Objective To investigate the ectopic bone formation of the chitosan/phosphonic chitosan sponge combined with human umbil ical cord mesenchymal stem cells (hUCMSCs) in vitro. Methods Phosphorous groups were introduced in chitosan molecules to prepare the phosphonic chitosan; 2% chitosan and phosphonic chitosan solutions were mixed at a volume ratio of 1 ∶ 1 and freeze-dried to build the complex sponge, and then was put in the simulated body fluid for biomimetic mineral ization in situ. The hUCMSCs were isolated by enzyme digestion method from human umbil ical cord and were cultured. The chitosan/phosphonic chitosan sponge was cultured with hUCMSCs at passage 3, and the cell-scaffoldcomposite was cultured in osteogenic medium. The growth and adhesion of the cells on the scaffolds were observed by l ight microscope and scanning electron microscope (SEM) at 1 and 2 weeks after culturing, respectively. The cell prol iferation was detected by MTT assay at 1, 2, 3, 4, 5, and 6 days, respectively. Bilateral back muscles defects were created on 40 New Zealand rabbits (3-4 months old, weighing 2.1-3.2 kg, male or female), which were divided into groups A, B, and C. In group A, cellscaffold composites were implanted into 40 right defects; in group B, the complex sponge was implanted into 20 left defects; and in group C, none was implanted into other 20 left defects. The gross and histological observations were made at 4 weeks postoperatively. Results The analysis results of phosphonic chitosan showed that the phosphorylation occurred mainly in the hydroxyl, and the proton type and chemical shifts intensity were conform to its chemical structure. The SEM results showed that the pores of the chitosan/phosphonic chitosan sponge were homogeneous, and the wall of the pore was thinner; the coating of calcium and phosphorus could be observed on the surface of the pore wall after mineral ized with crystal particles; the cells grew well on the surface of the chitosan/phosphonic chitosan sponge. The MTT assay showed that the chitosan/phosphonic chitosan sponge could not inhibit the prol iferation of hUCMSCs. The gross observation showed that the size and shape of the cell-scaffold composite remained intact and texture was toughened in group A, the size of the complex sponge gradually reducedin group B, and the muscle defects wound healed with a l ittle scar tissue in group C. The histological observation showed that part of the scaffold was absorbed and new blood vessels and new bone trabeculae formed in group A, the circular cavity and residual chitosan scaffolds were observed in group B, and the wound almost healed with a small amount of lymphocytes in group C. Conclusion The chitosan/phosphonic chitosan sponge has good biocompatibil ity, the tissue engineered bone by combining the hUCMSCs with chitosan/phosphonic chitosan sponge has the potential of the ectopic bone formation in rabbit.
【Abstract】 Objective To evaluate the effect on repairing composite defect of mandible and skin by pre fabricatedmusculocutaneous flap including ectopic bone induced by BMP-2 and collagen in rabbits’ latissimus dorsimuscle. Meth ods Twenty-four rabbits (4-6 weeks old) were randomly divided into 3 groups: experimental, control and blank control group (n=8 in each group). Composite carriers composed of BMP-2 and collagen I sponge were implanted into latissimus dorsi muscle pouches of rabbits. The bone formation was evaluated with roentgenography, ALP staining, Von Kossa staining, HE staining, toluidine blue staining and CD31 immunohistochemical labell ing of microvessels. After 6 weeks, the mandibular defect of 8 mm in diameter with local skin defect of 2 cm × 3 cm was made in experimental group, and a musculocutaneous flap including ectopic-induced bone was prefabricated to transfer and repair the composite defect. The mandibular defect of 8 mm in diameter without local skin defect was made in control and blank control group. Free ectopic-induced bone was used for the repair of mandibular defect in control group, but repairing was not performed in blank control group. All the samples were detected 6 weeks after operation for tetracycl ine fluorescent staining, X-ray, histological examination and bone quantity analysis to evaluate the effect. Results Bone formation induced by BMP-2/collagen composites were found as woven bone between 4 to 6 weeks. It showed that cartilaginous osteogenesis was the mainly type of bone formation. Microvessels could beseen in the bony tissues. The composite defects of mandible and skin were healed well in the experimental group. Major bony tissue were seen in the control group, while it still remained bony defect in the blank control group. The bone quantity analysis in the experimental, control, and blank control group were (1.594 ± 0.674), (0.801 ± 0.036), and (0.079 ± 0.010) mm2, there were significant differences between each groups (P lt; 0.05). Conclusion Prefabrication of musculocutaneous flap including boneinduced by the composite of BMP-2 and collagen is feasible and prevalent. It can be regarded as vascularized bone graft and used in repairing composite defect of bone and skin.