50

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5 9 14 28 56 168

Appendix 1

A 5 9 14 28 56 168

A. MicroCT images showing implant drift towards the shaft in the rat tibia after different time points (from 5 to 168 days). Liddell R et al., unpublished data. B

B. MicroCT images illustrate unicortical implant engagement at earlier time-points (5-28 days) transformed into bicortical contact with time (56 and 168 days). Liddell R et al., unpublished data.

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Appendix 2

Table 2: Mandibular diastema dimensions (intact rabbits) Age / Identification

Gender Distance 1st PM (mesial aspect of alveolar process) to lower incisor tip

Distance 1st PM (mesial aspect of alveolar process) to distal alveolar process of incisor

Distance 1st PM (mesial aspect of alveolar process) to anterior portion of mental foramen

Left Right Left Right Left Right 21 weeks

C1 Female 27.2mm 27.0mm 23.0mm 23.0mm 7.00mm 7.80mm C2 Female 28.0mm 28.0mm 24.0mm 24.0mm 6.50mm 6.50mm C3 Female 29.5mm 29.5mm 24.0mm 24.0mm 7.50mm 7.50mm Mean 28.2mm 23.7mm 7.10mm Standard deviation 1.10mm 0.51mm 0.55mm 95% CI 27.3-29.1mm 23.3-24.1mm 6.70-7.60mm

24 weeks C4 Female 27.7mm 27.7mm 23.5mm 23.5mm 7.50mm 7.50mm C5 Female NA NA 23.3mm 23.0mm 8.90mm 8.90mm C6 Female NA 28.0mm NA 23.8mm NA 7.80mm Mean 27.8mm 23.4mm 8.10mm Standard deviation 0.17mm 0.29mm 0.72mm 95% CI 27.6-28.0mm 23.2-23.7mm 7.50-8.80mm

Table 3: Distance from mesial alveolar process of mandibular 1st premolar to have 3mm of bone height above the lower incisor (intact rabbits)

Age / Identification

Gender Side Anterior Posterior Available mesial-distal span for 3mm of height

21 weeks C1 Female Right 16.89mm 9.21mm 7.68mm

Left 16.84mm 9.29mm 7.55mm C2 Female Right 18.85mm 6.1mm 12.75mm

Left 17.84mm 7.21mm 10.63mm C3 Female Right 17.24mm 10.86mm 6.38mm

Left 16.01mm 10.31mm 5.7mm Mean 17.28mm 8.83mm 8.44mm Standard deviation 0.97mm 1.83mm 2.70mm 95% CI 16.50-18.06mm 7.37-10.29mm 6.29-10.61mm 24 weeks C4 Female Right 19.30mm 8.02mm 11.28mm

Left NA NA NA C5

Female Right 17.02mm 9.78mm 7.24mm Left 16.16mm 8.23mm 7.93mm

C6 Female Right 19.90mm 8.05mm 11.85mm

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Left NA NA NA Mean 18.10mm 8.52mm 9.58mm Standard deviation 1.79mm 0.85mm 2.33mm 95% CI 16.34-19.85mm 7.69-9.35mm 7.29-11.86mm

Table 4: Maximal bone height and distance from mesial alveolar process of mandibular 1st premolar (intact rabbits)

Age / Identification

Gender Side Maximal height

Distance from mesial alveolar process crest of mandibular 1st PM

21 weeks C1 Female Right 3.40mm 13.42mm

Left 3.54mm 12.73mm C2 Female Right 4.5mm 13.86mm

Left 4.16mm 12.81mm C3 Female Right 3.09mm 15.36mm

Left 3.09mm 13.10mm Mean 3.63mm 13.55mm Standard deviation 0.58mm 0.98mm 95% CI 3.17-4.09mm 12.76-14.33mm 24 weeks C4 Female Right 4.18mm 15.08mm

Left 4.04mm NA C5

Female Right 3.31mm 14.62mm Left 3.20mm 12.92mm

C6 Female Right 4.00mm 15.22mm Left NA NA

Mean 3.75mm 14.46mm Standard deviation 0.45mm 1.06mm 95% CI 3.35-4.14mm 13.42-15.50mm

Table 5: Mandibular diastema dimensions (pilot study rabbits)

Age / Identification

Gender Distance 1st PM (mesial aspect of alveolar process) to lower incisor tip

Distance 1st PM (mesial aspect of alveolar process) to distal alveolar process of incisor

Distance 1st PM (mesial aspect of alveolar process) to anterior portion of mental foramen

Left Right Left Right Left Right 18 weeks

D1 Male 26.0cm 26.0mm 22.0 mm 22.0 mm 7.00 mm 7.00 mm D2 Male 27.2mm 27.5mm 23.1 mm 23.4 mm 7.70 mm 7.60 mm Mean 26.7mm 22.6mm 7.30mm Standard deviation 0.79mm 0.73mm 0.38mm 95% CI 25.9-27.4mm 21.9-23.3mm 7.00-7.70mm

21 weeks D4 Male 27.0mm 26.8mm 23.5mm 23.5mm 7.00 mm 7.00 mm D5 Male 27.5mm 27.5mm 24.2mm 23.8mm 8.20 mm 8.80 mm

66

Mean 27.2mm 23.8mm 7.80mm Standard deviation 0.36mm 0.33mm 0.90mm 95% CI 26.9-27.5mm 23.4-24.1mm 6.90-8.60mm

30 weeks D3 Male 29.0mm 29.0mm 24.5mm 24.5mm 7.00mm 7.00mm D6 Male 28.5mm 28.5mm 23.5mm 23.5mm 6.50mm 6.50mm Mean 28.8mm 24.0mm 6.80mm Standard deviation 0.29mm 0.58mm 0.29mm 95% CI 28.5-29.0mm 23.4-24.6mm 6.50-7.00mm

Table 6: Distance from mesial alveolar process of mandibular 1st premolar to have

3mm of bone height above the lower incisor (pilot study rabbits) Age / Identification

Gender Side Anterior Posterior Available mesial-distal span for 3mm of height

18 weeks D1 Male Right NA NA

Left NA NA D2 Male Right NA NA

Left NA NA 21 weeks D4 Male Right 17.49mm 8.45mm 9.05mm

Left 17.89mm 8.60mm 9.29mm D5 Male Right 19.34mm 6.72mm 12.62mm

Left 19.85mm 6.91mm 12.94mm Mean 18.64mm 7.67mm 10.98mm Standard deviation 1.13mm 0.99mm 2.09mm 95% CI 17.53-19.75mm 6.70-8.64mm 8.93-13.02mm 30 weeks D3 Male Right 22.45mm 7.5mm 14.95mm

Left 22.70mm 7.00mm 15.70mm D6 Male Right 21.61mm 6.22mm 15.39mm

Left 22.83mm 5.27mm 17.56mm Mean 22.40mm 6.50mm 15.90mm Standard deviation 0.55mm 0.97mm 1.15mm 95% CI 21.86-22.93mm 5.54-7.45mm 14.77-17.03mm

Table 7: Maximal bone height and distance from mesial alveolar process of mandibular 1st premolar (pilot study rabbits)

Age / Identification

Gender Side Maximal height

Distance from mesial alveolar process crest of 1st mandibular PM

18 weeks D1 Male Right 3.53mm NA

Left 3.30mm NA D2 Male Right 4.11mm NA

Left 4.11mm NA

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Mean 3.76mm NA Standard deviation 0.41mm NA 95% CI 3.36-4.17mm NA 21 weeks D4 Male Right 3.66mm 13.94mm

Left 3.64mm 12.93mm D5 Male Right 4.27mm 14.94mm

Left 4.56mm 14.12mm Mean 4.03mm 13.98mm Standard deviation 0.46mm 0.83mm 95% CI 3.58-4.48mm 13.17-14.79mm 30 weeks D3 Male Right 6.00mm 17.19mm

Left 5.53mm 17.20mm D6 Male Right 6.41mm 14.25mm

Left 6.43mm 14.00mm Mean 6.09mm 15.66mm Standard deviation 0.42mm 1.78mm 95% CI 5.68-6.51mm 13.92-17.40mm

Table 8: Mandibular diastema dimensions (all rabbits included) Age / Identification

Gender Distance 1st PM (mesial aspect of alveolar process) to lower incisor tip

Distance 1st PM (mesial aspect of alveolar process) to distal alveolar process of incisor

Distance 1st PM (mesial aspect of alveolar process) to anterior portion of mental foramen

Left Right Left Right Left Right 18 weeks

D1 Male 26.0mm 2.60mm 22.0 mm 22.0 mm 7.00 mm 7.00 mm D2 Male 27.2mm 27.5mm 23.1 mm 23.4 mm 7.70 mm 7.60 mm Mean 26.7mm 22.6mm 7.30mm Standard deviation 0.79mm 0.73mm 0.38mm 95% CI 25.9-27.4mm 21.9-23.3mm 7.00-7.70mm

21 weeks D4 Male 27.0mm 26.8mm 23.5mm 23.5mm 7.00 mm 7.00 mm D5 Male 27.5mm 27.5mm 24.2mm 23.8mm 8.20 mm 8.80 mm C1 Female 27.2mm 27.0mm 23.0mm 23.0mm 7.00mm 7.80mm C2 Female 28.0mm 28.0mm 24.0mm 24.0mm 6.50mm 6.50mm C3 Female 29.5mm 29.5mm 24.0mm 24.0mm 7.50mm 7.50mm Mean 27.8mm 23.7mm 7.40mm Standard deviation 0.98mm 0.43mm 0.74mm 95% CI 27.2-28.4mm 23.4-24.0mm 6.90-7.80mm

24 weeks C4 Female 27.7mm 27.7mm 23.5mm 23.5mm 7.50mm 7.50mm C5 Female NA NA 23.3mm 23.0mm 8.90mm 8.90mm

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C6 Female NA 28.0mm NA 23.8mm NA 7.80mm Mean 27.8mm 23.4mm 8.10mm Standard deviation 0.17mm 0.29mm 0.72mm 95% CI 27.6-28.0mm 23.2-2.37mm 7.50-8.80mm

30 weeks D3 Male 29.0mm 29.0mm 24.5mm 24.5mm 7.00mm 7.00mm D6 Male 28.5mm 28.5mm 23.5mm 23.5mm 6.50mm 6.50mm Mean 28.75mm 24.0mm 6.80mm Standard deviation 0.29mm 0.57mm 0.29mm 95% CI 28.5-29.0mm 23.4-24.6mm 6.50-7.00mm

Table 9: Distance from mesial alveolar process of mandibular 1st premolar to have 3mm of bone height above the lower incisor (all rabbits included)

Gender Side Anterior Posterior Available mesial-distal span for 3mm of height

18 weeks D1 Male Right NA NA

Left NA NA D2 Male Right NA NA

Left NA NA 21 weeks D4 Male Right 17.49mm 8.45mm 9.05mm

Left 17.89mm 8.60mm 9.29mm D5 Male Right 19.34mm 6.72mm 12.62mm

Left 19.85mm 6.91mm 12.94mm C1 Female Right 16.89mm 9.21mm 7.68mm

Left 16.84mm 9.29mm 7.55mm C2 Female Right 18.85mm 6.1mm 12.75mm

Left 17.84mm 7.21mm 10.63mm C3 Female Right 17.24mm 10.86mm 6.38mm

Left 16.01mm 10.31mm 5.7mm Mean 17.82mm 8.37 9.46mm Standard deviation 1.20mm 1.60mm 2.69mm 95% CI 17.08-18.57mm 7.38-9.36mm 7.79-11.12mm 24 weeks C4 Female Right 19.30mm 8.02mm 11.28mm

Left NA NA NA C5

Female Right 17.02mm 9.78mm 7.24mm Left 16.16mm 8.23mm 7.93mm

C6 Female Right 19.90mm 8.05mm 11.85mm Left NA NA NA

Mean 18.10mm 8.52mm 9.58mm Standard deviation 1.79mm 0.85mm 2.33mm 95% CI 16.34-19.85mm 7.69-9.35mm 7.29-11.86mm

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30 weeks D3 Male Right 22.45mm 7.5mm 14.95mm

Left 22.70mm 7.00mm 15.70mm D6 Male Right 21.61mm 6.22mm 15.39mm

Left 22.83mm 5.27mm 17.56mm Mean 22.40mm 6.50mm 15.90mm Standard deviation 0.55mm 0.97mm 1.15mm 95% CI 21.86-22.93mm 5.54-7.45mm 14.77-17.03mm

Table 10: Maximal bone height and distance from mesial alveolar process of mandibular 1st premolar (all rabbits included)

Gender Side Maximal height Distance from mesial alveolar process crest of mandibular 1st PM

18 weeks D1 Male Right 3.53mm NA

Left 3.30mm NA D2 Male Right 4.11mm NA

Left 4.11mm NA Mean 3.76mm NA Standard deviation 0.41mm NA 95% CI 3.36-4.17mm NA 21 weeks D4 Male Right 3.66mm 13.94mm

Left 3.64mm 12.93mm D5 Male Right 4.27mm 14.94mm

Left 4.56mm 14.12mm C1 Female Right 3.40mm 13.42mm

Left 3.54mm 12.73mm C2 Female Right 4.5mm 13.86mm

Left 4.16mm 12.81mm C3 Female Right 3.09mm 15.36mm

Left 3.09mm 13.10mm Mean 3.79mm 13.72mm Standard deviation 0.55mm 0.90mm 95% CI 3.45-4.13mm 13.16-14.28mm 24 weeks C4 Female Right 4.18mm 15.08mm

Left 4.04mm NA C5

Female Right 3.31mm 14.62mm Left 3.20mm 12.92mm

C6 Female Right 4.00mm 15.22mm Left NA NA

Mean 3.75mm 14.46mm Standard deviation 0.45mm 1.06mm

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95% CI 3.35-4.14mm 13.42-15.50mm 30 weeks D3 Male Right 6.00mm 17.19mm

Left 5.53mm 17.20mm D6 Male Right 6.41mm 14.25mm

Left 6.43mm 14.00mm Mean 6.09mm 15.66mm Standard deviation 0.42mm 1.78mm 95% CI 5.68-6.51mm 13.92-17.40mm

Table 11: Screws’ position from placement to 85 days D6 right Distance from 4mm below cusp of 1st mandibular PM to:

Incisor tip Mesial edge of posterior screw head

Mesial edge of anterior screw head

Screw placement 28mm 8.15mm 11.3mm 85 days later 28.5mm 10.4mm 13.6mm Difference: 0.5mm 2.25mm 2.3mm

Table 13: Reverse torque values Left Right

Anterior screw Posterior screw Anterior screw Posterior screw 6 days D1 Nano: 0.9Ncm Micro: 0.3Ncm* Micro: 1.4Ncm Nano: 1.3Ncm D2 Micro: 2.1Ncm Nano: 1.2Ncm Nano: 0.3Ncm* Micro: 1.6Ncm Mean Nano: 0.925Ncm

Micro: 1.35Ncm Standard deviation

Nano: 0.45Ncm Micro: 0.76Ncm

95% Confidence interval

Nano: 0.48-1.37Ncm Micro: 0.61-2.09Ncm

22 days D4 Micro: 0.3Ncm* Nano: 0.4Ncm* Nano: 1.3Ncm Micro: 1.5Ncm D5 Nano: 3.5Ncm Micro: 2.5Ncm* Micro: 0.9Ncm Nano: 4.4Ncm* Mean Nano: 2.4Ncm

Micro: 1.3Ncm Standard deviation

Nano: 1.86Ncm Micro: 0.94Ncm

95% Confidence interval

Nano: 0.57-4.23Ncm Micro: 0.38-2.22Ncm

85 days D3 Nano: 9.3Ncm Micro: 7.9Ncm Micro: 12Ncm* Nano: 10.2Ncm* D6 Micro: 7.3Ncm Nano: 7.7Ncm Nano: 7.8Ncm Micro: 5.8Ncm Mean Nano: 8.75Ncm

Micro: 8.25Ncm Standard Nano: 1.21Ncm

71

deviation Micro: 2.65Ncm 95% Confidence interval

Nano: 7.56-9.94Ncm Micro: 5.65-10.85Ncm

*Outliers (see power analysis)

Table 14: Relationship of mental foramen with posterior screw D1: 6d D2: 6d D4: 22d D5: 22d D3: 85d D6: 85d Left Lateral to

screw Posterior Lateral to

screw Posterior Posterior Posterior

Right Lateral to screw

Lateral to screw

Lateral to screw

Posterior Posterior Posterior

Table 15: Screw’s distance to lower incisor

D1: 6d D2: 6d D4: 22d D5: 22d D3: 85d D6: 85d Left Ant: Above PDL Above PDL Within PDL

Above PDL Above PDL Above PDL

Post: Above PDL Above PDL In contact with incisor

Above PDL Above PDL Above PDL

Right Ant: Above PDL Above PDL Above PDL

Above PDL Above PDL Above PDL

Post: Above PDL Above PDL Within PDL

Above PDL Above PDL Above PDL