【摘要】：由于腫瘤、外傷、炎癥、畸形等因素所造成骨破壞的治療一直是口腔醫(yī)學(xué)領(lǐng)域的難題，利用組織工程技術(shù)構(gòu)建人工骨進(jìn)行骨缺損的治療是目前主要的研究方向之一，而骨組織工程存在的主要問(wèn)題是所構(gòu)建的人工骨成骨能力有限。通過(guò)建立功能性微血管環(huán)境可以滿(mǎn)足種子細(xì)胞新陳代謝需求，進(jìn)一步促進(jìn)新骨形成以及參與細(xì)胞間的相互作用。由于血管在骨形成中具有重要作用，使得血管內(nèi)皮細(xì)胞（endothelial cells，ECs）在骨組織工程中應(yīng)用被廣泛關(guān)注，但其來(lái)源有限，且不易擴(kuò)增。骨髓基質(zhì)干細(xì)胞(bone mesenchymal stemcells，BMSCs)在骨髓中大量存在，容易獲取，且有向血管內(nèi)皮細(xì)胞等多向分化的潛能。因此，我們首先分離了兔的BMSCs作為種子細(xì)胞，經(jīng)內(nèi)皮細(xì)胞條件培養(yǎng)液中誘導(dǎo)培養(yǎng)，利用內(nèi)皮細(xì)胞表面標(biāo)記物CD34免疫組化染色證明其誘導(dǎo)的細(xì)胞為血管內(nèi)皮細(xì)胞。體外以單純BMSCs為對(duì)照組，ECs和BMSCs共培養(yǎng)為實(shí)驗(yàn)組，通過(guò)堿性磷酸酶活性檢測(cè)、考馬斯亮藍(lán)總蛋白測(cè)定、堿性磷酸酶和茜素紅染色，結(jié)果表明，ECs對(duì)BMSCs向成骨細(xì)胞分化具有促進(jìn)作用。將共培養(yǎng)的種子細(xì)胞接種在PLGA支架材料上，植入直徑為1cm的兔下頜骨缺損模型中，通過(guò)軟X線(xiàn)和組織化學(xué)染色分別對(duì)4周和8周的實(shí)驗(yàn)動(dòng)物進(jìn)行觀察，，結(jié)果顯示BMSCs可以在PLGA支架材料上生長(zhǎng)且材料具有較好的生物相容性且無(wú)細(xì)胞毒性，誘導(dǎo)的ECs與BMSCs共培養(yǎng)在PLGA支架材料上，同BMSCs對(duì)照組比具有明顯的促成骨作用及骨缺損修復(fù)能力。結(jié)果提示血管化組織工程骨有望成為修復(fù)大面積骨缺損的有效的途徑。 磷酸鈣是天然骨的主要組成成分，作為骨替代物、人工骨和組織工程的支架等具有較好的應(yīng)用前景。不同結(jié)構(gòu)的磷酸鈣中，羥基磷灰石（HA）和磷酸三鈣（TCP）由于對(duì)人體生理環(huán)境具有較強(qiáng)的生物學(xué)反應(yīng)而受到廣泛的關(guān)注。由于HA的化學(xué)結(jié)構(gòu)穩(wěn)定使其具有較強(qiáng)的抗吸收作用，但其密度高、脆性大、彈性差等原因限制了其廣泛應(yīng)用。TCP按結(jié)構(gòu)分為高溫相(α-TCP)和低溫相(β-TCP)，其中α-TCP相對(duì)于β-TCP具有更好的生物降解性和生物相容性，并且植入人體后反應(yīng)溫和，基本不產(chǎn)熱，降解后被生物體迅速吸收和取代，因此具有良好的生物醫(yī)學(xué)應(yīng)用前景。本研究通過(guò)共沉淀法合成了α-TCP，采用XRD、SEM、TEM等方法評(píng)價(jià)了其物理學(xué)及化學(xué)性能。通過(guò)與大鼠BMSCs共培養(yǎng)，檢測(cè)MTT以評(píng)價(jià)材料的生物安全性，通過(guò)Real Time PCR檢測(cè)ALPase、SP7、RUNX2、COL-Ⅰ等成骨細(xì)胞相關(guān)因子mRNA的表達(dá)，探討α-TCP在體外對(duì)BMSCs成骨作用的影響。結(jié)果表明，α-TCP能夠上調(diào)大鼠BMSCs向成骨細(xì)胞分化相關(guān)基因的表達(dá)，證明α-TCP可以促進(jìn)BMSCs向成骨細(xì)胞分化的作用，這就為其在骨組織工程中的應(yīng)用提供了一定的理論依據(jù)。
[Abstract]:The treatment of bone destruction caused by tumor, trauma, inflammation and deformity has always been a difficult problem in the field of stomatology. It is one of the main research directions to use tissue engineering technology to construct artificial bone to treat bone defect. The main problem in bone tissue engineering is the limited osteogenic capacity of the artificial bone. The establishment of functional microvascular environment can meet the metabolic needs of seed cells, further promote the formation of new bone and participate in intercellular interaction. Vascular endothelial cells (endothelial cells,ECs) have been widely used in bone tissue engineering due to the important role of blood vessels in bone formation. Bone marrow stromal cells (bone mesenchymal stemcells,BMSCs) are abundant in bone marrow, easy to obtain, and have the potential to differentiate into vascular endothelial cells. Therefore, we first isolated rabbit BMSCs as seed cells and cultured in conditioned medium of endothelial cells. The endothelial cells were identified as vascular endothelial cells by CD34 immunohistochemical staining. In vitro, BMSCs was used as control group, ECs and BMSCs co-cultured as experimental group, the activity of alkaline phosphatase, total protein of Coomassie brilliant blue, alkaline phosphatase and alizarin red staining were determined. ECs can promote the differentiation of BMSCs into osteoblasts. The co-cultured seed cells were seeded on PLGA scaffold and implanted into rabbit mandibular defect model with diameter of 1cm. The experimental animals were observed at 4 and 8 weeks by soft X-ray and histochemical staining, respectively. The results showed that BMSCs could grow on PLGA scaffold and the material had good biocompatibility and no cytotoxicity. The induced ECs and BMSCs co-cultured on PLGA scaffold. Compared with the control group of BMSCs, it had obvious effect of promoting bone and repairing bone defect. The results suggest that vascularized tissue engineered bone may be an effective way to repair large bone defects. Calcium phosphate is the main component of natural bone. As a substitute for bone, artificial bone and tissue engineering scaffold have a good application prospect. Hydroxyapatite (HA) and tricalcium phosphate (TCP) have attracted wide attention due to their strong biological responses to human physiological environment. HA has strong absorption resistance due to its stable chemical structure, but its high density, high brittleness and poor elasticity limit its wide application. TCP is divided into high temperature phase (偽-TCP) and low temperature phase (尾-TCP),) according to its structure. 偽-TCP has better biodegradability and biocompatibility than 尾-TCP, and the reaction is mild after implantation, and it is absorbed and replaced quickly by organism after degradation, so it has a good prospect of biomedical application. In this study, 偽-TCP, was synthesized by coprecipitation method and its physical and chemical properties were evaluated by XRD,SEM,TEM et al. The effects of 偽 -TCP on BMSCs osteogenesis in vitro were investigated by co-culture with rat BMSCs to evaluate the biological safety of the material, and the expression of mRNA, a factor related to osteoblasts such as ALPase,SP7,RUNX2,COL- 鈪

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