Cell preparation

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.

PRP preparation

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.

Patient selection

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.

Surgical technique

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/>