Engineered Bone by Autologous Osteoblasts on Polymeric Scaffolds in Maxillary Sinus Augmentation: Histologic Report

Maxillary sinus floor augmentation has been used for occlusal rehabilitation with dental implants in the posterior maxilla.1 Currently, several regenerative therapies, including synthetic bone grafts, allogenic and xenogenic bone matrix, and recombinant growth/differentiation factors, have been used for maxillary sinus grafting.1–8 

Modern bone tissue engineering techniques, through their use in combination with biomaterials and osteogenic cells, promise to obtain bone regeneration in difficult contexts, without harvesting autogenous bone from other anatomic sites. By manipulating 3 essential elements—biomaterials, 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.

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 nonsmoker 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). Maxillary sinus floor elevation was required to allow successful implant insertion. The patient received information on all proposed treatment, and he provided signed informed consent before undergoing treatment.

Six weeks before surgery, a specimen (2 × 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 solution, 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% collagenase CLSIII (Clostridium histolyticum, BioChrom, 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 µm/mL) were added prophylactically. Cells were cultured at 37°C with 5% carbon dioxide (CO₂) and 95% humidified air. Every 3 days, 75% of the culture medium was replaced. Reaching 70% confluence, cells were detached from culture flasks with 0.02% trypsin and 0.02% thylenediaminetetraacetic 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 osteogenic 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 (±25%) was reached, and was soaked into biodegradable scaffolds (Ethisorb Tamponade, Ethicon, Nordensted, Germany), with a volume of 100 mL each. The scaffolds were characterized by an unwoven, disk-shaped polyglycolid-polylactid structure (PLGA) with defined size of 8 mm diameter and 2 mm height. Scaffold porosity was very high (>90%). Every single disk was finally capable 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, Deisenhofen, Germany) made of DMEM/Ham's F-12 (1:1) enriched with 5% autogenous serum, ascorbic acid (0.3 mM), dexamethasone (10⁻⁸ mol/L), and beta-glycerophosphate (10 mM). After 6 to 9 days of three-dimensional (3D) culture, cellular vitality was tested by measurement of cellular glucose consumption (mg glucose consumption/5 mL of culture medium/48 h). When glucose consumption rates suggested 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.

The patient received antibiotics (2 g amoxicillin) prophylactically 1 hour before surgery. Local anesthesia (2 mL articaine hydrochloride 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 × 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 × 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.

Implant placement surgery was performed after a 6 month healing period. Bone cores were harvested through a transcrestal using a 2.0 × 10 mm diameter trephine bur under sterile saline solution irrigation. Three implants with sandblasted acid-etched surfaces (2 implants with 3.75 mm diameter and 13 mm length, and 1 with a 3.75 mm × 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.

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 (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). After polymerization, the specimens was sectioned lengthwise along the larger axis of the specimen, using a high-precision diamond disk, to about 150 µm, and were ground down to about 30 µm. Two slides obtained from this specimen were stained with basic fuchsin and toluidine blue. Histomorphometry of newly formed bone and marrow spaces was carried out on the whole sample at low magnification (×25). These measurements were obtained using a light microscope (Laborlux S, Leitz, Wetzlar, Germany) 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, Oppenweiler, Germany) and a histometry software package with image capturing capabilities (Image-Pro Plus 4.5, Media Cybernetics Inc, Bethesda, Md).

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 presence of mature bone with cancellous areas. The cancellous bone exhibits as compact areas with incremental basophilic lines mixed with interposed reversion lines (Figure 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 interposing 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.

Most studies in maxillary sinus floor augmentation 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 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 technique presented in this report has been previously described with interesting results in an earlier study on maxillary sinus augmentation in humans,16 this strategy for engineered bone transplant creation may not ensure sufficient dental implant anchorage. 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 augmentation in 20 patients. The authors demonstrated that extended or rapid resorption of the synthetic polymeric scaffolds could jeopardize bone regeneration, thus making it impossible to guarantee adequate mechanical 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 mineralization 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 regenerations 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.

CO₂

carbon dioxide

3D

3-dimensional

PBS

phosphate-buffered saline solution

PLGA

polyglycolid-polylactid