【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,在臨床的應用受到很大限制。隨著材料科學的不斷進步和發(fā)展,人工合成骨修復體成為解決臨床使用骨替代物的最佳方法。在人工合成骨修復體的材料中,β-磷酸三鈣(β-TCP)由于與人體骨骼相似無機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,骨修復要求修復體材料具有多孔的結(jié)構(gòu)便于細胞和血管的長入。為進一步提高β-TCP修復體材料的成骨誘導能力,促進骨組織再生修復,本研究基于3D打印制備的β-TCP多孔修復體,采用物理包被、負載和細胞工程技術(shù)方法對修復體進行功能化改性。主要研究內(nèi)容和結(jié)果如下:(一)β-TCP多孔骨修復體的制備及生物安全性評價利用3D打印技術(shù)構(gòu)建孔結(jié)構(gòu)精確可控的β-TCP多孔修復體,對其進行體內(nèi)和體外的生物安全性評價。研究發(fā)現(xiàn),在體外,β-TCP多孔修復體有利于骨髓間充質(zhì)干細胞(mBMSCs)的粘附與增殖;在體內(nèi),β-TCP多孔修復體生物相容性好,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,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多孔修復體通過物理包被的方法在β-TCP多孔修復體表面構(gòu)建重組Ⅰ型膠原納米纖維。研究發(fā)現(xiàn)β-TCP多孔修復體表面的Ⅰ型膠原納米纖維能促進mBMSCs的增殖與成骨分化,同時,刺激mBMSCs基質(zhì)小泡的分泌,促進基質(zhì)礦化。(三)Ⅰ型膠原納米纖維負載生長因子PIGF功能化β-TCP多孔修復體采用Ⅰ型膠原納米纖維負載生長因子PIGF修飾β-TCP多孔修復體表面,實現(xiàn)PIGF的有效緩釋。研究發(fā)現(xiàn)PIGF的負載能促進mBMSC細胞增值及成血管相關(guān)生長因子的分泌,更重要的是,PIGF還能刺激mBMSC細胞成骨相關(guān)基因的表達,誘導其成骨分化。因此,該修復體具有同時促成血管及促成骨的雙重誘導調(diào)控作用。(四)轉(zhuǎn)基因pigf-tk-CHO細胞功能化β-TCP多孔修復體構(gòu)建基于四環(huán)素誘導表達系統(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自殺系統(tǒng)(GCV/HSV-tk suicide gene system)的pIRES2-ZsGreen1-TK質(zhì)粒,同時轉(zhuǎn)染到CHO細胞中。經(jīng)過轉(zhuǎn)基因CHO修飾的β-TCP多孔修復體,可實現(xiàn)誘導表達PIGF生長因子和細胞凋亡。這種“智能雙安全調(diào)控表達系統(tǒng)”骨修復體提高了轉(zhuǎn)基因治療骨修復的生物安全性和有效性,是一種理想的基于基因工程對骨修復體功能化方法。
[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：2203540