摘要
背景:研究证明脱蛋白松质骨具有良好的孔隙结构、生物可降解性和力学特性,但是应用于临床还要考虑其安全性。目的:观察异种脱蛋白松质骨支架与经诱导骨髓间充质干细胞的相容性以及对其生物学行为的影响。设计、时间及地点:体外观察实验,于2008-02/10在黑龙江省纤维化生物治疗重点实验室完成。材料:取成年猪新鲜股骨远端松质部分,初制为3cm×0.5cm×0.5cm大小。抽取山羊髂骨骨髓,梯度密度离心法分离提纯骨髓间充质干细胞,传至第2代时换含1×10-7mol/L地塞米松、1×10-2mol/Lβ-甘油磷酸钠、100mg/L维生素C的培养液进行成骨诱导。方法:无菌条件下将支架材料置入6孔板内,每孔1块,重复3孔。调整细胞悬液浓度为2×109L-1,向每块支架滴加细胞悬液1mL,培养5h后将骨块翻面后沿6孔板内壁缓慢加入诱导培养液10mL。另3孔接种相同数量细胞单纯培养做对照分析。主要观察指标:倒置相差显微镜下观察细胞贴附及基质分泌情况,MTT法分析种子细胞与支架复合培养1,3,5,7d后的存活和增殖能力,复合培养1,3,5,7d后检测碱性磷酸酶活性,流式细胞仪分析复合培养7d后细胞周期、DNA倍体水平及细胞凋亡率。结果:复合培养3d时接种于支架上的细胞在孔隙内表面贴壁生长,并分泌较多的细胞外基质,复合培养5,7d,细胞基质分泌旺盛,几乎充满支架孔隙。在各个时间点复合材料组骨髓间充质干细胞增殖峰值高于单纯培养组,但差异无显著性意义(P>0.05)。复合材料组倍增时间为3d,单纯培养组为3.15d。各时间点两组碱性磷酸酶活性差异无显著性意义(P>0.05)。两组细胞周期大致相同,未见异倍体细胞。结论:异种脱蛋白松质骨与骨髓间充质干细胞有良好的细胞相容性并促进细胞生长分化。
BACKGROUND： Deproteinized cancellous bone has been previously shown to be characterized by well pore structure, biodegradability, and mechanical ability; however, the safety still needs to be further studied in clinical application. OBJECTIVE： To study the biocompatibility of induced bone mesenchymal stem cells on xenogeneic deproteinized cancellous bone, and investigate the effect on biological behavior. DESIGN, TIME AND SETTING： In vitro observational study was performed at the Key Laboratory of Fibrous Biotherapy of Heilongjiang Province between February and October 2008. MATERIALS： Deproteinized bone was derived from femoral cancellous bone of adult pig, making the size of 3 cm× 0.5 cm × 0.5 cm. Bone marrow derived from ilium of goat underwent density gradient centrifugation so as to separate and purity bone rnesenchymal stem cells. At the second passage, bone mesenchymal stem cells were induced with culture media containing Dexamethasone （1 ×10^-7 mol/L）,β -sodium glycerophosphate （1 × 10.2 tool/L）, and vitamin C （100 mg/L）. METHODS： Xenogeneic deproteinized cancellous bone was put in a 6-well plate （bone/well） and re-put in three random wells. 1 m/suspension at the concentration of 2×10^9/L was added in each well, and the samples were culture for 5 hours. Thereafter, the bone was turned over, and 10 mL inducing culture media was slawly added along inner wall of the 6-well plate. The same number of cells was inoculated in the three wells for control analysis. MAIN OUTCOME MEASURES： Cell adhesion and matrix secretion were observed under inverted phase contrast microscope; survival and proliferation as well as alkaline phosphatase activities of coultured cell and stent were analyzed at days 1, 3, 5, and 7 using MTT method; cell cycle, DNA ploid, and apoptotic rate were analyzed using flow cytometry at day 7 after co-culture, RESULTS： Cells inoculated on the stent exhibited adherent growth and excreted a lot of extracellular matrixes at day 3 after co-culture; pore space of the stent was nearly full of matrixes at day 5 and 7 after co-culture; proliferative peak of bone mesenchymal stem cells in the co-culture group was higher than single culture group at different time points, but there was no significant difference （P 〉 0.05）. Culture time was prolonged for 3 days in the co-culture group and 3.15 days in the single culture group. There were still no significant differences in alkaline phosphatase activities between the two groups at different time points （P 〉 0.05）. The cell cycles of the two groups were similar, and heteroploid cells were not found out in both groups. CONCLUSION： Xenogeneic deproteinized cancellous bone has a good biocompatibility to bone mesenchymal stern cells and promotes cell growth and differentiation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第12期2217-2221,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
黑龙江省卫生厅科研项目(2007-062)~~
