磷酸钙骨水泥胶原支架复合P物质对大鼠骨髓间充质干细胞特性影响研究

Study on the effect of CPC collagen scaffold combined with P substance on the characteristics of rats BMSCs

目的因各种原因导致的牙槽骨甚至颌骨的缺损、缺失在临床上十分常见,对患者口腔功能和美观均可造成严重影响。磷酸钙骨水泥(CPC)是公认的具有良好生物相容性的可降解骨移植材料,与Ⅰ型胶原混合后可塑性增强,孔隙率增加且弹性模量更接近于人体骨。本研究通过人工合成神经递质P物质(SP)结合于CPC胶原支架材料,体外培养SD大鼠骨髓间充质干细胞(BMSCs),观察材料表面形貌及对BMSCs生物学行为的影响。方法2015年9月至2016年1月在第四军医大学口腔医院选取4周龄雌性大鼠20只,进行BMSCs体外培养。研究材料分为3组,A组:纯CPC组;B组:CPC+Ⅰ型胶原组;C组:CPC+Ⅰ型胶原+SP组。扫描电镜(SEM)观察样本表面形貌及BMSCs形态、黏附与增殖。通过测定细胞增殖对数、成骨及成脂诱导对BMSCs进行鉴定,荧光染色定量分析BMSCs的黏附与增殖,成骨诱导液培养14 d后经茜素红染色,并通过碱性磷酸酶(ALP)试剂盒检测细胞ALP活性。结果原代BMSCs生长旺盛,生长曲线呈典型"S"形。成骨和成脂诱导液培养3周,行茜素红和油红O染色,镜下可见钙化结节及脂滴形成,BMSCs多向分化能力良好。SEM下B、C组可见胶原与CPC联结紧密,胶原表面呈条索样间隙,表面BMSCs细胞与胶原附着数量多,突触长并彼此相联,细胞伸展良好。荧光显微镜下观察,C组表面活性细胞比例高于A、B组。成骨诱导14 d后茜素红染色,肉眼观C组颜色较深,ALP检测C组BMSCs的ALP表达高于A、B 2组(P<0.05)。结论本研究构建的SP加载的CPC胶原复合材料支架对SD大鼠BMSCs的黏附、增殖和成骨分化均具有促进作用,为今后临床牙槽外科修复重建工作提供了新的思路方法和研究基础。

ObjectiveThe alveolar bone defect and loss caused by various reasons are very common in clinic,which can cause serious effects on the function and appearance of the oral cavity. Calcium phosphate cemen（tCPC）is recognized as a biodegradable bone graft material with good biocompatibility,and it is more similar to the human bone with the increase of the plasticity and porosity ofⅠcollagen after mixing. In this study,the artificial neural transmitter sp（substance P）was integrated in collagen CPC scaffold and SD rat bone marrow mesenchymal stem cells（BMSCs）were cultured in vitro to observe the surface morphology and the effects on biological behavior of BMSCs.MethodsFrom September2015 to January 2016,20 female rats of 4 weeks old were selected from School of Stomatology,the Fourth Military Medical University to culture BMSCs in vitro. Materials were divided into 3 groups,group A：pure CPC group;group B：CPC ＋typeⅠcollagen group;group C：CPC ＋ typeⅠcollagen＋P substance group. In the 4-week old female rats,BMSCs were（）was performed to observe the morphology,adhesion and proliferation of the sample surface and BMSCs. Through the determination of the logarithm of cell proliferation,osteogenic and adipogenic differentiation of BMSCs wereidentified;fluorescence staining was used to make quantitative analysis of BMSCs adhesion and proliferation;osteogenic induction medium was used for culture for 14 days and stained by alizarin red,and the ALP kit was used for the detection of ALP activity.ResultsThe growth of the primary BMSCs was strong,and the growth curve was in typical＂S＂shape.Three weeks after the culture with osteogenic and adipogenic induction medium,alizarin red and oil red O was used for staining,and microscopy showed formation of calcified nodule and lipid droplet. BMSCs differentiation capacity was good. Under SEM group B and C showed that the collagen and CPC were closely connected,and there was a kind of gap on the surface. On the surface there were many BMSCs and collagen attached,the synapse was long and connected with each other,and the cells extended well. Under the fluorescence microscope,the ratio of living cells in group C was higher than group A and B. After osteogenic induction of 14 days,alizarin red was used for staining,and group C was darker with naked eyes. With ALP detection,the expression of ALP of BMSCs in group C was higher than that of group A and B（P〈0.05）.ConclusionIn this study,we constructed the substance P loading of CPC collagen composite scaffold played an important role in adhesion,proliferation and bone differentiation of SD rats BMSCs,provided a new method and research foundation for future clinical alveolar surgical repair and reconstruction work.