發(fā)布時(shí)間：2018-08-21 09:33

【摘要】：第一部分納米鈦表面促進(jìn)骨髓間充質(zhì)干細(xì)胞的粘附和增殖的研究 [摘要]目的探討納米顆粒鈦表面對(duì)大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)粘附和增殖的作用及機(jī)制。方法第3代的MSCs分別接種于納米鈦表面和純鈦表面對(duì)照培養(yǎng)32小時(shí)。分別通過(guò)掃描電子顯微鏡（SEM）和MTT法觀測(cè)細(xì)胞形態(tài)和存活數(shù)，通過(guò)免疫組化檢測(cè)粘附的骨髓間充質(zhì)干細(xì)胞的有絲分裂率，應(yīng)用實(shí)時(shí)聚合酶鏈反應(yīng)（實(shí)時(shí)PCR），以確定粘附相關(guān)的，CD44V6和Integrinβ1信使核糖核酸（mRNA）的表達(dá)變化。結(jié)果培養(yǎng)4小時(shí)后，干細(xì)胞在納米鈦表面比純鈦表面更快鋪展，并于16小時(shí)后更早地融合。而且，初始接種16個(gè)小時(shí)后，納米鈦表面細(xì)胞的增殖活性比純鈦表面顯著增加（P0.01），32小時(shí)后，納米鈦表面粘附的間充質(zhì)干細(xì)胞的有絲分裂率也高于純鈦表面（P0.01）。更有趣的是，接種4小時(shí)后，納米鈦表面組CD44V6和Integrinβ1的mRNA表達(dá)高于純鈦表面組（P0.01），通過(guò)統(tǒng)計(jì)分析發(fā)現(xiàn)CD44V6和Integrinβ1的mRNA表達(dá)呈正相關(guān)（rs=0.98，P0.01）。結(jié)論納米鈦表面可以顯著地促進(jìn)大鼠骨髓間充質(zhì)干細(xì)胞的粘附和增殖，提高鈦表面活性。 第二部分含納米硅鈦表面促進(jìn)大鼠骨髓間充質(zhì)干細(xì)胞成骨分化的研究 [摘要]目的探討含納米硅（Si）鈦表面對(duì)大鼠骨髓間充質(zhì)干細(xì)胞（MSCs）成骨分化的影響及初步探討可能的機(jī)制。方法第3代骨髓間充質(zhì)干細(xì)胞分別培養(yǎng)于純鈦表面和含納米硅鈦表面12天。Cell Counting Kit-8試劑盒檢測(cè)細(xì)胞數(shù)，熒光顯微鏡和Western印跡檢測(cè)細(xì)胞增殖和向成骨細(xì)胞分化的細(xì)胞活力。茜素紅S染色和定量測(cè)定分析含納米硅鈦表面對(duì)間充質(zhì)干細(xì)胞的鈣鹽礦化沉積的影響。結(jié)果含納米硅鈦表面培養(yǎng)的間充質(zhì)干細(xì)胞的增殖較純鈦表面強(qiáng)（P0.05）。納米硅鈦表面的間充質(zhì)干細(xì)胞的堿性磷酸酶分泌，I型膠原和骨鈣素表達(dá)和鈣鹽礦化沉積量均較純鈦表面增多（P0.05）。結(jié)論含納米硅鈦表面能促進(jìn)大鼠骨髓間充質(zhì)干細(xì)胞成骨分化，推測(cè)采用納米硅涂層技術(shù)對(duì)醫(yī)用鈦假體表面進(jìn)行改性，有利于促進(jìn)骨整合。 第三部分大鼠人工膝關(guān)節(jié)置換模型的建立 [摘要]目的設(shè)計(jì)并制作一個(gè)鈦金屬非骨水泥型大鼠膝關(guān)節(jié)置換手術(shù)，探討建立大鼠人工膝關(guān)節(jié)置換模型的可行性。方法將21只體重300~325克的SD大鼠隨機(jī)分為關(guān)節(jié)置換組、假手術(shù)組和無(wú)功能組，每組7只。每組動(dòng)物均行手術(shù)處理。關(guān)節(jié)置換組大鼠行股骨髁間植入鈦螺絲釘假體后分層縫合；假手術(shù)組只經(jīng)右膝髕腱內(nèi)側(cè)暴露股骨關(guān)節(jié)面后分層縫合；無(wú)功能組經(jīng)右膝髕腱內(nèi)側(cè)暴露股骨遠(yuǎn)端，切除膝半月板、前交叉韌帶、內(nèi)、外側(cè)副韌帶，切斷髕腱后分層縫合。然后評(píng)估術(shù)后4周內(nèi)大鼠疼痛相關(guān)的飲食和行走站立行為，檢查右膝關(guān)節(jié)活動(dòng)度，X線片檢查膝關(guān)節(jié)病變情況。所有數(shù)值用平均值±標(biāo)準(zhǔn)差表示。采用SPSS12.0軟件包對(duì)各組實(shí)驗(yàn)數(shù)據(jù)進(jìn)行t檢驗(yàn)或方差分析并進(jìn)行多重比較。P0.05表示差異有統(tǒng)計(jì)學(xué)意義。結(jié)果術(shù)后4周內(nèi)關(guān)節(jié)置換組與假手術(shù)組之間的飲食量和行走站立指數(shù)差異無(wú)統(tǒng)計(jì)學(xué)意義，于術(shù)后1周開(kāi)始這兩組站立指數(shù)優(yōu)于無(wú)功能組（P0.05）。術(shù)后各組膝關(guān)節(jié)被動(dòng)活動(dòng)度均良好，各組之間差異無(wú)統(tǒng)計(jì)學(xué)意義。X線片顯示關(guān)節(jié)置換組假體位置良好，周圍無(wú)骨溶解現(xiàn)象。結(jié)論本實(shí)驗(yàn)設(shè)計(jì)的大鼠人工膝關(guān)節(jié)置換模型操作簡(jiǎn)單，術(shù)后活動(dòng)功能好，，可用于模擬臨床人工膝關(guān)節(jié)置換術(shù)的實(shí)驗(yàn)研究。 第四部分含納米硅鈦假體在大鼠膝關(guān)節(jié)置換模型中的機(jī)械穩(wěn)定性和骨整合的研究 [摘要]目的探討鈦和含納米硅鈦假體材料，植入到大鼠膝關(guān)節(jié)置換模型中，承重下的機(jī)械穩(wěn)定性和骨整合情況。方法分別將鈦和含納米硅鈦假體植入大鼠膝關(guān)節(jié)中，術(shù)后第3天開(kāi)始每周給術(shù)側(cè)膝關(guān)節(jié)腔內(nèi)注射超高分子量聚乙烯(UHMW-PE)微顆粒，1個(gè)月后處死動(dòng)物。術(shù)后每周在X線片上觀察假體位置和周圍骨質(zhì)變化情況。用拔釘試驗(yàn)評(píng)估假體機(jī)械穩(wěn)定性。Micro-CT(微型CT)掃描重建股骨遠(yuǎn)端骨質(zhì)。顯微組織病理學(xué)檢測(cè)假體周圍骨、軟骨生長(zhǎng)變化和無(wú)菌性炎癥反應(yīng)。采用SPSS12.0軟件包對(duì)各組實(shí)驗(yàn)數(shù)據(jù)進(jìn)行t檢驗(yàn)或方差分析并進(jìn)行多重比較，P0.05表示差異有統(tǒng)計(jì)學(xué)意義。結(jié)果鈦和含納米硅鈦假體均無(wú)下沉，兩組假體均機(jī)械穩(wěn)定，無(wú)松動(dòng)。但是，含納米硅組假體骨界面的骨整合增強(qiáng)，螺絲釘最大抗拔出力較鈦骨溶解誘導(dǎo)組增大（P0.05）。含納米硅組假體周圍新骨形成較鈦骨溶解誘導(dǎo)組增多，假體周圍成骨細(xì)胞增殖增多，無(wú)明顯炎癥細(xì)胞浸潤(rùn)。結(jié)論大鼠膝關(guān)節(jié)置換模型中，含納米硅鈦表面比鈦表面更有利于骨整合，可減輕聚乙烯微顆粒誘導(dǎo)的無(wú)菌性炎癥反應(yīng)。當(dāng)利用生物型假體置換關(guān)節(jié)時(shí)，本文為推薦使用含納米硅表面改性的鈦金屬假體打下基礎(chǔ)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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