骨形态发生蛋白-2-磷酸钙共沉淀支架与人脂肪间充质干细胞构建新型组织工程化骨  

A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2

作　　者：姜蔚然 张晓[1] 刘云松[1] 吴刚 葛严军[1] 周永胜[1] 

机构地区：[1]北京大学口腔医学院·口腔医院,修复科口腔数字化医疗技术和材料国家工程实验室,口腔数字医学北京市重点实验室,北京100081 [2]Department of Oral Implantology and Prosthetic Dentistry,Academic Centre for Dentistry Amsterdam(ACTA),Research Institute MOVE,VU University and University of Amsterdam,Amsterdam,the Netherlands 1081 LA

出　　处：《北京大学学报（医学版）》2017年第1期6-15,共10页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81400484);北京大学口腔医院青年科研基金项目(YS020213)资助~~

摘　　要：目的:构建可缓释骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)的仿生磷酸钙(biomimetic calcium phosphate,BioCaP)共沉淀三维支架(BMP-2-coprecipitated biomimetic calcium phosphate,BMP-2-BioCaP),检测其理化特性,探究其对人脂肪间充质干细胞(human adipose-derived stem cells,h ASCs)体内外成骨分化的影响,最终构建以h ASCs和BMP-2-BioCaP为基础的新型组织工程化骨。方法:构建BMP-2-BioCaP三维缓释支架,扫描电子显微镜观察表面形貌,体外检测其缓释能力。将BMP-2-BioCaP颗粒分别浸泡于增殖培养基(proliferation medium,PM)与成骨诱导培养基(osteogenic medium,OM)中,每2天提取上清液用于h ASCs的体外培养。CCK-8实验检测各组h ASCs的体外增殖能力,诱导7 d及14 d后进行碱性磷酸酶(alkaline phosphatase,ALP)染色及活性定量检测,14 d及21 d进行茜素红染色及矿化沉积定量分析,4 d及14 d检测成骨相关基因的表达情况。体内实验使用6只裸鼠,于其背部正中做皮肤切口,向两侧共分离出4个皮下植入腔,分别植入:(1)单纯BioCaP支架,(2)BioCaP支架+h ASCs,(3)BMP-2-BioCaP缓释支架,(4)BMP-2-BioCaP缓释支架+h ASCs(实验组)。植入4周后取材,标本制成组织学切片进行HE染色观察。结果:BioCaP表面由不规则晶体组成三维立体多孔结构,孔直径约为5~10μm,加入BMP-2后,不影响BioCaP原有的立体结构。缓释曲线结果显示,蛋白质在前2天释放速度较快,随后释放速度放缓并于5 d后趋于平稳,之后每天释放量较稳定,至第21天仍有少量释放,累积释放量达20%。CCK-8结果显示,BMP-2-BioCaP缓释支架不会影响h ASCs的早期增殖。诱导7 d与14 d后,OM+BMP-2-BioCaP组ALP染色及活性定量检测均显著高于其他组(P<0.01)。诱导21 d后,OM+BMP-2-BioCaP组矿化结节染色及钙沉积定量检测均高于其他组(P<0.01)。诱导4 d时OM+BMP-2-BioCaP组的Runt相关转录因子2基因(Runt-related transcription factor 2,RUNX2)与ALP基因表达水平较对Objective： To construct a novel biomimetic calcium phosphate（ BioCaP） scaffold loaded with bone morphogenetic protein-2（ BMP-2）,and to investigate its role in the osteogenesis of human adipose-derived stem cells（ h ASCs） in vitro and in vivo. Methods： The BioCaP scaffold coprecipitated with BMP-2（ BMP-2-BioCaP） was constructed in this study. Field emission scanning electron microscopy（ SEM） was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium（ PM） or osteogenic medium（ OM）,respectively. The supernatants were collected and used to culture h ASCs in vitro. Cell numbers were determined using the cell-counting kit-8（ CCK-8） to assess the cell proliferation. After 7 and 14 days,alkaline phosphatase（ ALP） staining and quantification were performed to test the activity of ALP. After 14 and 21 days,the calcification deposition was determined by alizarin red S（ ARS） staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study,6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups：（ 1） BioCaP scaffold only,（ 2） BioCaP scaffold ＋ h ASCs,（ 3） BMP-2-BioCaP scaffold,（ 4） BMP-2-BioCaP scaffold ＋ h ASCs（ test group）. After 4 weeks of implantation,hematoxylin-eosin（ HE） staining was performed to evaluate the in vivo osteogenesis of h ASCs. Results： SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight,plate-like and sharp-edged crystal units,and the length of the crystal units varied between 5 and 10 μm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days,and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly

关 键 词：骨形态发生蛋白质类 磷酸钙类 沉淀 人脂肪间充质干细胞 组织工程 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]