兔骨髓基质干细胞在不同脱钙程度骨基质材料上的增殖

Proliferation of rabbit bone marrow stromal stem cells on varying degrees of decalcified bone matrix materials

背景:脱钙骨基质具有天然网状孔隙结构,有较好的可塑性和生物相容性,但目前报道对制备脱钙骨基质所用的脱钙时间各异。目的:比较不同脱钙时间的脱钙骨基质对兔骨髓基质干细胞增殖的影响,为组织工程软骨筛选最佳支架材料。方法:取兔髂骨制成条块状,经反复脱脂后,分别脱钙处理6,12,24h,乙醇浸泡2d制得脱钙骨基质,置于24孔培养板中。取浓度为5×109L-1的第3代兔骨髓基质干细胞悬液40μL,接种于上述培养板不同脱钙时间的预湿材料上。扫描电镜下观察脱钙骨基质形态及其孔隙率、孔径,MTT法测定骨髓基质干细胞在脱钙骨基质上的增殖情况,扫描电镜下观察骨髓基质干细胞在脱钙骨基质上的黏附情况。结果与结论:脱钙骨基质呈天然的高密度孔隙网架结构,骨小梁、小梁间隙和骨内管腔系统同时存在,脱钙6,12,24h的脱钙骨基质其孔隙率、孔径大小无明显差异(P>0.05)。与脱钙12,24h的脱钙骨基质比较,骨髓基质干细胞与脱钙6h的脱钙骨基质复合更为紧密,细胞相容性最好。扫描电镜下,脱钙6h的脱钙骨基质上生长的骨髓基质干细胞培养8d后增殖稳定,适合移植,且细胞数量明显多于脱钙12,24h的脱钙骨基质(P<0.01)。

BACKGROUND：Demineralized bone matrix （DBM） has natural mesh pores,good plasticity and biocompatibility.However,the decalcification time in preparation of DBM remains controversial.OBJECTIVE：To compare effects of varying degrees of decalcification with DBM on the proliferation of bone marrow stromal stem cells （BMSCs） so as to provide the best DBM scaffold for cartilage tissue engineering METHODS：Rabbit iliac bones were prepared into strips,defatted,followed by 6,12 and 24 hours of decalcification and 2 days of soaking in alcohol to prepare DBM.DBMs were placed in 24-well plates.The third passage of BMSCs at a density of 5x109/L were incubated on 24-well plate with DBMs.The DBM porosity and pore size were observed by scanning electron microscopy;BMSCs proliferation on the DBM was determined by MTT,and cell attachment on DBM was observed by scanning electron microscopy.RESULTS AND CONCLUSION：DBM displayed natural high-density porous grid structure,in the presence of bone trabecula,trabecular space and bone luminal system.The porosity and pore size of DBM decalcified for 6,12 and 24 hours were similar （P 0.05）.Compared with decalcified for 12 and 24 hours,BMSCs attached to DBM decalcified for 6 hours more closely and the DBM showed better compatibility.SEM observation showed the BMSCs on DBMs decalcified for 6 hours proliferated stably after 8 days and applicable for transplantation.Moreover,the number of cells were significantly more than DBMs decalcified for 12 and 24 hours （P 0.01）.