纳米羟基磷灰石—胶原—重组人骨形成蛋白-2复合支架的制备及细胞相容性

发布时间：2018-04-20 17:59

本文选题：骨组织工程 + 羟基磷灰石　；参考：《山西医科大学》2012年硕士论文

【摘要】：背景：利用骨组织工程学的理论,通过成骨细胞与支架共培养,测量细胞在材料上的增殖、分化情况,以此来评估生物支架材料的细胞相容性。目的：从细胞学角度评价复合材料纳米羟基磷灰石/胶原/重组人骨形成蛋白-2对成骨细胞增殖、分化的影响,了解其细胞相容性,并与同种异体骨比较,为该材料的临床应用提供实验依据。方法：采用改良胶原酶消化法处理新生24h内Wistar大鼠的颅盖骨,并进行成骨细胞原代培养。通过碱性磷酸酶染色、苏木精-伊红染色、钙结节茜素红染色、倒置相差显微镜对提取的成骨细胞进行观察与鉴定。然后将培养状况良好的第3代细胞与纳米羟基磷灰石／胶原/重组人骨形成蛋白-2支架材料体外复合培养。培养第3,6,9d后,采用倒置相差显微镜观察细胞形态,碱性磷酸酶活性测定法、MTT法测定支架材料对成骨细胞分化、增殖的影响。结果：1、通过原代培养提取的成骨细胞呈圆形悬浮于混合培养液中,2小时后开始逐渐贴壁。培养3天后,显微镜下可见细胞呈梭形及不规则形。碱性磷酸酶染色大部分细胞(95%以上)呈阳性,经茜素红染色可见红色矿化结节。2、ALP法和MTT法：该材料对成骨细胞增殖分化效果良好,并可促进成骨作用。3、倒置相差显微镜下见纳米羟基磷灰石/胶原/重组人骨形成蛋白-2复合材料周围聚集大量成骨细胞,并随时间增殖。4、扫描电镜观察发现：纳米羟基磷灰石/胶原/重组人骨形成蛋白-2复合支架表面有大量成骨细胞矿化结节,与对照组比较差异明显,进一步证实有利于骨形成。结论：纳米羟基磷灰石/胶原/重组人骨形成蛋白-2材料较同种异体骨材料对于成骨细胞的黏附、生长、分化、增殖效果更好,证明其具备良好的细胞相容性,有望为骨组织工程提供一种新的骨科修复材料。
[Abstract]:Background: by using the theory of bone tissue engineering, the cell proliferation and differentiation were measured by co-culture of osteoblasts and scaffolds to evaluate the cytocompatibility of scaffold materials. Objective: to evaluate the effect of nano-hydroxyapatite / collagen / recombinant human bone morphogenetic protein-2 on the proliferation and differentiation of osteoblasts from the cytological point of view, and to understand its cytocompatibility and compare it with that of allogeneic bone. To provide experimental basis for the clinical application of the material. Methods: calvaria of neonatal Wistar rats was treated with modified collagenase digestion method and osteoblasts were cultured in primary culture. The extracted osteoblasts were observed and identified by alkaline phosphatase staining hematoxylin eosin staining calcium nodule alizarin red staining and inverted phase contrast microscope. The third passage cells were cultured with nano-hydroxyapatite / collagen / recombinant human bone morphogenetic protein-2 scaffold in vitro. The cell morphology was observed by inverted phase contrast microscope and the effect of scaffold material on the differentiation and proliferation of osteoblasts was measured by MTT assay. Results the osteoblasts cultured in primary culture were suspended in the mixed medium for 2 hours and then gradually adhered to the wall. After 3 days of culture, the cells were spindle-shaped and irregular under microscope. Alkaline phosphatase staining was positive in most cells (> 95%). By alizarin red staining, red mineralized nodule. 2ALP and MTT showed that the material had a good effect on the proliferation and differentiation of osteoblasts. Under inverted phase contrast microscope, a large number of osteoblasts were observed around nano-hydroxyapatite / collagen / recombinant human bone morphogenetic protein-2 composites. The results of scanning electron microscopy showed that there were a large number of mineralized osteoblast nodules on the surface of nano-hydroxyapatite / collagen / recombinant human bone morphogenetic protein-2 composite scaffold, which was significantly different from that of the control group. Conclusion: the effect of nano-hydroxyapatite / collagen / recombinant human bone morphogenetic protein-2 on osteoblast adhesion, growth, differentiation and proliferation is better than that of homologous bone graft. It is expected to provide a new orthopedic repair material for bone tissue engineering.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

【相似文献】