新短肽P17-骨形态发生蛋白2/胶原基质矿化磷灰石材料生物活性的评价

Bioactivity of the peptide P17-bone morphogenetic protein-2/nano-hydroxyapatite-mineralized collagen composite

背景:胶原基质矿化磷灰石材料具有仿生的化学组成及良好的生物学性能,已被用于某些骨缺损修复;新短肽P17-骨形态发生蛋白2具有良好的生物相容性和成骨诱导生物活性,因此将新短肽P17-骨形态发生蛋白2与胶原基质矿化磷灰石材料制备成复合支架材料可望提升骨修复效率和效果。目的:探讨新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料的生物活性。方法:将兔骨髓间充质干细胞分别接种于新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料与胶原基质矿化磷灰石材料上,培养3,7 d后,利用RT-PCR检测细胞碱性磷酸酶mRNA相对表达。将新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料(实验组)与胶原基质矿化磷灰石材料(对照组)分别埋置于SD大鼠皮下,植入12,35 d后进行Masson染色后组织学分析。将新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料(实验组)与胶原基质矿化磷灰石材料(对照组)分别植入日本大耳白兔下颌骨箱状缺损处,植入5,15周后进行大体与X射线检查。实验经中国医科大学附属口腔医院伦理委员会批准。结果与结论:①复合材料组培养7 d的碱性磷酸酶mRNA表达高于胶原基质矿化磷灰石组(P<0.05);②皮下埋植实验显示两组材料和组织界面均未引起明显的急性炎症反应,植入后35d实验组可见更多的纤维细胞与材料嵌合;③骨缺损修复实验中,大体观察显示两种材料均具有良好的骨修复能力,植入5周时缺损区已有缩小趋势,植入15周缺损表面比较平整;X射线检查显示与对照组相比,实验组缺损区缩小趋势更明显;④结果表明,新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合支架材料具有比胶原基质矿化磷灰石更为优良的生物活性与骨缺损修复能力。

BACKGROUND:A mineralized collagen composite,i.e.nano-hydroxyapatite/collagen(nHAC)has biomimetic three-dimensional structure and good bioactive properties.As a bone tissue engineering material,it is widely used in bone defect repair.A newly designed P17-bone morphogenetic protein-2(P17-BMP2)has good biocompatibility and osteogenic capacity.Therefore,the composite scaffold material was prepared by combining the new P17-BMP-2 and nHAC,which might be used for the enhancement of osteogenic capacity in the treatment of bone defects.OBJECTIVE:To investigate the bioactivity of the P17-BMP-2/nHAC composite.METHODS:Rabbit bone marrow mesenchymal stem cells were seed on the P17-BMP-2/nHAC composite and nHAC.After 3 and 7 days of culture,the relative expression level of alkaline phosphatase was detected by RT-PCR.The subcutaneous implantation of P17-BMP-2/nHAC(experimental group)and nHAC(control group)into Sprague-Dawley rats was performed.Masson staining was performed for histological analysis at 12 and 35 days of implantation.P17-BMP-2/nHAC(experimental group)and nHAC(control group)were implanted into the white rabbit mandibular box-shaped bone defect,respectively.At 5 and 15 weeks,gross observation and X-ray were performed.The study was approved by the Medical Ethics Committee of China Medical University School&Hospital of Stomatology.RESULTS AND CONCLUSION:(1)The relative expression level of alkaline phosphatase in the P17-BMP-2/nHAC group was significantly higher than that in the nHAC group(P<0.05).(2)The result of subcutaneous implantation showed that the acute inflammatory response initiated by the P17-BMP-2/nHAC or nHAC was not found.More activated fibroblasts growing into the implants could be found on the sections of P17-BMP-2/nHAC compared to that of nHAC at 35 days after implantation.(3)In the bone defect repair test,gross observation showed that both materials held good defect repair ability,the defect area began to reduce at 5 weeks after implantation,and the defect surface became flat at 15 weeks after implantation.X-ray examination showed that compared with the control group,the defect area was more significantly reduced in the experimental group.(4)These results indicate that P17-BMP-2/nHAC composite scaffold has higher bioactivity and a stronger ability to repair bone defect.