本文關(guān)鍵詞： 海藻酸鈉 磷酸鈣骨水泥 山羊骨髓間充質(zhì)干細(xì)胞　出處：《廣州醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：背景：經(jīng)皮椎體微創(chuàng)手術(shù)，在治療骨質(zhì)疏松性椎體壓縮性骨折（OsteoporoticVertebral Compressive Fractures,OVCF）方面取得令人鼓舞的成就，椎體后凸成形術(shù)(Percutaneous Kyphoplasty，PKP)作為一種微創(chuàng)OVCFs的一種微創(chuàng)新技術(shù)，不僅可以有效緩解甚至能消除疼痛，而且還可以恢復(fù)椎體高度和改善脊柱后突畸形。但該技術(shù)在推廣的同時(shí)也出現(xiàn)了一系列并發(fā)癥，主要是目前最常用的充填材料聚甲基丙烯酸甲酯(Polymethyacrylate，，PMMA)骨水泥存在諸多不足：黏滯度低，易滲漏；聚合溫度高、易損傷周圍組織；無生物活性；不能降解、成為永久異物；無骨誘導(dǎo)性和骨傳導(dǎo)性；聚合時(shí)單體有毒性.有研究發(fā)現(xiàn)磷酸鈣骨水泥（CPC）固化后多孔結(jié)構(gòu)的許多孔徑超過了100μm，這對細(xì)胞長入、工程化組織在體內(nèi)的物質(zhì)交換以及骨的再生都是有利的。磷酸鈣礦物質(zhì)為細(xì)胞粘附和成骨細(xì)胞表型表達(dá)提供了更合適的基質(zhì)。CPC具有極好的骨傳導(dǎo)性，生物可吸收性而且可被新生骨替代。鈣鹽（CPC）的植入可明顯提高成骨細(xì)胞的成骨能力，有利于新骨的生成。Lee SK,等認(rèn)為CPC雖然會(huì)降低細(xì)胞的增殖能力，但其增加了堿性磷酸酶的水平，從而增強(qiáng)了礦化結(jié)節(jié)的形成，并上調(diào)了骨橋蛋白、骨涎蛋白、細(xì)胞外基質(zhì)磷酸糖的信使RNA水平。然而CPC凝固反應(yīng)的pH值變化和離子活性可能對直接位于CPC表面上的hBMSCs有不利影響。而幾種不影響細(xì)胞活性的可注射水凝膠聚合載體又都不能應(yīng)用于承重部分的骨修復(fù)。例如曾有結(jié)論指出：水凝膠支架不具備機(jī)械強(qiáng)度，不能應(yīng)用于承受負(fù)荷較大的部位.承受壓力而避免支架破裂以及維持組織結(jié)構(gòu)使重建組織外形符合要求是至關(guān)重要的。找到一種方法保護(hù)細(xì)胞免受CPC的影響變得越來越迫切。海藻酸鹽是從海藻中提取的天然多糖，因?yàn)樗糜诜庋b和保護(hù)細(xì)胞且無細(xì)胞毒性，能夠在一個(gè)溫和的條件下形成交聯(lián)凝膠。本實(shí)驗(yàn)將為新型人工骨材料的應(yīng)用提供前期的實(shí)驗(yàn)基礎(chǔ) 目的： 1、觀察包裹在海藻酸鈉微球中的骨髓間充質(zhì)干細(xì)胞在有磷酸鈣骨水泥的環(huán)境中的成活及成骨情況。 2、對比包裹與未包裹的骨髓間充質(zhì)干細(xì)胞在有磷酸鈣骨水泥的環(huán)境中的成活情況證明海藻酸鈉微球?qū)?xì)胞的保護(hù)作用。 方法： 1、無菌條件下從山羊髂骨處骨穿收集肝素抗凝骨髓，采用密度梯度離心或貼壁法分離培養(yǎng)出骨髓間充質(zhì)干細(xì)胞。將0.3g海藻酸鈉加入25ml155mmmol/l的氯化鈉中制備1.2%的海藻酸鈉溶液。細(xì)胞封裝入海藻酸鈉中，密度為1x10的6次方/ml。通過將細(xì)胞和海藻酸鈉混合物通過25號(hào)針頭注入含3ml100mmol/l氯化鈉溶液的12孔板中促進(jìn)交聯(lián)，形成細(xì)胞-海藻酸鈉微球。將微球置于培養(yǎng)板中，上層覆蓋磷酸鈣骨水泥，加培養(yǎng)基沒過骨水泥，定期換液。分別于7,14,21天后移去細(xì)胞培養(yǎng)液，將hBMSC-海藻酸鹽珠取出，用PBS沖洗。live/dead試劑盒染色、DAPI染色，普通光學(xué)顯微鏡和倒置熒光顯微鏡下觀察細(xì)胞成活情況。 2、制成細(xì)胞-海藻酸鈉微球后置于培養(yǎng)板中，上層覆蓋磷酸鈣骨水泥，加成骨誘導(dǎo)培養(yǎng)基沒過骨水泥，定期換液。分別于7,14,21天后二甲酚橙染色，普通顯微鏡及倒置熒光顯微鏡下觀察細(xì)胞成骨情況。 結(jié)果： 1．21天后live/dead試劑盒染色，倒置熒光顯微鏡下課件綠色熒光，且DAPI染色見相同位置出現(xiàn)藍(lán)色熒光。 2．7,14天時(shí)二甲酚橙染色，熒光顯微鏡下未見明顯染色痕跡，21天時(shí)見部分區(qū)域出現(xiàn)紅色熒光 結(jié)論： 1、DAPI染色DNA物質(zhì)出現(xiàn)藍(lán)色熒光，live/dead試劑盒染色后活細(xì)胞產(chǎn)生綠色熒光，二者發(fā)出熒光區(qū)域重合相互印證。說明海藻酸鈉微球中確有活細(xì)胞存在。 2、在7,14天時(shí)，微球中的存活細(xì)胞在向成骨細(xì)胞轉(zhuǎn)化時(shí)還未出現(xiàn)礦物顆粒，21天時(shí)已有部分區(qū)域可見礦物顆粒，說明骨髓間充質(zhì)干細(xì)胞的成骨分化功能良好。
[Abstract]:Background: percutaneous vertebroplasty and minimally invasive surgery in the treatment of osteoporotic vertebral compression fractures (OsteoporoticVertebral Compressive, Fractures, OVCF) has made encouraging achievements, kyphoplasty (Percutaneous Kyphoplasty PKP) is a minimally invasive OVCFs micro innovation technology, not only can effectively alleviate and even eliminate the pain, and can improve the recovery of the vertebral height and kyphosis. But the technology in the promotion has also appeared in a series of complications, is currently the most commonly used filling materials of polymethacrylic acid methyl ester (Polymethyacrylate, PMMA) bone cement has many disadvantages: low viscosity, easy leakage; high polymerization temperature, easy to damage the surrounding tissue; no biological activity; can not become a permanent foreign body; no degradation, osteoinduction and osteoconduction; polymerization of monomer toxicity. Studies have found that calcium phosphate Cement (CPC) after curing many porous structure more than 100 m, the cell growth, tissue engineering is advantageous in the exchange of material and bone regeneration in vivo. Calcium phosphate minerals for cell adhesion and osteogenic phenotype expression of bone conduction matrix provides excellent.CPC more appropriate, biodegradable and can be replaced by the new bone. Calcium salt (CPC) implantation can significantly improve the osteogenic ability of osteoblasts, in favor of the new bone formation in.Lee SK, such that CPC may reduce the cell proliferation ability, but the increase of alkaline phosphatase levels, thereby enhancing the formation of the mineralized nodules, and upregulation of osteopontin, bone sialoprotein, extracellular matrix phosphate sugar messenger RNA levels. However, CPC coagulation reaction pH value change and ion activity may have adverse effect on the CPC is located directly on the surface of hBMSCs. And some do not affect Cell activity of injectable hydrogel polymeric carrier and can not be applied to the bearing part of the bone repair. For example there had been concluded that the hydrogel scaffolds do not have enough mechanical strength, can not be applied to load large regions. Under pressure and avoid stent rupture and maintain organizational structure to meet the requirements of the shape reconstruction of organization is very important. Find the effect a method of protecting the cell from CPC becomes more and more urgent. Alginate is a natural polysaccharide extracted from seaweed, as it is used for encapsulation and protection of cells and cell toxicity, can form a gel in a mild condition. This experiment will provide the experimental basis for the application of the new artificial bone material
Objective:
1, the survival and osteogenesis of bone marrow mesenchymal stem cells (MSCs) wrapped in sodium alginate microspheres were observed in the environment with calcium phosphate cement.
2, comparing the survival of encapsulated and unencapsulated bone marrow mesenchymal stem cells in calcium phosphate cement environment, it is proved that alginate microspheres protect cells.
Method錛

本文編號(hào)：1548097