| 1. Cultivation and identification of bone marrow mesenchymal stem cells from rabbitsPurpose: To isolate and culture bone marrow mesenchymal stem cells (BMSCs) and identify them. Methods: The growth and morphological characteristics were observed. To identify BMSCs'multipotential ability, alizarin red, toluidine blue,and oil red O staining were performed after specific inducing culture for osteogenesis, chondrogenesis, and adipogenesis respectively. Results: The fibrous adherent cells have proliferation ability. These BMSCs can be made to differentiate into osteoblasts, chondrocytes, adipocytes under special culture conditions. Conclusions: There are multipotential mesenchymal stem cells in rabbit bone marrow and they can be isolated and cultured using adherence screening method.2. Fabrication and osteogenic differentiation of BMSC SheetPurpose: To investigate the feasibility of constructing bone BMSC sheets in a standard culture plate using a simple method and characterized the potential of osteogenic differentiation. Methods: We first harvested a cell sheet from rabbit BMSCs using a continuous culture method. Histological and immunohistochemical examination, electron microscopy examination, energy- dispersive analysis by X-rays, quantitative analysis of alkaline phosphatase activity, and gene expression analysis were performed to evaluate the harvested sheet. Results: The BMSCs proliferated fast and formed an intact sheet that is robust enough to be detached from the culture plate with a cell scraper and to be handled easily with forceps. The cell sheet, which was composed of multipale layers of cells and their extracellular matrix showed evident mineralized nodules, high ALP activities, high calcium and phosphorase concentrations, and up-regulated gene expression of osteogenic markers after osteoblastic differentiation. (p < 0.05). Conclusions: Our study indicates BMSC sheet can be fabricated in a standard culture plate using a scrape technique and the obtained sheet maintain the potential of osteogenic differentiation.3.Engineering injectable bone using osteogenic BMSC sheet fragmentsPurpose: To circumvent these limitations associated with the use of isolated cells in cell therapy, we investigated the feasibility of constructing injectable bone tissue using BMSC sheet fragments, without use of alien carriers. Methods: The osteogenic differentiated BMSC sheet was cut into small fragments. Then serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of cell sheet fragments (the sheet fragment group) were injected subcutaneously on the back of the nude mice to evaluate the ectopic bone formation. Gross examination, CT scanning, and histological examination were performed to evaluate the harvested specimens. Results: The results revealed that the sheet fragment group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. There were no significant differences detected between the density for the 8-week engineered bone and that for native spine bone of mice (p < 0.05). Conclusions: Our study indicates that BMSC sheet fragments can be used to engineer injectable bone tissue without use of alien carriers and the engineered bone might be considered as a promising strategy for bone repair.4. Injection of cell sheet fragments enhances delayed mandibular bone healing in a rabbit modelPurpose: To investigate whether injections of osteogenic BMSC sheet fragments are more effective than BMSCs in treatment of delayed mandibular bone healing. Methods: According to the material injected in the fracture gap, 18 semimandibles of nine rabbits were divided randomly into 3 groups as follows (n =6 for each group): group A (serum-free medium), group B (dissociated cell suspension), and group C (suspension of cell sheet fragments). The mandibles were harvested and evaluated by gross examination, radiography, micro-CT scanning, histologic and histomorphometric examination at 6 weeks after injection. Results: Both the radiographic evaluation and micro-CT scanning showed a significant increase in bony union in the group C, compared with the group A and the group B. The histologic examination showed more intensive bone formation in the group C than that in the other two groups. Conclusions: The results show that injection of cell sheet fragments promotes new bone formation in the fracture gap and indicate a promising approach to treat the delayed bone healing.5. Locally injection of cell sheet fragments enhances bew bone formation in mandibular distraction osteogenesis: a rabbit modelPurpose: To investigate whether injections of osteogenic BMSC sheet fragments could be used to promote new bone formation during distraction osteogenesis. Methods: Thirty two New Zealand rabbits underwent bilateral mandibular osteotomy and their mandibles were lengthened 9 mm after a 5-day latency period. According to the material injected in the distracted gap and the distraction rate, rabbits were divided randomly into 4 groups as follows (n = 8 for each group): for group A, group B, and group C, bilateral distraction osteogenesis was performed at a rate of 0.75 mm per 12 h for 6 days and 0.3 mL serum-free medium, dissociated cell suspension, and suspension of cell sheet fragments were injected into the distracted gap in each group respectively; For group D, distraction osteogenesis was performed at a rate of 0.5 mm per 12 h for 9 days without any injections. Rabbits were killed at 3 and 6 weeks after injection. The distracted areas were harvested and evaluated by gross examination, radiography, micro-CT scanning, histologic and histomorphometric examination, and three-point bending testing. Results: Radiographic evaluation and micro-CT scanning indicated a significant increase in bony union of the distraction regenerate in the group C and the group D, compared with the group A and the group B. Corresponding to the radiographic findings, the histologic examination showed more intensive bone formation in the group C and the group D after 3 and 6 weeks as compared to the other two groups. The maximum load was significantly higher in the group C and the group D than those in the others. Conclusions: The results show that injection of cell sheet fragments promotes new bone formation in the distracted gap and indicate a promising approach to shorten the treatment period of osteodistraction.6. Engineering scaffold-free three-dimensional bone tissue using osteogenic BMSC sheetPurpose: To circumvent these limitations associated with the use of alien scaffolds and isolated cells in bone tissue engineering, we investigated the feasibility of constructing functional three-dimensional bone tissue using BMSC sheets, without alien scaffolds. Methods: The osteogenic pre-differentiated bone marrow stromal sheets were rolled and fabricated into a large three-dimensional construct. The construct was then implanted into the subcutaneous pockets of nude mice for in vivo experiments. Gross examination, CT scanning, histological examination, and biomechanical test were performed to evaluate the harvested specimens. Results: CT scanning and histological examination confirmed new bone formation in vivo. There were no significant differences detected between the density for the 8-week engineered bone and that for native spine bone of mice (p < 0.05). Additionally, the engineered bone exhibited enhanced compressive strength. Conclusions: Our study indicates that mineralized osteogenic cell sheet can be used to engineer functional three-dimensional bone tissue without use of alien scaffold and the engineered construct might be considered as a promising substitute for bone repair.
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