【摘要】：由于炎癥、外傷與腫瘤等引起的骨缺損是臨床常見的疾病。傳統(tǒng)的自體/異體骨移植由于來源短缺等原因,在臨床的應(yīng)用受到很大限制。隨著材料科學(xué)的不斷進(jìn)步和發(fā)展,人工合成骨修復(fù)體成為解決臨床使用骨替代物的最佳方法。在人工合成骨修復(fù)體的材料中,β-磷酸三鈣(β-TCP)由于與人體骨骼相似無機(jī)成份和可生物降解性成為較為理想的材料。在結(jié)構(gòu)方面,骨修復(fù)要求修復(fù)體材料具有多孔的結(jié)構(gòu)便于細(xì)胞和血管的長(zhǎng)入。為進(jìn)一步提高β-TCP修復(fù)體材料的成骨誘導(dǎo)能力,促進(jìn)骨組織再生修復(fù),本研究基于3D打印制備的β-TCP多孔修復(fù)體,采用物理包被、負(fù)載和細(xì)胞工程技術(shù)方法對(duì)修復(fù)體進(jìn)行功能化改性。主要研究?jī)?nèi)容和結(jié)果如下:(一)β-TCP多孔骨修復(fù)體的制備及生物安全性評(píng)價(jià)利用3D打印技術(shù)構(gòu)建孔結(jié)構(gòu)精確可控的β-TCP多孔修復(fù)體,對(duì)其進(jìn)行體內(nèi)和體外的生物安全性評(píng)價(jià)。研究發(fā)現(xiàn),在體外,β-TCP多孔修復(fù)體有利于骨髓間充質(zhì)干細(xì)胞(mBMSCs)的粘附與增殖;在體內(nèi),β-TCP多孔修復(fù)體生物相容性好,不刺激結(jié)締組織增生,降解產(chǎn)物不引起主要臟器的毒性反應(yīng),具有良好的生物相容性和生物安全性。(二)Ⅰ型膠原納米纖維功能化β-TCP多孔修復(fù)體通過物理包被的方法在β-TCP多孔修復(fù)體表面構(gòu)建重組Ⅰ型膠原納米纖維。研究發(fā)現(xiàn)β-TCP多孔修復(fù)體表面的Ⅰ型膠原納米纖維能促進(jìn)mBMSCs的增殖與成骨分化,同時(shí),刺激mBMSCs基質(zhì)小泡的分泌,促進(jìn)基質(zhì)礦化。(三)Ⅰ型膠原納米纖維負(fù)載生長(zhǎng)因子PIGF功能化β-TCP多孔修復(fù)體采用Ⅰ型膠原納米纖維負(fù)載生長(zhǎng)因子PIGF修飾β-TCP多孔修復(fù)體表面,實(shí)現(xiàn)PIGF的有效緩釋。研究發(fā)現(xiàn)PIGF的負(fù)載能促進(jìn)mBMSC細(xì)胞增值及成血管相關(guān)生長(zhǎng)因子的分泌,更重要的是,PIGF還能刺激mBMSC細(xì)胞成骨相關(guān)基因的表達(dá),誘導(dǎo)其成骨分化。因此,該修復(fù)體具有同時(shí)促成血管及促成骨的雙重誘導(dǎo)調(diào)控作用。(四)轉(zhuǎn)基因pigf-tk-CHO細(xì)胞功能化β-TCP多孔修復(fù)體構(gòu)建基于四環(huán)素誘導(dǎo)表達(dá)系統(tǒng)(Tet-on induce expression system)的pLVX-Tet-On-Tight-myc-PIGF-PGK-Puro質(zhì)粒和基于更昔洛韋誘導(dǎo)自殺系統(tǒng)(GCV/HSV-tk suicide gene system)的pIRES2-ZsGreen1-TK質(zhì)粒,同時(shí)轉(zhuǎn)染到CHO細(xì)胞中。經(jīng)過轉(zhuǎn)基因CHO修飾的β-TCP多孔修復(fù)體,可實(shí)現(xiàn)誘導(dǎo)表達(dá)PIGF生長(zhǎng)因子和細(xì)胞凋亡。這種“智能雙安全調(diào)控表達(dá)系統(tǒng)”骨修復(fù)體提高了轉(zhuǎn)基因治療骨修復(fù)的生物安全性和有效性,是一種理想的基于基因工程對(duì)骨修復(fù)體功能化方法。
[Abstract]:Bone defect caused by inflammation, trauma and tumor is a common clinical disease. Traditional autologous / allogeneic bone transplantation is limited in clinical application due to shortage of sources. With the progress and development of material science, artificial bone prosthesis has become the best method to solve the clinical use of bone substitutes. Among the synthetic bone restoration materials, 尾 -TCP (尾 -TCP) is an ideal material due to its inorganic composition and biodegradability similar to human bone. In terms of structure, bone repair requires porous structures to facilitate the growth of cells and blood vessels. In order to further improve the osteogenic induction ability of 尾 -TCP restorative material and promote the regeneration of bone tissue, a 尾 -TCP porous prosthesis based on 3D printing was prepared with a physical coating. The functional modification of the prosthesis was carried out by loading and cell engineering techniques. The main contents and results are as follows: (1) preparation and biosafety evaluation of 尾 -TCP porous bone prosthesis. 3D printing technique was used to construct 尾 -TCP porous prosthesis with precise and controllable pore structure and to evaluate its biosafety in vivo and in vitro. It was found that 尾 -TCP porous repair was beneficial to the adhesion and proliferation of bone marrow mesenchymal stem cells (mBMSCs) in vitro, and that 尾 -TCP porous repair had good biocompatibility, did not stimulate connective tissue proliferation, and the degradation products did not cause toxic reactions in main organs. It has good biocompatibility and safety. (2) Recombinant type 鈪，

本文編號(hào)：2203540