复合BMSCs包芯结构骨支架材料修复兔桡骨骨缺损的实验研究

发布时间：2018-04-12 00:18

本文选题：包芯结构 + Ⅰ型胶原　；参考：《第四军医大学》2012年博士论文

【摘要】：骨科临床上造成大段骨缺损的疾病非常常见，例如：严重的创伤、骨质疏松病人的骨折、肿瘤、肌肉骨骼系统的先天性畸形等。治疗上述疾病需要切除病损的骨质，必然导致骨的大段缺损，骨缺损后的重建问题成为治疗的最大挑战。近些年，骨组织工程不断取得突破性进展，逐渐成为治疗大段骨缺损最有前景的方法。骨组织工程主要包括3个主要因素，其中骨支架材料作为种子细胞和活性因子的载体，为新生骨的生成提供支撑，成为骨组织工程最关键的一个因素。合适的结构和物理性能以及良好的生物相容性是作为理想的骨支架材料两个最重要的要求：作为临时的支撑结构，骨支架材料决定新生骨最终的生成形状，，并影响复合在支架材料上的细胞间的相互作用，支架材料表面的亲水性和粗糙程度等不同，在其表面生长的细胞也会表现出不同的生物活性。在以前的研究中，通过低温成型技术，我们已经制备出具有三维结构的PLGA/β-TCP复合支架材料，而且通过体内和体外实验均已经证实PLGA/β-TCP复合支架材料具有良好的加工和成型特性、较高的空隙率、良好的机械强度和合适的降解速度，能够满足骨组织工程理想支架材料的要求。但是，PLGA/β-TCP复合支架材料表面具有强烈的疏水性，不利于细胞的粘附、增殖和成骨分化，大大影响了其作为支架材料的修复能力。支架材料具有较好的亲水性和良好的生物相容性，不但可以确保细胞在支架材料表面的粘附、增殖和分化，而且能够促进氧气和细胞必须的营养物质顺利进入支架材料，这对于骨缺损的成功修复至关重要。因此，为了使PLGA/β-TCP复合支架材料更适合细胞的粘附、增殖和分化，改变材料的表面特性非常重要。研究证实，多孔支架材料表面覆盖一层胶原以后，材料的吸水率会得到有效提高，而且有研究表明，骨髓基质干细胞如果在I型胶原上培养，其粘附、增殖和成骨分化能力也会得到显著提高。基于以上原因，我们制备出了内PLGA/β-TCP外I型胶原的包芯结构骨支架材料，此材料内层为PLGA/β-TCP复合支架材料，在其表面覆盖一层I型胶原。本实验主要包括以下几部分的研究： 1基于仿生学原理，模拟人体骨组织真实结构，结合低温沉积技术，构建仿生结构组织工程骨构建修复方案，开发可控的环形套管喷头，通过快速成型的方法，制造出所需的材料、结构、细胞分布高度仿生的复杂的三维结构骨支架-包芯结构骨支架材料。 2以PLGA/β-TCP复合支架材料作为对照组，通过体外和骨髓基质干细胞（BMSCs）共培养的观察实验，对包芯结构骨支架材料的物理性能和生物相容性进行评价。外观形态通过扫描电镜观测；物理性能通过如下指标测定：孔隙率、孔径、压缩强度和杨氏模量；亲水性通过吸水率评价；骨髓基质干细胞在支架材料上的粘附率通过细胞计数测定，增殖率通过MTT方法测定，成骨能力通过碱性磷酸酶活性的检测进行评价；细胞在支架材料上的生长情况通过扫描电镜观测；实验结果证实，包芯结构骨支架材料和对照组都有良好的物理性能，包芯结构骨支架材料的亲水性较对照组显著提高(p0.01)，细胞在包芯结构骨支架材料上的粘附、增值及成骨分化能力明显优于(p0.05)对照组。 3兔桡骨大段骨缺损修复实验：把各组支架材料和骨髓基质干细胞复合，随后植入到兔桡骨大段骨缺损模型里进行兔桡骨骨缺损修复的对比研究。通过X线、MicroCT、组织学和荧光双标等方法观察支架材料的成骨情况、降解情况和修复兔桡骨大段骨缺损的情况；实验结果证实，48周后，包芯结构骨支架材料在骨缺损部位完全降解，骨塑型完成，成功修复了兔桡骨大段骨缺损，而且支架材料的降解速度和成骨速度匹配良好，相对于PLGA/β-TCP复合支架材料，包芯结构骨支架材料表现出更好的成骨活性和修复大段骨缺损的能力。包芯结构骨支架材料具有良好的物理性能及生物相容性，在复合BMSCs的条件下，能很好的修复兔桡骨大段骨缺损，包芯结构骨支架材料作为骨组织工程理想的支架材料，具有良好的临床应用前景。
[Abstract]:The Department of orthopedics clinical cause of large bone defect diseases, such as severe trauma, patients with osteoporosis fracture, tumor, congenital malformation of the musculoskeletal system. The treatment of these diseases requires removal of bone, will inevitably lead to the large segment defect of bone, reconstruction of bone defect after treatment has become the greatest challenge in recent years, bone tissue engineering has made breakthrough progress, has gradually become a method for treatment of large bone defects. The most promising bone tissue engineering mainly includes 3 main factors, including bone scaffold material as the carrier of seed cells and active factors, to provide support for the formation of new bone, become one of the most critical factor of bone tissue engineering. Appropriate structure and physical properties and good biocompatibility as bone scaffolds are two of the most important requirements: as a temporary support structure, bone scaffold materials The final generation form of new bone is determined, and the cell interaction on composite scaffolds is affected. The hydrophilic and roughness of the scaffolds are different. The cells grown on the surface also show different biological activities.
In a previous study, through the low temperature molding technology, we have prepared the PLGA/ beta -TCP composite scaffold has a three-dimensional structure, and through in vivo and in vitro experiments have confirmed that PLGA/ beta -TCP composite scaffolds have good characteristics of processing and molding, high void rate, good mechanical strength and appropriate degradation speed, can meet the ideal bone tissue engineering scaffold material requirements. However, with the strong hydrophobic surface of PLGA/ beta -TCP composite scaffold material, is not conducive to cell adhesion, proliferation and osteogenic differentiation, greatly affects the ability of the repair scaffold. Scaffold has good compatibility and good hydrophilicity life, not only can ensure the cells in the scaffold surface adhesion, proliferation and differentiation, but also can promote the oxygen and nutrients necessary to successfully enter the cell scaffold material for bone defect, this Successful repair is critical. Therefore, in order to make PLGA/ beta -TCP composite scaffolds more suitable for cell adhesion, proliferation and differentiation, it is very important to change the surface properties of materials.
The research confirmed that after surface covered with a layer of collagen scaffold material, the water absorption rate can be effectively improved, and studies have shown that bone marrow stromal stem cells cultured in collagen type I if, on the adhesion, proliferation and osteogenic differentiation ability can be improved. Based on the above reasons, the core structure of bone scaffold materials we prepared the PLGA/ beta -TCP type I collagen, the inner layer of PLGA/ beta -TCP composite scaffold, on its surface covered with a layer of type I collagen. This study mainly includes the following parts of the research:
1 based on the principle of bionics, simulate the structure of human bone tissue, combined with low temperature deposition technology, biomimetic structure construction of tissue engineered bone repair scheme, a ring sleeve nozzle developed controllable, through the method of rapid prototyping and manufacturing the required materials, structure, three-dimensional structure of bone scaffold complex cells distributed highly bionic core the structure of bone scaffold materials.
2 to PLGA/ beta -TCP composite scaffolds as control group by in vitro and bone marrow stromal stem cells (BMSCs) were co cultured, the core structure of bone scaffold material physical properties and biocompatibility were evaluated. Morphology was observed by scanning electron microscope; physical properties through the following indexes: the porosity, pore size. The compression strength and modulus; hydrophilic by water absorption rate; bone marrow stromal cell counts were determined by cell adhesion on scaffolds was determined by the MTT method, the proliferation rate, evaluate the osteogenic ability by alkaline phosphatase activity assay; cell growth in the scaffold by SEM observation; experimental results confirmed that the core structure of bone scaffold materials and the control group have good physical properties, hydrophilic core structure of bone scaffold was significantly higher than the control group (P0.01), fine The adhesion, value added and osteogenic differentiation of the cell on the bone scaffold material of the core structure were obviously better than that of the control group (P0.05).
3 segmental bone defect repair experiment: the group of scaffold materials and bone marrow stem cells combined, then implanted into the segmental bone defect model in the comparative study of repairing bone defect of rabbit radius. By X-ray, MicroCT, augementation histological and fluorescent double labeling methods such as observation of scaffold, degradation and in repairing segmental bone defect; experimental results show that, after 48 weeks, completely degraded in the bone defect site core structure of bone scaffold material package, bone modeling completed, the repair of segmental bone defect, and the degradation rate of scaffold materials and bone formation rate, good, relative to the PLGA/ beta -TCP composite scaffolds, core structure scaffolds exhibit better osteogenic activity and repair of large segmental bone defects.
The core structure bone scaffold material has good physical and biocompatibility. Under the condition of compound BMSCs, it can repair the large radius bone defect of rabbit well. The core structure bone scaffold material is an ideal scaffold material for bone tissue engineering. It has good clinical application prospect.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

【参考文献】