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

摘要

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 (hASCs) 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 hASCs 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 + hASCs,(3) BMP-2-BioCaP scaffold,(4) BMP-2-BioCaP scaffold + hASCs (test group).After 4 weeks of implantation,hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs.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 affected by BMP-2BioCaP.ALP activity was higher by the induction of OM + BMP-2-BioCaP than of the other groups (P ＜0.01).More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2),ALP,osteopontin (OPN) and osteocalcin (OC) were determined in the OM + BMP-2-BioCaP group at different time points (P ＜0.01).HE staining showed that,in the test group and BMP-2-BioCaP scaffold group,the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs,and newly-formed bone-like tissues could be found in ECM around the scaffold materials.Moreover,compared with the BMP-2-BioCaP scaffold group,more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups.No obvious positive results were found in the other groups.Conclusion:BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo.The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.%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,hASCs)体内外成骨分化的影响,最终构建以hASCs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hASCs的体外培养.CCK-8实验检测各组hASCs的体外增殖能力,诱导7d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,4d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+hASCs,(3) BMP-2-BioCaP缓释支架,(4) BMP-2-BioCaP缓释支架+hASCs(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5～10 μm,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5d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hASCs的早期增殖.诱导7d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4d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基因表达水平较对照组显著升高(P＜0.01),诱导14 d时RUNX2、ALP、骨桥蛋白(osteopontin,OPN)和骨钙素(osteocalcin,OC)基因表达量均显著高于其他组(P＜0.01).HE染色分析可见,实验组和BMP2-BioCaP缓释支架组中,细胞外基质呈强嗜酸性,出现类似骨陷窝的结构,并可见包含其中的类骨细胞.与BMP-2-BioCaP缓释支架组相比,实验组的细胞外基质嗜酸性更强,类骨组织的面积更大,结构更加典型,其他组未见矿化基质和类骨组织形成.结论:BMP-2-BioCaP支架能够实现BMP-2的良好缓释,并能显著促进hASCs的体内外成骨分化,以hASCs和BMP-2-BioCaP为基础构建的新型组织工程化骨具有潜在的应用前景.