【作者】 严安；

【导师】 廖前德；

【作者基本信息】 中南大学 ， 外科学， 2009， 硕士

【摘要】 目的:探讨富血小板血浆凝胶复合材料经成骨诱导后对比格犬颈椎缺损的修复能力,为组织工程化骨的建立和修复颈椎缺损中的应用提供实验依据。方法:(1)比格犬骨髓间充质干细胞的分离、纯化、扩增,富血小板血浆及凝胶的制备。(2)将传至第3代的骨髓间充质干细胞分别接种于富血小板血浆凝胶/强化磷酸三钙复合支架材料和单纯强化磷酸三钙支架上并体外成骨诱导培养1周。(3)经前路制作比格犬颈椎缺损模型,并分别将制备好的骨髓间充质干细胞复合单纯强化磷酸三钙支架(Ⅰ组)、犬自体髂骨(Ⅱ组)、骨髓间充质干细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料(Ⅲ组)植入颈椎缺损处。观察植入物周边组织反应、局部缺损修复和骨痂形成情况和表面变化情况。(4)术后4、8、12周行X线片检查,观察X线片变化情况。(5)术后4、8、12周取材行HE染色及骨钙素蛋白免疫组织化学染色观察表达情况。(6)RT-PCR检测体内各组在4、8、12周时OCN、ALP、Col1A1的mRNA的表达情况。结果:(1)各组材料复合体植入比格犬颈椎缺损后,伤口愈合好,没有全身和局部的炎症和毒性反应。植入材料部位均未见明显炎症及排斥反应。(2)术后4、8、12周摄X线片结果显示Ⅱ组及Ⅲ组12周时基本达到骨质愈合,而Ⅰ组仍未见材料完全吸收、骨性愈合情况,材料-骨质界面清晰,材料周缘出现负影。(3)术后4、8、12周取材行HE染色及骨钙素蛋白免疫组织化学染色可见各组均可观察到新生软骨成骨情况,强化磷酸三钙/富血小板血浆凝胶复合材料组表达较强化磷酸三钙材料组更强。(4)各组在4、8、12周各组均可见OCN、ALP和Col1A1的mRNA表达条带出现。比较4、8、12周各组目的条带和内参灰度值,8、12周时Ⅱ组和Ⅲ组各项表达强于Ⅰ组,Ⅱ组和Ⅲ组之间无明显区别。结论:富血小板血浆凝胶/强化磷酸三钙复合支架材料在体内显著促进骨髓间充质干细胞成骨效果,并能持续稳定表达成骨特异性表型,是一种较为理想的骨组织工程支架材料,在组织相容性、降解速度及成骨效果等方面均比单纯强化磷酸三钙支架优秀。富血小板血浆凝胶/强化磷酸三钙复合支架材料能够有效的修复犬颈椎缺损,为组织工程骨在颈椎缺损修复中的应用提供了一定的实验基础和论依据。更多还原

【Abstract】 Objective: To investigate the capacity of repairing cervical bone defect of beagle dogs with platelet-rich plasma gel composite materials after being osteogenic induced , so as to provide experimental basis for establishing tissue-engineered bone and repairing cervical defects.Method: (1) Separation, purification, amplification of the beagle bone marrow mesenchymal stem cells, and preparation of platelet-rich plasma gel. (2) Choose the 3rd generation bone marrow mesenchymal stem cells respectively combine with platelet-rich plasma gel / beta-tricalcium phosphate composite scaffold material, simply beta-tricalcium phosphate composite scaffold and cultured with osteogenesis induced in vitro for 1 week. (3) Produced Beagle cervical bone defect model by anterior operative approach, and combined the prepared bone marrow mesenchymal stem cells with tricalcium phosphate alone enhanced stent (Ⅰgroup), canine autologous bone (Ⅱgroup), BMSCs compound strengthen the tricalcium phosphate / platelet-rich plasma gel scaffold (Ⅲgroup) implanted cervical defect respectively. Then observe the tissue reaction around the implant, partial defect repair and bone formation and surface changes. (4) X-ray inspection was used at 4,8,12 week. Observe any changes in X-ray film. (5)Obtain the sample at 4,8,12 weeks and use HE staining and immunohistochemical to observe the osteocalcin protein expression. (6) RT-PCR was used to detect the expression of mRNA regarding OCN, ALP, CollA1 in the body by each groups at 4,8,12 week.Result: (1) There was no systemic and local inflammatory and toxicity response in each group of complex materials which implanted in beagle dogs with cervical bone defects,and the wound healing in each group was well. No significant inflammation and rejection occurred in implant sites. (2) The postoperative X-ray at 4,8,12 weeks displayed that groupⅡand groupⅢreached bone healing in 12 weeks, but the group I did not show complete absorption, bone healing, the materials - bone interface clear, material negative impact peripheral. (3) HE staining and osteocalcin protein immunohistochemical staining showed that endochondral bone emerged in each group, and the expression of enhanced tricalcium phosphate / autologous platelet-rich plasma gel composite material was stronger than the group of tricalcium phosphate group in 4,8,12 weeks. (4) The OCN, ALP and the mRNA expression of CollA1 emerging bands could be seen in 4,8,12 weeks. Compared the purpose band and the reference strip gray value of of each group, the expression of the groupⅡand groupⅢwere stronger than group I in 8,12 weeks .there is no any significant difference between groupsⅡandⅢ.Conclusion: Platelet-rich plasma gel / beta-tricalcium phosphate composite scaffold is an ideal material for bone tissue engineering scaffold material which has a satisfactory effect especially in histocompatibility, degradation rate and the capacity of osteogenics. Platelet-rich plasma gel / beta-tricalcium phosphate composite scaffold material can effectively promote osteoblastic effect of BMSCs after revulsant in vivo, in which osteoblastic phenotype are stably expressed . Platelet-rich plasma gel / beta-tricalcium phosphate composite scaffold material can effectively repair the bone defect of cervical in dogs which provide a experimental and theoretical basis for tissue engineering to repair bone defects in the cervical spine.更多还原