Osteoperiosteal Tissue-Engineered Injectable Bone
Opportunities multiply as they are seized.
The use of dental implants in oral rehabilitation is becoming a standard method of care in den-A- tistry. When alveolar bone volume is insufficient to allow implant placement, augmentation is needed. The ability to augment the alveolar ridge has gradually expanded the scope of implant dentistry. During the past 10 years, alveolar augmentation techniques have become established treatment modalities. Dahlin et al1 reported an experimental study in rabbits involving the formation of new bone around titanium implants after use of a barrier membrane technique. Various bone grafting materials have been used for augmentation, including autogenous grafts, freeze-dried bone grafts, hydroxyapatite, and xenografts.2,3 Although the results of these reports indicate that various augmentations are clinically successful, this conclusion is questionable because only autogenous bone has osteogenic potential.4 However, use of autogenous bone is limited by availability for harvesting.
To avoid the need for graft harvesting of autogenous bone as well as the use of alloplasts alone, the authors attempted to regenerate bone in significant osseous defects with minimal invasiveness by providing a clinical alternative to the aforementioned graft materials that is both autologous and inductive. The new technology, termed tissue-engineered injectable bone,5,6 is based on tissue engineering concepts7 and involves the morphogenesis of new tissue using constructs formed from isolated autologous cells with biocompatible scaffolds and growth factors. It was previously reported that tissue-engineered bone induces excellent bone regeneration and promotes bone formation in a grafted area treated with platelet-rich plasma (PRP), which contains various growth factors.6
Recently, a two-step procedure for dental implant treatments, which involves placement of the implant after primary healing and remodeling of the graft, has been recommended for patients with less than 5 mm of alveolar bone height in the posterior maxilla or alveolar ridge. On the other hand, a one-step procedure, which allows dental implants to be placed simultaneously with grafts for patients who have at least 5 mm of alveolar bone to stabilize the implants,8–10 offers the advantages of fewer surgical treatments and the coordinated consolidation of the graft around the implants during healing, thus reducing both the number of surgical procedures and the healing time.
Therefore, the application of tissue-engineered bone plasticity for one-step procedures was explored by using mesenchymal stem cells (MSCs), PRP, and fibrin to increase the rate of bone formation and to enhance bone regeneration.
Methods and Materials
One month before surgery, MSCs (stromal stem cells) were isolated from the patient’s iliac crest marrow aspirate (10 mL) according to a previously described method.11 Briefly, the basal medium, low-glucose Dulbecco’s modified Eagle’s medium (Sigma-Aldrich), and growth supplements (50 mL of serum, 10 mL of 200 mM L-glutamine, and 0.5 mL of penicillin-streptomycin mixture containing 25 units of penicillin and 25 µg of streptomycin) were obtained from BioWhittaker. Three supplements for inducing osteogenesis—dexamethasone, sodium β-glycerophosphate, and L-ascorbic acid-2-phosphate—were obtained from Sigma Chemical. Cells were incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO2. The MSCs were replated at densities of 3.1 × 103 cells/cm2 in 0.2 mL/cm2 of control medium.
In culture, MSCs were trypsinized prior to implanting. For safety, the culture media was examined for contaminations of bacterium and for fungus prior to transplantation.
Preoperative hematologic assessments included a complete blood count with platelet levels. PRP was extracted 1 day prior to surgery. The PRP was isolated in a 200-mL collection bag containing the anticoagulant citrate under sterile conditions at the blood transfusion department of Nagoya University Hospital, Japan. The blood was centrifuged for 10 minutes at 1,500 rpm. Subsequently, the yellow plasma (containing the buffy coat, which contains platelets and leukocytes) was taken up. A second centrifugation (at 3,500 rpm for 5 minutes) was performed to combine the platelets into a single pellet. The plasma supernatant, platelet-poor plasma and relatively few cells, was removed. The resulting pellet of platelets, the buffy coat/plasma fraction (PRP), was resuspended in the residual 20 mL of plasma for use as platelet gel.
Injectable bone preparation
The PRP was stored at 22°C in a conventional shaker until used. Human thrombin in a powder form (5,000 units) was dissolved in 5.0 mL of 10% calcium chloride in a separate sterile cup. Next, 3.5 mL of PRP, MSCs (1.0 × 107 cell/mL), and 0.5 mL of air were aspirated into a 5.0-mL sterile syringe. In a second 2.5-mL syringe, 500 µL of the thrombin–calcium chloride mixture was aspirated. The cells were resuspended directly into the PRP. The two syringes were connected with a T connector, and the plungers of the syringes were pushed and pulled alternately, allowing air bubbles to traverse the two syringes. Within 5 to 30 seconds, the contents assumed a gel-like consistency as the thrombin affected the polymerization of fibrin to produce an insoluble gel.
Thirteen patients aged 44 to 74 years (mean age of 54.6 years) were treated, and their responses were analyzed to determine the efficiency of tissue-engineered bone formation for alveolar ridge augmentation.
Six patients with partial or total edentulism received sinus floor grafting, and seven patients underwent concurrent onlay plasty in which vertical alveolar augmentation was performed. All patients had conventional denture retention problems because of severe anterior or posterior alveolar ridge atrophy. In the maxilla, some patients had a residual sinus floor of less than 5 mm in height so that sinus grafting and implants could resolve the problem (Table 21-1)12; in other patients, the residual alveolar arch was markedly atrophied in both a horizontal and sagittal dimension.
Patients were selected for injectable bone grafting because they preferred not to undergo any surgery for harvesting of autogenous bone. In all cases, the reconstruction included sinus floor grafting or onlay plasty with simultaneous implant replacement. All patients were healthy and free of disease that might influence treatment outcome (eg, diabetes, immunosuppressive chemotherapy, chronic sinus inflammation, or rheumatoid arthritis).
The patients were extensively informed about the risks and benefits of the prescribed procedures, including surgery, graft materials, implants, and uncertainties of using a new bone regenerative method (Fig 21-1). They consented to cooperate during treatment, and the research protocol was approved by the university ethics committee.
Sinus floor augmentation
In all six patients, surgery was carried out while the patient was under general anesthesia. The sinus grafting procedure followed Tatum’s classic description.13 In brief, after elevation of a mucoperiosteal flap, a round hollow bur was used to create a door in the lateral maxillary sinus wall. After mobilization, the door was reflected inward. The space created by this procedure was filled with 1.5 to 5.8 g of tissue-engineered injectable bone. Simultaneous implant placement was performed. Care was taken to keep the sinus membrane intact to avoid spilling of the graft material. The mucoperiosteal flap was repositioned and sutured in the usual manner.
Vertical ridge augmentation
Standard titanium implants were placed in the atrophied maxilla or mandible at a depth of at least 5 mm so that part of the implant was exposed. Injectable bone was applied around the implant to cover exposed threads. After coagulation of the tissue-engineered bone, the grafted area was covered with a guided bone regeneration membrane (Gore-Tex) to prevent flap compression. The membrane was fixed with cover screws and/or microscrews. Finally, the buccal and labial periosteum was released to close the wound in a tension-free manner.
The MSCs were trypsinized at day 7 and used for the implants at a concentration of 1.0 × 107 cells/mL. The mean platelet count of the PRP was 972,269 (range of 524,480 to 2,033,000). These values confirmed the platelet sequestration ability of the process, which showed that the mean concentration was 446% above baseline.
None of the patients had postoperative problems besides normal swelling and inflammation at the surgical sites. The main complications during surgery were sinus membrane perforation and wound separation. Perforation of the sinus mucosa occurred in four procedures and resulted in only minor postoperative nasal bleeding without severe inflammatory signs during the observation period.
Sinus floor augmentation
Patients who underwent sinus floor augmentation were evaluated 6 months to 3 years after the first surgery. A total of 23 implants were placed with injectable bone. The clinical observation was carried out on the grafted area. The cumulative survival and success rates for implants placed in conjunction with injectable bone were 100%. Postoperative radiographic findings were consistent with integration between the implant and the regenerated bone; no bone loss or peri-implant radiolucency was observed. Comparison of preoperative and postoperative radiographs showed a mean increase in mineralized tissue of 8.7 mm.
Vertical ridge augmentation
Patients who underwent vertical ridge augmentation procedures were evaluated. The clinical conditions of the 26 implants placed in conjunction with ridge augmentation using injectable bone were also analyzed. The cumulative survival and success rates for implants placed in conjunction/>