人骨髓间充质干细胞结合多孔β-TCP构建生物相容性人工骨

Human Bone Marrow Derived Mesenchymal Stem Cells Combined with Porous Beta-tricalcium Phosphate Promote Bone Formation in vivo

研究人骨髓间充质干细胞(HBMSCs)结合多孔β磷酸三钙(β-TCP)的生物相容性与体内成骨作用。密度梯度离心结合差异贴壁法分离HBMSCs,常规扩增传代,相差显微镜形态学观察和流式细胞仪检测细胞表面标记CD13、CD29、HLA-2、CD34、CD45和HLA-DR;分别在成骨、成软骨和成脂肪细胞培养基中定向诱导分化,验证其多向分化能力。将HBMSCs在低压下载入多孔β-TCP立方块,形成MSCs/β-TCP复合物,电镜观察材料内部与细胞结合情况。继续成骨诱导培养2周后植入裸鼠背部皮下,于植入4周和8周后取出复合物做组织学检查。设非成骨诱导培养复合物为对照。原代和传代细胞呈梭形外观,生长增殖能力良好;流式细胞仪检测间充质细胞来源表面标记物CD13、CD29,HLA-2阳性,造血细胞来源表面标记物CD34、CD45和HLA-DR阴性;能成功高效定向分化为成骨细胞、软骨细胞、脂肪细胞;细胞在β-TCP材料表面贴附、增殖良好;MSCs/β-TCP复合物植入皮下4周后即有少量新骨生成,至8周时更明显;对照组新骨生成量较少。本方法快速、高效分离扩增HBMSCs;HRMSCs与多孔可降解β-TCP材料生物相容性良好;二者结合能显著提高体内新骨生成,提示其可用于临床作为骨移植替代物,可提高骨移植修复骨缺损的疗效。

The aim of this study is to investigate in vivo bone formation using human bone marrow derived mesenchymal stem cells （HBMSCs） seeded in the porous β-tricalcium phosphate （β-TCP）. HBMSCs were isolated from human bone marrow by combination of gradient centrifugation and different adherent time. Morphology and growth characteristics were examined by phase contrast microscopy. Cell surface markers CD13, CD29, HLA-2, CD34, CD45 and HLA-DR were tested by flow cytometry. HBMSCs were induced respectively to osteoblasts, chondrocytes and adipocytes in vitro to verify their multi-differentiation potential. MSCs β-TCP constructs were constituted by loading HBMSCs into β-TCP blocks in a low pressure system, center area of the construct was viewed by scanning electron microscope. The constructs were transplanted into subcutaneous sites of the rats＇ back after 2 weeks of oteogenic culture. The constructs were taken 4 and 8 weeks later for histological examination. β-TCP was set as a control. Cells were spindle-shaped and present active proliferation in primary and passage cultures. The Cell surface markers CD13, CD29, HLA-2 were positive expressed and CD34, CD45 and HLA-DR were negative. Cells were successfully induced into osteoblasts, chondrocytes and adipocytes. New bone formation in the MSCs/β-TCP constructs were observed after 4 weeks of transplantation, which became much more obvious after 8 weeks. New bone formation in the control was less. HBMSCs combined with β-TCP greatly enhanced the bone formation in vivo, indicating its potential application in clinical orthopaedics as a bone graft.