CPC与BMSCs-海藻酸钠的共培养研究

发布时间：2018-02-28 16:35

  本文关键词： 海藻酸钠 磷酸钙骨水泥 山羊骨髓间充质干细胞　出处：《广州医学院》2012年硕士论文　论文类型：学位论文

【摘要】：背景：经皮椎体微创手术，在治疗骨质疏松性椎体压缩性骨折（OsteoporoticVertebral Compressive Fractures,OVCF）方面取得令人鼓舞的成就，椎体后凸成形术(Percutaneous Kyphoplasty，PKP)作为一种微创OVCFs的一种微创新技术，不仅可以有效缓解甚至能消除疼痛，而且还可以恢复椎体高度和改善脊柱后突畸形。但该技术在推广的同时也出现了一系列并发症，主要是目前最常用的充填材料聚甲基丙烯酸甲酯(Polymethyacrylate，，PMMA)骨水泥存在诸多不足：黏滞度低，易渗漏；聚合温度高、易损伤周围组织；无生物活性；不能降解、成为永久异物；无骨诱导性和骨传导性；聚合时单体有毒性.有研究发现磷酸钙骨水泥（CPC）固化后多孔结构的许多孔径超过了100μm，这对细胞长入、工程化组织在体内的物质交换以及骨的再生都是有利的。磷酸钙矿物质为细胞粘附和成骨细胞表型表达提供了更合适的基质。CPC具有极好的骨传导性，生物可吸收性而且可被新生骨替代。钙盐（CPC）的植入可明显提高成骨细胞的成骨能力，有利于新骨的生成。Lee SK,等认为CPC虽然会降低细胞的增殖能力，但其增加了碱性磷酸酶的水平，从而增强了矿化结节的形成，并上调了骨桥蛋白、骨涎蛋白、细胞外基质磷酸糖的信使RNA水平。然而CPC凝固反应的pH值变化和离子活性可能对直接位于CPC表面上的hBMSCs有不利影响。而几种不影响细胞活性的可注射水凝胶聚合载体又都不能应用于承重部分的骨修复。例如曾有结论指出：水凝胶支架不具备机械强度，不能应用于承受负荷较大的部位.承受压力而避免支架破裂以及维持组织结构使重建组织外形符合要求是至关重要的。找到一种方法保护细胞免受CPC的影响变得越来越迫切。海藻酸盐是从海藻中提取的天然多糖，因为它用于封装和保护细胞且无细胞毒性，能够在一个温和的条件下形成交联凝胶。本实验将为新型人工骨材料的应用提供前期的实验基础 目的： 1、观察包裹在海藻酸钠微球中的骨髓间充质干细胞在有磷酸钙骨水泥的环境中的成活及成骨情况。 2、对比包裹与未包裹的骨髓间充质干细胞在有磷酸钙骨水泥的环境中的成活情况证明海藻酸钠微球对细胞的保护作用。 方法： 1、无菌条件下从山羊髂骨处骨穿收集肝素抗凝骨髓，采用密度梯度离心或贴壁法分离培养出骨髓间充质干细胞。将0.3g海藻酸钠加入25ml155mmmol/l的氯化钠中制备1.2%的海藻酸钠溶液。细胞封装入海藻酸钠中，密度为1x10的6次方/ml。通过将细胞和海藻酸钠混合物通过25号针头注入含3ml100mmol/l氯化钠溶液的12孔板中促进交联，形成细胞-海藻酸钠微球。将微球置于培养板中，上层覆盖磷酸钙骨水泥，加培养基没过骨水泥，定期换液。分别于7,14,21天后移去细胞培养液，将hBMSC-海藻酸盐珠取出，用PBS冲洗。live/dead试剂盒染色、DAPI染色，普通光学显微镜和倒置荧光显微镜下观察细胞成活情况。 2、制成细胞-海藻酸钠微球后置于培养板中，上层覆盖磷酸钙骨水泥，加成骨诱导培养基没过骨水泥，定期换液。分别于7,14,21天后二甲酚橙染色，普通显微镜及倒置荧光显微镜下观察细胞成骨情况。 结果： 1．21天后live/dead试剂盒染色，倒置荧光显微镜下课件绿色荧光，且DAPI染色见相同位置出现蓝色荧光。 2．7,14天时二甲酚橙染色，荧光显微镜下未见明显染色痕迹，21天时见部分区域出现红色荧光 结论： 1、DAPI染色DNA物质出现蓝色荧光，live/dead试剂盒染色后活细胞产生绿色荧光，二者发出荧光区域重合相互印证。说明海藻酸钠微球中确有活细胞存在。 2、在7,14天时，微球中的存活细胞在向成骨细胞转化时还未出现矿物颗粒，21天时已有部分区域可见矿物颗粒，说明骨髓间充质干细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锛

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