兔下颌骨来源骨髓间充质干细胞生物学行为及其体内成骨的研究

Biological Behaviour and Osteogenesis in vivo of Bone Marrow Mesenchymal Stem Cells Derived from Rabbit mandible

目的:探讨兔下颌骨来源的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)体外分离、培养及其生物学行为,评价下颌骨BMMSCs体内成骨的效果。方法:取兔下颌骨中的松质骨,贴壁培养,取第P2或P3代检测:1)倒置显微镜下观察细胞生长状态;2)MTT法测定细胞增殖并绘制细胞生长曲线;3)向成骨细胞和脂肪细胞诱导分化;4)将P3代BMMSCs与羟基磷灰石/磷酸三钙(Hydroxyapatite/tricalcium phosphate,HA/TCP)复合,自体回植;单纯HA/TCP植入做对照。术后8周和20周,通过组织学观察评价体内成骨的效果。结果:1)细胞经传代后形态一致;2)细胞生长曲线显示下颌骨BMMSCs经历潜伏期、对数生长期和平台期;3)成骨诱导、成脂诱导后形成钙结节及脂滴,茜素红、油红O染色呈阳性;4)体内成骨显示术后8周BMMSCs-HA/TCP组空隙内新骨形成;术后20周大量新骨形成,HA/TCP大部已降解。单纯HA/TCP植入组术后8周未见新骨形成;20周大部HA/TCP降解,仍未见新骨形成。结论:兔下颌骨分离出的BMMSCs,具有强大的自我复制、增殖能力及多向诱导分化的潜能,并能体内成骨。

Objective： To investigate the methods of isolation and cultivation and biological behaviour of BMMSCs derived from rabbit mandible, and to evaluate the effect of osteogenesis in vivo. Methods： BMMSCs were collected from rabbit mandible and isolated by density gradient centrifugation. Cells were adherently cultured in vitro, and P2 or P3 BMMSCs were collected and examined： 1） Ceils growth were observed by inverted microscopy; 2）The propa- gation of BMMSCs were tested by the methods of MTT and drew a growth curve; 3） The potential of multi-direc- tional differentiation into osteoblasts and adipocytes were estimated by related methods; 4）Autologous P3 BMMSCs were combined with HA/TCP to implant into 15ram bone defect. HA/TCP alone was used as control. The bone formation was observed with histological examination 8 and 20 weeks after operation. Results： 1） The majority of adherent cells were long fusiform, and few were small trangle. 2） The growth curve of BMMSCs showed that every passages were experienced incubation period, log phase and platform period. 3） BMMSCs after induction showed osteogenic and adipogenic potential. The staining of mineralized nodules and lipid drops were positive by alizarin red S and oil red O. 4） Histological examination demonstrated that new bone was distributed throughout the HA/TCP after 8 weeks. 20 weeks after operation, the bone defect were repaired fully by new bone in experimental group. New bone was not formed after 8 and 20 weeks in control group. Conclusion： The highly uniformed BMMSCs de rived from rabbit mandible can be collected. These cells have the ability of self-replication and propagation, the po- tential of multi-directional differentiation and osteogenesis in vivo.