含纳米硅钛表面促进骨整合及其机理的实验研究

发布时间：2018-08-21 09:33

【摘要】：第一部分纳米钛表面促进骨髓间充质干细胞的粘附和增殖的研究 [摘要]目的探讨纳米颗粒钛表面对大鼠骨髓间充质干细胞(MSCs)粘附和增殖的作用及机制。方法第3代的MSCs分别接种于纳米钛表面和纯钛表面对照培养32小时。分别通过扫描电子显微镜（SEM）和MTT法观测细胞形态和存活数，通过免疫组化检测粘附的骨髓间充质干细胞的有丝分裂率，应用实时聚合酶链反应（实时PCR），以确定粘附相关的，CD44V6和Integrinβ1信使核糖核酸（mRNA）的表达变化。结果培养4小时后，干细胞在纳米钛表面比纯钛表面更快铺展，并于16小时后更早地融合。而且，初始接种16个小时后，纳米钛表面细胞的增殖活性比纯钛表面显著增加（P0.01），32小时后，纳米钛表面粘附的间充质干细胞的有丝分裂率也高于纯钛表面（P0.01）。更有趣的是，接种4小时后，纳米钛表面组CD44V6和Integrinβ1的mRNA表达高于纯钛表面组（P0.01），通过统计分析发现CD44V6和Integrinβ1的mRNA表达呈正相关（rs=0.98，P0.01）。结论纳米钛表面可以显著地促进大鼠骨髓间充质干细胞的粘附和增殖，提高钛表面活性。第二部分含纳米硅钛表面促进大鼠骨髓间充质干细胞成骨分化的研究 [摘要]目的探讨含纳米硅（Si）钛表面对大鼠骨髓间充质干细胞（MSCs）成骨分化的影响及初步探讨可能的机制。方法第3代骨髓间充质干细胞分别培养于纯钛表面和含纳米硅钛表面12天。Cell Counting Kit-8试剂盒检测细胞数，荧光显微镜和Western印迹检测细胞增殖和向成骨细胞分化的细胞活力。茜素红S染色和定量测定分析含纳米硅钛表面对间充质干细胞的钙盐矿化沉积的影响。结果含纳米硅钛表面培养的间充质干细胞的增殖较纯钛表面强（P0.05）。纳米硅钛表面的间充质干细胞的碱性磷酸酶分泌，I型胶原和骨钙素表达和钙盐矿化沉积量均较纯钛表面增多（P0.05）。结论含纳米硅钛表面能促进大鼠骨髓间充质干细胞成骨分化，推测采用纳米硅涂层技术对医用钛假体表面进行改性，有利于促进骨整合。第三部分大鼠人工膝关节置换模型的建立 [摘要]目的设计并制作一个钛金属非骨水泥型大鼠膝关节置换手术，探讨建立大鼠人工膝关节置换模型的可行性。方法将21只体重300~325克的SD大鼠随机分为关节置换组、假手术组和无功能组，每组7只。每组动物均行手术处理。关节置换组大鼠行股骨髁间植入钛螺丝钉假体后分层缝合；假手术组只经右膝髌腱内侧暴露股骨关节面后分层缝合；无功能组经右膝髌腱内侧暴露股骨远端，切除膝半月板、前交叉韧带、内、外侧副韧带，切断髌腱后分层缝合。然后评估术后4周内大鼠疼痛相关的饮食和行走站立行为，检查右膝关节活动度，X线片检查膝关节病变情况。所有数值用平均值±标准差表示。采用SPSS12.0软件包对各组实验数据进行t检验或方差分析并进行多重比较。P0.05表示差异有统计学意义。结果术后4周内关节置换组与假手术组之间的饮食量和行走站立指数差异无统计学意义，于术后1周开始这两组站立指数优于无功能组（P0.05）。术后各组膝关节被动活动度均良好，各组之间差异无统计学意义。X线片显示关节置换组假体位置良好，周围无骨溶解现象。结论本实验设计的大鼠人工膝关节置换模型操作简单，术后活动功能好，，可用于模拟临床人工膝关节置换术的实验研究。第四部分含纳米硅钛假体在大鼠膝关节置换模型中的机械稳定性和骨整合的研究 [摘要]目的探讨钛和含纳米硅钛假体材料，植入到大鼠膝关节置换模型中，承重下的机械稳定性和骨整合情况。方法分别将钛和含纳米硅钛假体植入大鼠膝关节中，术后第3天开始每周给术侧膝关节腔内注射超高分子量聚乙烯(UHMW-PE)微颗粒，1个月后处死动物。术后每周在X线片上观察假体位置和周围骨质变化情况。用拔钉试验评估假体机械稳定性。Micro-CT(微型CT)扫描重建股骨远端骨质。显微组织病理学检测假体周围骨、软骨生长变化和无菌性炎症反应。采用SPSS12.0软件包对各组实验数据进行t检验或方差分析并进行多重比较，P0.05表示差异有统计学意义。结果钛和含纳米硅钛假体均无下沉，两组假体均机械稳定，无松动。但是，含纳米硅组假体骨界面的骨整合增强，螺丝钉最大抗拔出力较钛骨溶解诱导组增大（P0.05）。含纳米硅组假体周围新骨形成较钛骨溶解诱导组增多，假体周围成骨细胞增殖增多，无明显炎症细胞浸润。结论大鼠膝关节置换模型中，含纳米硅钛表面比钛表面更有利于骨整合，可减轻聚乙烯微颗粒诱导的无菌性炎症反应。当利用生物型假体置换关节时，本文为推荐使用含纳米硅表面改性的钛金属假体打下基础。
[Abstract]:The first part is to study the adhesion and proliferation of bone marrow mesenchymal stem cells promoted by nano titanium surface.
[ABSTRACT] Objective To investigate the effect and mechanism of titanium nanoparticles on the adhesion and proliferation of rat bone marrow mesenchymal stem cells (MSCs). Methods The 3rd generation MSCs were inoculated on the surface of titanium nanoparticles and cultured on the surface of pure titanium for 32 hours. The mitotic rate of adherent BMSCs was measured. Real-time polymerase chain reaction (real-time PCR) was used to determine the expression of adhesion-related, CD44V6 and Integrin beta 1 messenger ribonucleic acid (mRNA). After 16 hours of inoculation, the proliferation activity of mesenchymal stem cells on the surface of nano-titanium was significantly higher than that on the surface of pure titanium (P 0.01). After 32 hours, the mitotic rate of mesenchymal stem cells adhered to the surface of nano-titanium was also higher than that on the surface of pure titanium (P 0.01). In the face group (P 0.01), the expression of CD44V6 and Integrin beta 1 mRNA was positively correlated (rs=0.98, P 0.01). Conclusion Nano-titanium surface can significantly promote the adhesion and proliferation of rat bone marrow mesenchymal stem cells and enhance the surface activity of titanium.
The second part of the study is to promote the osteogenic differentiation of rat bone marrow mesenchymal stem cells with nano-si-ti surface.
[Abstract] Objective To investigate the effect of titanium containing nano-silicon (Si) on osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs). Methods The 3rd generation MSCs were cultured on pure titanium and titanium containing nano-silicon for 12 days, respectively. Alizarin red S staining and quantitative analysis of the effect of nano-silica-titanium surface on calcium mineralization of mesenchymal stem cells. Results The proliferation of mesenchymal stem cells cultured on the surface of nano-silica-titanium was stronger than that on the surface of pure titanium (P 0.05). The secretion of alkaline phosphatase, the expression of collagen type I and osteocalcin, and the deposit of calcium mineralization were all increased (P 0.05) compared with pure titanium surface.
The third part is the establishment of artificial knee replacement model in rats.
[ABSTRACT] Objective To design and fabricate a titanium cementless knee replacement operation in rats and explore the feasibility of establishing a rat model of artificial knee replacement.Methods 21 SD rats weighing 300-325 grams were randomly divided into joint replacement group, sham operation group and non-function group with 7 rats in each group. Titanium screw prosthesis was implanted in the femoral condyle of rats and then sutured in different layers; sham operation group only exposed the femoral articular surface through the medial patellar tendon of the right knee; nonfunction group exposed the distal femur through the medial patellar tendon of the right knee, resected the knee meniscus, anterior cruciate ligament, medial and lateral collateral ligament, cut the patellar tendon and sutured in different layers. Pain-related behaviors of diet and walking, right knee movement and X-ray examination of knee joint lesions were performed in rats. There was no significant difference in dietary intake and walking and standing index between the two groups within a week. The standing index of the two groups was superior to that of the non-functional group (P 0.05) from 1 week after operation. Conclusion The model of knee arthroplasty in rats can be used to simulate the experimental study of clinical knee arthroplasty.
Part IV Mechanical stability and osseointegration of nano-silicon-titanium prosthesis in rat knee joint replacement model
[ABSTRACT] Objective To investigate the mechanical stability and osseointegration of titanium and nano-silicon-titanium prosthesis implanted into the knee joint replacement model of rats.Methods Titanium and nano-silicon-titanium prosthesis were implanted into the knee joint of rats respectively.UHMW-PE was injected into the knee joint cavity weekly from the third day after operation. Granules, one month later, were sacrificed. The position of the prosthesis and the changes of surrounding bone were observed on X-ray every week after surgery. The mechanical stability of the prosthesis was evaluated by nail pulling test. The distal femoral bone was reconstructed by micro-CT scanning. The bone around the prosthesis, cartilage growth and aseptic inflammation were examined by histopathology. SPSS12.0 software was used. Results Titanium and nano-silicon-titanium prostheses did not sink, and the prostheses in both groups were mechanically stable without loosening. The formation of new bone around the prosthesis in the group containing nano-silicon was more than that in the group induced by titanium osteolysis, and the proliferation of osteoblasts around the prosthesis was increased without obvious inflammatory cell infiltration. This article will lay a foundation for recommending the use of titanium metal prostheses containing nano-silicon surface modification when using biological prostheses to replace joints.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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