骨髓基质干细胞成骨能力的实验研究

Osteogeneic ability of bone marrow stromal stem cells

目的:研究以骨髓基质干细胞(Bonemarrowstemcells,BMSCs)为种子细胞,海绵状脱钙骨基质(Demineralizedbonematrix,DBM)为细胞载体的组织工程块修复骨缺损的能力。方法:从兔股骨分离出的骨髓细胞经体外诱导分化条件培养,检测碱性磷酸酶、Ⅰ型胶原的表达及钙结节的形成。将培养分化的兔BMSCs经5-溴-2'-脱氧尿嘧啶核苷(5-Bromo-2'-dexyouridine,BrdU)标记后接种到兔海绵状脱钙骨基质(spongeofDBM,sDBM)支架上,细胞接种密度为每块DBM种植(4～6)×106个细胞,然后分别植入兔背部肌肉内及兔桡骨中段10mm长的骨膜骨缺损内。对照组为骨缺损内单纯植入sDBM及空白组。术后观察6周。结果:经条件培养的BMSCs体外表达碱性磷酸酶、Ⅰ型胶原及形成钙结节。体内异位植入含BMSCs的组织工程骨块可形成新骨组织,成骨方式类似软骨化骨。体内骨缺损修复实验结果显示组织工程骨块与单独sDBM均在6周完全修复骨缺损。新生骨骨密度测量组织工程骨块植入组虽然均值高于单独sDBM植入组,但差异无显著性意义(t=2.289,P=0.071)。极限压缩强度测量显示,组织工程骨块植入组新生骨生物力学强度已达正常桡骨的90%,与正常桡骨段差异无显著性意义(t=2.893,P=0.0623);单纯sDBM植入组新生骨在生物力学强度为正常桡骨的86%。新生骨矿化率结果显示?

AIM:To study the ability of tissue engineering section to repair bone defects with bone marrow stromal stem cells(BMSCs) as seed cells and demineralized bone matrix(DBM) as cell carrier. METHODS:BMSCs derived from the bone shaft of femurs of a two month old New Zealand white rabbit were cultured under the condition of induction and differentiation in vitro.The expressions of alkaline phosphatase(ALP) and type Ⅰcollagen as well as the formation of calcium nodules were detected.Five weeks later,the cultured BMSCs were collected and marked by 5 Bromo 2' dexyouridine(BrdU),and then inoculated on the scaffold of sponge of demineralized bone matrix(sDBM) with the inoculation density of (4-6)×106 cells/pieces.The composites were transplanted into muscles of back and a 10 mm length segmental bone defect of rabbit periosteum respectively(trial group).The control group was implanted with sDBM into the defects,and the blank group were implanted nothing.Postperative observation lasted for 6 weeks. RESULTS:The cultured BMSCs expressed ALP and type Ⅰcollagen and calcium nodules were formed in vitro.After ectopic implantation of the tissue engineered section in vivo,new bone tissue was formed,whose osteogenic type was similar to cartilaginous bone.The experiment of repair of bone defect in vivo showed that both tissue engineered section and sDBM could repair the bone defects completely during 6 weeks.The bone mineral density(BMD) of new bone of the trial group was higher than that of the control group,but no significant difference was found between them(t=2.289,P=0.071).The measurement of compressive ultimate strength(CUS) showed that the biomechanical strength of new bone was 90%of normal radius,without significant difference(t=2.893,P=0.0623),and that in the control group was 86%of normal radius.The mineralization rate was significantly higher in the trial group than in the control group(t=2.965,P=0.017). CONCLUSION:The BMSCs induced in vitro have the ability of osteogenesis,and in vivo osteogenesis was found after they were combined with sDBM.The bone defects can be repaired during 6 weeks.Biomechanical strength and mineralization rate of tissue engineered new bone are higher in the trial group than in the control group.