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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。
Purpose For a variety of reasons, alveolar bone and even the jaw bone defects, the loss is very common in clinical practice, patients with oral function and beauty can have a serious impact. Calcium phosphate cement (CPC) is recognized as a biodegradable bone graft material with good biocompatibility. When mixed with type I collagen, plasticity increases, porosity increases and elastic modulus is closer to the human bone. In this study, SD rat bone marrow mesenchymal stem cells (BMSCs) were cultured in vitro by combining the neurotransmitter substance P (SP) with CPC collagen scaffold material. The surface morphology of the material and the effect on the biological behavior of BMSCs were observed. Methods From September 2015 to January 2016, 20 female 4-week-old rats were selected at the Stomatological Hospital of the Fourth Military Medical University to conduct BMSCs culture in vitro. The study materials were divided into 3 groups: group A: pure CPC group; group B: CPC + type Ⅰ collagen group; group C: CPC + type Ⅰ collagen + SP group. Scanning electron microscopy (SEM) was used to observe the surface morphology and morphological, adhesion and proliferation of BMSCs. BMSCs were identified by measuring cell proliferation logarithm, osteogenic and adipogenic induction, and the adhesion and proliferation of BMSCs were quantitatively analyzed by fluorescence staining. Alveolar red staining was performed 14 days after osteogenic induction liquid culture, and alkaline phosphatase (ALP ) Kit to detect cell ALP activity. Results The primary BMSCs grew vigorously and the growth curve was typical “S” shape. Osteoblasts and adipogenic medium were cultured for 3 weeks. Alizarin red and oil red O staining were performed. The calcified nodules and lipid droplets were observed under microscope. The multi-directional differentiation of BMSCs was good. Under the SEM, the collagen and CPC were closely linked in the B and C groups. The surface of the collagen was in the form of a cord-like gap. The number of BMSCs and collagen attached to the surface was large, the length of the synapse was connected with each other, and the cell stretched well. Fluorescent microscope observation, C group of surface active cells was higher than the A, B group. Alizarin red staining was performed 14 days after osteogenic induction. The color of C group was darker than that of C and B groups (P <0.05). CONCLUSIONS: The SP-loaded CPC collagen composite scaffold constructed in this study can promote the adhesion, proliferation and osteogenic differentiation of BMSCs in SD rats, and provide a new way of thinking and research foundation for clinical alveolar surgery.