接种密度对磷酸钙骨水泥支架骨髓基质干细胞增殖和成骨分化的影响  

Effects of cell seeding density on proliferation and osteodifferentiation of bone mesenchymal stem cells on calcium phosphate cement scaffolds

作　　者：李广军[1] 赵亮[1] 邱耿韬 李伦超 王吉兴[1] 

机构地区：[1]南方医科大学南方医院脊柱骨科, 广州510515

出　　处：《中华创伤骨科杂志》2014年第6期527-532,共6页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金（31100695）,教育部青年教师基金（20114433120003）,广东省自然科学基金（S2011010003918）,广东省科技计划项目（20128010200024）,广州市科技计划项目（2012J2200049）.

摘　　要：目的 探讨不同初始接种密度的骨髓基质干细胞（BMSCs）接种至磷酸钙骨水泥（CPC）支架上对细胞增殖和成骨分化效果的影响,并验证BMSCs生长在CPC上的生物黏附性能.方法 培养小型猪的BMSCs至第4代,按1、5、25 mol/mL 3种细胞接种密度接种至CPC支架上,成骨诱导培养1、4、8d时采用扫描电镜观察细胞黏附形态及性能,EdU法检测细胞增殖率,活/死细胞染色试剂盒检测活细胞百分比及黏附性能、单位面积活细胞数,茜素红染色法检测矿物质合成,实时聚合酶链式反应（RT-PCR）检测成骨基因碱性磷酸酶（ALP）、骨钙素（OC）、Ⅰ型胶原（Col-Ⅰ）、RUNT相关转录因子-2（Runx-2）的mRNA的表达.结果 BMSCs在CPC支架表面黏附扩展良好,细胞接种密度从l mol/mL上升至25 mol/mL,活细胞百分比、单位面积活细胞数、矿物质合成、成骨分化基因也相应升高.ALP表达量在4d时达峰值,5 mol/mL组和25 mol/mL组的ALP表达量均高于1 mol/mL组,差异有统计学意义（P＜0.05）,5 mol/mL组与25 mol/mL组之间差异无统计学意义（P＞0.05）.OC、Col-Ⅰ、Runx-2的mRNA表达量在培养8d时明显增高,此时5 mol/mL组与25 mol/mL组差异均无统计学意义（P＞0.05）.培养4、8d时5 mol/mL组与25 mol/mL组的活细胞百分比、单位面积活细胞数比较差异均无统计学意义（P＞0.05）.结论 BMSCs的初始接种密度并非越高越好,在5 mol/mL至25mol/mL之间时,细胞接种至复合生物功能化CPC支架上,更利于细胞的增殖、分化和细胞外基质的合成,且具有良好的生物黏附性能.Objective To investigate the effects of cell seeding density on proliferation and osteodifferentiation of bone marrow mesenchymal stem cells （BMSCs） on calcium phosphate cement （CPC） scaffolds and on the bioadhesive property of BMSCs on CPC as well.Methods BMSCs were isolated from minipigs and cultured.The fourth passage BMSCs were seeded on CPC scaffolds at 3 different densities （1,5,25 mol/mL） for osteodifferentiation.On days 1,4 and 8,scanning electron microscopy was used to detect cell bioadhesive property and extension,EdU assay to detect cell proliferation,live/dead fluorescent staining assay to detect cell viability,and RT-PCR to detect mRNA expression of alkaline phosphatase （ALP）,osteocalcin （OC）,collagen type Ⅰ （Col-Ⅰ）,and Runt-related transcription factor 2 （Runx-2）.Results The BMSCs seeded on the CPC scaffolds showed fine bioadhesive property and extension.When the cell seeding density was increased from 1 mol/mL to 25 mol/mL,the percentage of live cells,osteodifferentiation,and bone mineral synthesis also increased.On day 4 when ALP gene peaked,ALP expression levels in the 5 mol/mL and 25 mol/mL groups were significantly higher than in the 1 mol/mL group （P 〈 0.05）,but there was no significant difference between the 5 mol/mL and 25 mol/mL groups （P 〉 0.05）.The expression levels of OC,Col-Ⅰ,and Runx-2 were significantly higher on day 8,but the differences between the 5 mol/mL and 25 mol/mL groups were not significant （P 〉 0.05）.There were no significant differences on day 4 or 8 between the 5 mol/mL and 25 mol/mL groups regarding the percentage of live cells or the number of live cells per specimen surface area （P 〉 0.05）.Conclusions BMSCs seeded on the CPC have good bioadhesive property.A too high seeding density may not be advantageous,for an intermediate seeding density can yield the best proliferation,differentiation and extracellular matrix formation.The best seeding density of BMSCs on CPC may vary between 5 mol/mL and 25 mol/mL.

关 键 词：间质干细胞 磷酸钙类 接种 细胞增殖 成骨分化 

分 类 号：R318.08[医药卫生—生物医学工程]