Engineered Bone by AutologousOsteoblasts on Polymeric Scaffolds inMaxillary Sinus Augmentation:Histologic ReportCarlo Mangano, DDS, MD1

Adriano Piattelli, DDS, MD2

Lucia Tettamanti, DDS1

Francesco Mangano, DDS3

Alessandro Mangano, DDS4

Fabio Borges, DDS5

Giovanna Iezzi, DDS, PhD6

Susana d’Avila, DDS, MS, PhD7

Jamil Awad Shibli, DDS, MS, PhD8*

Several regenerative therapies have been used for maxillary sinus grafting. However, recent

advances in modern bone tissue engineering techniques have been evaluated. The aim of this

histologic report was to evaluate the bone obtained by a culture of autogenous osteoblasts

seeded on polyglycolic-polylactid scaffolds in maxillary sinus augmentation. A 56-year-old

partially edentulous male with severe atrophy of the posterior maxilla received 6 polyglycolid-

polylactid disks (8 mm diameter 3 2 mm depth, Oral Bone), each carrying 1.5 million autogenous

osteoblasts into the depth of the sinus cavity. After 6 months healing, a bone core was harvested

and histologically evaluated. The augmented maxillary sinus with engineered bone presented a

mean of 28.89% and 71.11% of bone and medullary spaces, respectively. Data from this case

report demonstrate that the newly formed bone provided by engineered bone tissue allowed

proper initial stability for dental implant placement. However, the role of this new bone in the

long-term success of dental implant anchorage needs further investigation.

Key Words: maxillary sinus augmentation, engineered bone tissue, human histology,dental implants

INTRODUCTION

Maxillary sinus floor aug-

mentation has been used

for occlusal rehabilitation

with dental implants in the

posterior maxilla.1 Current-

ly, several regenerative therapies, including

synthetic bone grafts, allogenic and xeno-

genic bone matrix, and recombinant growth/

differentiation factors, have been used for

maxillary sinus grafting.1–8

1 Department of Biomaterial Sciences, Insubria Univer-sity, Varese, Italy.2 Department of Oral Pathology and Oral Medicine,University of Chieti-Pescara, Chieti, Italy.3 Private practice, Gravedona (COMO), Italy.4 University of Milan, Milan, Italy.5 Department of Periodontology, Dental ResearchDivision, Guarulhos University, Guarulhos, SP, Brazil.6 University of Chieti, Chieti, Italy.7 Oral Implantology Clinic, Guarulhos University, Guar-ulhos, SP, Brazil.8 Oral Implantology Clinic, Department of Periodontol-ogy, Dental Research Division, Guarulhos University,Guarulhos, SP, Brazil.* Corresponding author, e-mail: jashibli@yahoo.comDOI: 10.1563/AAID-JOI-D-09-00028

CASE REPORT

Journal of Oral Implantology 491

Modern bone tissue engineering tech-

niques, through their use in combination

with biomaterials and osteogenic cells,

promise to obtain bone regeneration in

difficult contexts, without harvesting autog-

enous bone from other anatomic sites. By

manipulating 3 essential elements—bioma-

terials, growth factors, and osteogenic

cells—bone tissue engineering seeks to

construct the ideal bone graft material,

characterized by the same biological and

structural properties of native bone.9,10

Therefore, the purpose of this case report

was to evaluate the histologic behavior of

the engineered bone tissue, obtained by a

culture of autogenous osteoblasts seeded on

polyglycolic-polylactid scaffolds (Oral Bone,

BioTissue, Friburg, Germany) in maxillary

sinus augmentation.

CASE REPORT

Patient and engineered bone

A 56-year-old partially edentulous male was

referred by his general dentist for implant

therapy for oral rehabilitation with dental

implants. The patient was a healthy non-

smoker with no significant medical history.

After clinical and radiographic examination,

the patient presented missing teeth in the

premolar and molar regions caused by

periodontal disease. The posterior maxilla

presented severe atrophy11 (Figure 1). Max-

illary sinus floor elevation was required to

allow successful implant insertion. The pa-

tient received information on all proposed

treatment, and he provided signed informed

consent before undergoing treatment.

Six weeks before surgery, a specimen (2

3 5 mm) of bone marrow was taken from

the posterior area of the mandible, together

with 100/150 mL of venous blood sample.

The specimen, preserved in a medium

containing antibiotics and antimycotic solu-

tion, was then transferred to BioTissue

Technologies Laboratories to be processed

in a clean room. In the first 28 hours, cells

were enzymatically detached by 0.1% colla-

genase CLSIII (Clostridium histolyticum, Bio-

Chrom, Berlin, Germany) in DMEM/Ham’s F12

(Dulbecco’s substratum modified by Eagle

1:1, Invitrogen GmbH, Karlsruhe, Germany).

After 3 hours, the cellular suspension was

strained and filtered through a 100-mm

mesh, washed 2 times using phosphate-

buffered saline solution (PBS, Invitrogen

GmbH), and seeded as primary culture in

polystyrene culture flasks (Corning, Acton,

Mass). The medium consisted of DMEM/

Ham’s F12 (1:1) with 10% of autologous

patient serum. During the first 2 steps,

penicillin (10 U/mL) and streptomycin

(10 mm/mL) were added prophylactically.

Cells were cultured at 37uC with 5% carbon

dioxide (CO2) and 95% humidified air. Every

3 days, 75% of the culture medium was

FIGURE 1. Preoperatory view of severe atrophy ofthe posterior maxilla. (a) Axial view. (b) Panoramicview. (c) Detail of each slice.

Engineered Bone by Autologous Osteoblasts in Maxillary Sinus Augmentation

492 Vol. XXXVI/No. Six/2010

replaced. Reaching 70% confluence, cells were

detached from culture flasks with 0.02%

trypsin and 0.02% thylenediaminetet-

raacetic acid, then were subcultivated until

a cell number of 16 to 32 million units was

reached, in 3 to 4 passages. A fraction of

these cells was tested separately for oste-

ogenic reproducibility.12 Cell suspension

was subsequently mixed with fibrinogen

at a ratio of 3:1 (Tissucol Duo S, Baxter,

Vienna, Austria), until a cellular density of

15 million cells/mL (625%) was reached,

and was soaked into biodegradable scaf-

folds (Ethisorb Tamponade, Ethicon, Nor-

densted, Germany), with a volume of

100 mL each. The scaffolds were character-

ized by an unwoven, disk-shaped polygly-

colid-polylactid structure (PLGA) with de-

fined size of 8 mm diameter and 2 mm

height. Scaffold porosity was very high

(.90%). Every single disk was finally capa-

ble of carrying 1.5 million autogenous cells.

The fibrinogen was polymerized by adding

thrombin (diluted 1:10 with PBS). After

polymerization was completed, cell-seeded

constructs were cultured for 1 week in a

specific osteogenic medium (Sigma, Dei-

senhofen, Germany) made of DMEM/Ham’s

F-12 (1:1) enriched with 5% autogenous

serum, ascorbic acid (0.3 mM), dexametha-

sone (1028 mol/L), and beta-glycerophos-

phate (10 mM). After 6 to 9 days of three-

dimensional (3D) culture, cellular vitality

was tested by measurement of cellular

glucose consumption (mg glucose con-

sumption/5 mL of culture medium/48 h).

When glucose consumption rates suggest-

ed sufficient viability, constructs were

stored in sterile transport medium and

were transferred to the clinic for the sinus

floor elevation procedure precisely 6 weeks

after biopsy was performed.

Maxillary sinus augmentation

The patient received antibiotics (2 g amox-

icillin) prophylactically 1 hour before surgery.

Local anesthesia (2 mL articaine hydrochlo-

ride 4% with 1/100 000 adrenaline) was

administered. A horizontal crestal incision

and 2 vertical incisions extending beyond

the mucogingival junction were made, and a

full-thickness flap was reflected to expose

the maxillary sinus lateral bone wall. Under

constant irrigation with saline solution, an

osseous window of approximately 1 cm 3

1 cm was demarked and isolated, using a

round diamond-coated bur. The isolated

osseous window was subsequently removed

and conserved in saline solution. The

Schneiderian membrane was exposed and

carefully isolated, using specially designed

elevators, to avoid undesired perforations.

Engineered bone transplants were used for

augmentation in the maxillary sinus. Six

polyglycolid-polylactid disks (8 mm diameter

3 2 mm depth, Oral Bone), each carrying 1.5

million autogenous osteoblasts, were used,

placed, and condensed into the depth of the

sinus cavity. After the sinus augmentation

procedure was completed, the previously

isolated osseous window was repositioned

to close the sinus lateral wall. Sutures were

placed (Supramid, Novaxa Spa, Milan, Italy)

to ensure complete flap closure. Amoxicillin

500 mg capsules was given 3 times daily for

7 days, and ibuprofen 400 mg tablets to be

taken as needed were prescribed for the

patient.

Bone core harvesting

Implant placement surgery was performed

after a 6 month healing period. Bone cores

were harvested through a transcrestal using

a 2.0 3 10 mm diameter trephine bur under

sterile saline solution irrigation. Three im-

plants with sandblasted acid-etched surfaces

(2 implants with 3.75 mm diameter and

13 mm length, and 1 with a 3.75 mm 3

10 mm length) were inserted; primary

stability ranged from 20–40 N/cm. The

second-stage surgery was carried out after

a 5 month healing period.

Mangano et al

Journal of Oral Implantology 493

Specimen processing andhistometric analyses

The biopsies were processed (Precise 1

Automated System, Assing, Rome, Italy) to

obtain thin ground sections as previously

described.13 The specimen was dehydrated in

an ascending series of alcohol rinses and

embedded in glycol methacrylate resin (Tech-

novit 7200 VLC, Kulzer, Wehrheim, Germany).

After polymerization, the specimens was

sectioned lengthwise along the larger axis of

the specimen, using a high-precision dia-

mond disk, to about 150 mm, and were

ground down to about 30 mm. Two slides

obtained from this specimen were stained

with basic fuchsin and toluidine blue. Histo-

morphometry of newly formed bone and

marrow spaces was carried out on the whole

sample at low magnification (325). These

measurements were obtained using a light

microscope (Laborlux S, Leitz, Wetzlar, Ger-

many) connected to a high-resolution video

camera (3CCD, JVC KY-F55B, JVC, Yokohama,

Japan) and interfaced to a monitor and PC

(Intel Pentium III 1200 MMX, Intel, Santa Clara,

Calif). This optical system was linked to a

digitizing pad (Matrix Vision GmbH, Oppen-

weiler, Germany) and a histometry software

package with image capturing capabilities

(Image-Pro Plus 4.5, Media Cybernetics Inc,

Bethesda, Md).

RESULTS

The patient presented no complications

following sinus augmentation. The maxillary

sinus filled with engineered bone transplants

revealed a mean vertical bone gain of 6.4 mm

(Figure 2).

Histologic evaluation revealed the pres-

ence of mature bone with cancellous areas.

The cancellous bone exhibits as compact

areas with incremental basophilic lines

mixed with interposed reversion lines (Fig-

ure 3). The medullary spaces were ample

and were almost filled with well vascularized

connective tissue, showing no signs of

inflammation or foreign body reaction. These

spaces were filled with fatty marrow inter-

posing areas of fibrosis that were sometimes

dense. The augmented maxillary sinus with

bone engineered contained a mean of

28.89% and 71.11% of bone and medullary

space, respectively.

DISCUSSION

Most studies in maxillary sinus floor aug-

mentation have focused on new bone

formation around several graft materials.1–7

Modern bone tissue engineering techniques

have the goal of obtaining a bone substitute

with ideal properties from the structural and

biological point of view. In addition, this

material should reproduce the same features

of autogenous bone: It has to be osteogenic,

osteoconductive, and even osteoinductive.14

At the same time, it must be structurally and

mechanically able to sustain cell activity with

the advantage of unlimited availability.

Recent progress in molecular biology now

FIGURE 2. Postoperative view of the augmented maxillary sinus. (a) Coronal view. (b) Lateral view.

Engineered Bone by Autologous Osteoblasts in Maxillary Sinus Augmentation

494 Vol. XXXVI/No. Six/2010

permits the clinician to harvest and culture

osteogenic cells and to seed on biomaterials,

then differentiate the cells into functional

osteoblasts, and finally to implant the

osteoblasts into bone defects.

This case report demonstrates that the

use of bone tissue engineered in a maxillary

sinus augmentation procedure and bone

formation appear to be related.

It has been pointed out15 that an ideal

delivery scaffold to sustain cellular activity

in the bone graft site has not yet been

achieved. Notwithstanding that the tech-

nique presented in this report has been

FIGURE 3. (a and b) Ground sections of the bone biopsies retrieved after a 6 month healing period (Basicfuchsin and toluidine blue staining, original magnification 312). (c) Larger magnification of the biopsyspecimen. Arrows show the incremental basophilic lines mixed with interposed reversion lines (Basicfuchsin and toluidine blue staining, original magnification 3140). (d) Presence of several osteocytes innewly formed bone (Basic fuchsin and toluidine blue staining, original magnification 3140). (e) Someempty spaces are filled with connective tissue without signs of inflammation (Basic fuchsin andtoluidine blue staining, original magnification 3140).

Mangano et al

Journal of Oral Implantology 495

previously described with interesting re-

sults in an earlier study on maxillary sinus

augmentation in humans,16 this strategy for

engineered bone transplant creation may

not ensure sufficient dental implant anchor-

age. Polyglycolid-polylactid (PLGA) synthetic

polymeric scaffolds are characterized by a

rapid resorption rate, which could represent

an unfavorable factor for bone regeneration.

These data have been confirmed in a recent

clinical study17 on maxillary sinus augmenta-

tion in 20 patients. The authors demonstrated

that extended or rapid resorption of the

synthetic polymeric scaffolds could jeopar-

dize bone regeneration, thus making it

impossible to guarantee adequate mechani-

cal stability for osteoblasts delivered to the

graft site. Osteoblasts must adhere to a stable

structure to produce new bone matrix. New

matrix has to subsequently undergo mineral-

ization and maturation processes. To this

issue, fast or extended degradation of the

supporting scaffold determines the inevitable

failure of bone regeneration caused by the

collapse of newly formed bone matrix.

However, an ideal scaffold for bone regener-

ations is currently under study.17 The Oral

Bone material, with polyglycolid-polylactid

scaffolds, showed efficacy in promoting

cellular activity and bone regeneration as

presented in this case report.

In conclusion, data from this case report

demonstrate that the newly formed bone

provided by bone tissue engineering allowed

proper initial stability for dental implant

placement. However, the role of this new bone

in the long-term success of dental implant

anchorage needs further investigation.

ABBREVIATIONS

CO2: carbon dioxide

3D: 3-dimensional

PBS: phosphate-buffered saline solution

PLGA: polyglycolid-polylactid

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