Study On Osteogenesis Of Nacre Artificial Bone Combined With Osteoblasts In Vivo

BackgroundNacre is the inner layer of the shell of the bivalve marine mollusks, such as pearl shell, oyster, abalone and fan mussel. It's mainly composed of calcium carbonate, besides little organic matrix and microelement. It has good compatibility, degradable, osteocoduction and osteogenesis. Poly-DL-lactic acid(PDLLA),which has the character of easily degradable and shape-able, and nacre powder of Pteria martensii were selected to prepared nacre/PDLLA (N/P) artificial bone. Nacre was confirmed as an appropriate bone graft substitutes by both experiments of rabbit radium bone defect repairing and canine lumbar fusion. So, nacre's compatibility, osteoinduction and osteogenesis of N/P artificial bone combined with allogeneic osteoblasts in the repairing of rabbit radius critical-size bone defect will be researched in this experiment.Objective1. To explore the biocompatibiliry of N/P artificial bone by the study of the proliferation of the osteoblasts loaded on N/P artificial bone in vitro and in vivo.2. To explore the osteoinduction of nacre by the study of the mechanism of ectopic osteogenesis of N/P artificial bone combined with allogeneic osteoblasts.3. To survey the osteogenic effect of N/P composite artificial bone combined with allogeneic osteoblasts in the repairing rabbit radius segmental critical-size defect.Methods1. The osteoblasts from illiac bone of New Zealand white rabbits were isolated, cultured, expanded and then seeded into the N/P artificial bone in vitro. The appearance of osteoblasts was observed under the phase contrast microscope. The proliferation of the osteoblasts were detected by MTT assay, observation of scanning electron microscope and histological examination in vitro.2. After the complexes of osteoblasts and the N/P artificial bone were implanted subcutaneously in allografts of the New Zealand white rabbits, the inflammatory reactions and immunoreactions were observed by histological examination. The osteoblasts composite with the N/P artificial bone were labeled by thymidine analogue bromodeoxyuridine(BrdU) in vitro, then implanted subcutaneously into the back sites of rabbits. The proliferation of the labeled osteoblasts were analysis by immunohistochemical staining after implanted.3.The allogeneic osteoblasts were incubated with N/P artificial bone in vitro, then implanted subcutaneously into the back sites of the New Zealand white rabbits. N/P artificial bone which didn't combine with osteoblasts served as a control. The complexes which harvested at 1, 4 and 8 weeks after implanted respectively were examined by gross observation, histological examination and immunohistochemical investigation.4. The allogeneic osteoblasts seeded onto N/P artificial bone were co-cultured after 1 week in vitro, then the complexes were implanted into the radii segmental defect sites of the New Zealand white rabbits. The defects of 15 mm long were produced on bilateral radii of 18 adult rabbits by surgery. PDLLA alone artificial bone combined with allogeneic osteoblasts and N/P artificial bone which didn't combine with osteoblasts served as a control. The repairing effect was evaluated at 4,8 and 12 weeks after operation respectively by gross observation, radiograph examination, four-point-bending experiment (biomechanical testing),-6-histological examination and tetracycline tracing method. Results1. Under the phase contrast microscope, the appearance of the osteoblasts was spindle or triangle like. The adhesion, growth and proliferation of osteoblasts were seen on the surface and pore of the material by MTT assay, observation of scanning electron microscope and histological examination in vitro.2. It was confirmed by histological examination that the inflammatory reactions and immunoreactions of the N/P artificial bone were low. Osteoblasts labeled by BrdU could grow and proliferate, which confirmed by immunohistochemical staining after implanted in vivo.3. In experimental group, the osteoid was fo...