双相沉降法重构同种异体脱钙颗粒骨/Ⅰ型胶原的制备及细胞相容性

Preparation and biocompatibility of allogeneic decalcified bone granules with typeⅠcollagen using biphasic precipitation technique

背景:同种异体脱钙颗粒骨的重构塑型是目前材料开发的热点研究,但至今仍未找到理想的方法。目的:采用双相沉降法用Ⅰ型胶原将同种异体脱钙颗粒骨黏合制备成重构同种异体骨,并体外考察其与骨髓间充质干细胞的生物相容性。设计、时间及地点:对比观察实验,于2007-09/12在山西省医用组织库完成。材料:成年健康Wistar大鼠18只。Ⅰ型胶原为美国Sigma公司生产。方法:取10只大鼠四肢骨,制备同种异体脱钙颗粒骨,用双相沉降法将Ⅰ型胶原和同种异体脱钙颗粒骨制备成凝胶样复合体;取8只大鼠股骨,分离培养骨髓间充质干细胞;取第5代细胞,分别与同种异体脱钙颗粒骨/Ⅰ型胶原、同种异体脱钙颗粒骨共培养,另以单纯骨髓间充质干细胞为空白对照组。主要观察指标:双色流式细胞仪分析细胞表型特征。复合培养第7天倒置显微镜观察细胞的黏附和生长状况。复合培养第5天扫描电镜观察同种异体脱钙颗粒骨/Ⅰ型胶原的表面结构和细胞生长情况。共培养第4,7天时收集并计数细胞量,用全自动生化分析仪测定碱性磷酸酶含量。结果:①细胞呈CD45-CD90+CD44+CD71+,符合骨髓间充质干细胞表面抗原特征。②共育7d时,倒置显微镜观察各组细胞形态无明显差异,生长状态良好。③共育4,7d时,活细胞数和碱性磷酸酶活性测定结果显示,同种异体脱钙颗粒骨/Ⅰ型胶原组显著高于同种异体脱钙颗粒组和空白对照组(P<0.01)。④扫描电镜观察重构同种异体骨由胶原联结成团块状,表面粗糙。同种异体脱钙颗粒骨/Ⅰ型胶原组共育5d时,细胞紧密贴附于材料表面,有长突起伸入材料内部;同种异体脱钙颗粒组细胞贴附较少。结论:双相沉降法制备的重构同种异体骨(同种异体脱钙颗粒骨/I型胶原)具有良好的细胞相容性。

BACKGROUND： The reconstruction of allogeneic decalcified bone granules （ADBG） has been placed in the hot centre of material study. However, the ideal procedure remains need to be explored. OBJECTIVE： To combine ADBG with collagen type Ⅰ （CG） to prepare the reconstructed allogeneic decalcified bone （ADBG/CG） via the method of biphasic precipitation, in addition, to evaluate the biocompatibility of this new composite. DESIGN, TIME AND SETTING： A control observation study was conducted at Shanxi Provincial Tissue Bank between September 2007 and December 2007. MATERIALS： A total of 18 healthy, adult, Wistar rats. Type I collagen was produced by American Sigma Company. METHODS： Femora of 10 rats were used to prepare ADBG and the gel-like composite of ADBG was obscured via the method of biphasic precipitation. Femora of 8 rats were randomly selected for the isolation and culture of bone marrow mesenchymal stem cells （BMSCs）. The fifth passage of cells was co-cultured with （BMSCs/ADBG/CG）, BMSCs/ADBG or BMSCs,respectively. MAIN OUTCOME MEASURE： The phenotypic markers of cells were determined by double-color flow cytometry. The cell growth and adhesion were observed by inverted microscope at 7 days of co-culture. Scanning electron-microscope was performed to observe the superficial construction and cell adhesive growth at 5 days. Living cell number and alkaline phosphatase （ALP） content were detected for the analysis of cell viability at days 4 and 7. RESULTS： These Cells displayed CD45-CD90＋CD44＋CD71＋, which was in accordance with BMSCs. At days 7 of co-culture, there was no significant shape changes could be found under inverted microscope, all cells grew well. The number of living cell and ALP content in the BMSCs/ADBG/CG group was significantly higher than that of the BMSCs/ADBG and blank control groups （P〈 0.01）. Under scanning electron-microscope, ADBG displayed mass shape binding with CG with irregular surface. At 5 days of co-culture, living cells with long processes tightly adhered to the surface and grew toward the deep inner of the BMSCs/ADBG/CG group. There were fewer amounts of adherence cells in the BMSCs/ADBG group. CONCLUSION： The reconstructed allogeneic bone （ADBG/CG） obscured via the method of biphasic precipitation shows better biocompatibility.