孔隙结构互补的磷灰石多孔支架的研究
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摘要
由创伤或疾病所引起的超过临界尺寸的骨缺损完全依靠自身的修复是非常困难的,需要在骨缺损的部位植入一个连接材料以加快修复。目前常用的大段骨缺损修复手段主要有：自体移植、异体移植和人工合成材料以及骨组织工程支架。但自体移植材料的来源有限,在取材部位也造成了骨缺损,而且患者要承受二次手术的痛苦；异体移植往往伴随着严重的感染和炎症反应。因而,组织工程骨的发展也就成为了必然。骨组织工程中多孔支架的作用是为新骨形成提供一个基质,应满足以下几个基本的要求：较高的孔隙率,合适的孔隙大小,三维贯通的孔隙结构,有利于细胞粘附生长的表面特性、一定的力学性能以维持支架的预制形态,无细胞毒性等。多孔支架大孔的作用主要是为营养供应、气体扩散及代谢废物排出提供通道。为了在体外构建组织工程骨,需要将细胞接种到合适的三维多孔支架上,实现细胞的体外扩增,这就需要种子细胞的体外三维培养。作为细胞体外三维培养的第一步,将种子细胞均匀的接种到支架内部可以有效地促进骨重建的发生。在构建骨组织工程支架的材料中,羟基磷灰石的组成和结构都与自然骨极为相似,并具有很好的骨传导性和骨诱导性,因而被认为是构建骨组织工程支架的理想材料。
实验中以羟基磷灰石为原料,用溶胶-凝胶法制备了HA球状颗粒,将其堆积到多孔管中后组成HA颗粒堆积支架,并对支架的孔隙结构进行了表征；设计了三种不同的成骨细胞接种方法：直接法、混合法和转移法,考察了接种方法不同对成骨细胞生物学特性的影响。用相同的HA浆料添加石蜡球作为造孔剂后制备了颗粒造孔支架,并通过对石蜡球用二甲苯进行处理以改善多孔支架的孔隙贯通性,考察了孔隙贯通性对成骨细胞生物学特性的影响。对孔隙结构互补的这两种HA多孔支架进行体内和体外生物学评价,以考察支架大孔形状对成骨细胞生物学性能的影响和对体内异位成骨的影响。本实验获得的主要结论如下：
1.HA颗粒堆积多孔支架的大孔孔隙完全贯通,并且孔径大小可通过HA颗粒的大小控制,颗粒表面和内部均匀分布着大量微孔；HA颗粒堆积支架具有良好的生物相容性；三种细胞接种方法中,混合法和转移法接种可以使细胞在颗粒堆积支架内部均匀分散,而且转移法可以将更多的细胞接种到颗粒表面,具有更高的接种效率；不同的细胞接种方法对成骨细胞的增殖和分化没有明显影响。
2.通过二甲苯处理用作造孔剂的石蜡球可明显改善支架孔隙的贯通性,并同时提高支架的孔隙率,而支架的抗压强度有所降低；多孔支架的收缩率取决于所配制的HA浆料,与孔隙率无关；支架表面均匀分布着微米和亚微米级的微孔和粗糙结构；HA多孔支架具有良好的细胞相容性,并且成骨细胞可进入多孔支架内部正常生长；贯通性好的支架可以为细胞增殖提供更充分的空间。
3.两种不同的孔隙结构对体外成骨细胞在支架内部粘附和生长的影响没有明显区别；两种支架在动物体内非骨部位都可以很好的诱导成骨发生,并且所生成的新骨沿着孔隙的边缘正常生长,颗粒造孔支架的孔隙结构更有利于体内异位成骨的发生及血管化的产生。
Critical size bone defects due to trauma or disease are very difficult to repair via the natural growth of host tissue. Therefore, there exists a nccessity to replace these defects with bridging materials. Multiple approaches have been attempted by using autografts, allografts, artificial materials and bone tissue engineering scaffolds. The major problem with autografts is the insufficient supply and significant surgical morbidity associated with donor site pain and loss of function. Moreover, patients have to suffer a second procedure. Allografts are associated with infection and inflammation. Therefore, engineered bone tissue has been developed recently. In bone tissue engineering, scaffold served as the matrices of tissue formation plays a pivotal role, and has to fulfill a few basic requirements, that is, high porosity, pore interconnection, proper pore size, required surface properties permitting cell adhesion, and proliferation, desirable mechanical integrity to maintain the predesigned tissue structure, non-cytotoxicity. Large pores in the scaffold can allow effective nutrient supply, gas diffusion and metabolic waste removal. To engineer synthetic bone tissue, cells should be seeded into an appropriate three-dimensional (3D) porous scaffold matrix, and expand the cells in vitro. Recently,3D cultures have been widely adopted for tissue engineering to obtain satisfying artificial bone grafts. Cell seeding is the first step in establishing 3D cultures and achieving the seeding of cells with a uniform distribution throughout the porous scaffold could establish a basis for homogeneous tissue generation. Hydroxyapatite (HA) has a composition and structure very close to natural bone mineral and therefore has been considered to be the ideal material to build bone tissue engineering scaffold due to its osteoconductivity and osteoinductivity.
HA spheres were prepared by the method of sol-gel and water/oil emulsification technology, then accumulated in a porous tube as a porous scaffold. The porosity, macro and micro porous structures of the scaffold were characterized. Three cell seeding methods were designed to distribute osteoblasts into HA spherule scaffolds:direct load, admixture load and transferring load in vitro. The effects on biological property of osteoblasts seeded in the porous scaffold by the three cell seeding methods were investigated. Wax spheres were prepared as porogen to prepare HA porous scaffolds with the same HA slurry, and the wax spheres were treated with dimethyl benzene to improve the interconection of the HA scaffold pores. The effects of interconection on biological property of osteoblasts seeded in the porous scaffold were investigated. The two kinds of HA porous scaffolds with complementary porous structures were evaluated to reseach the influence on osteoblasts in vitro and ectopia osteoinduction in vivo of macropore structures. The main conclusions were obtained as follows:
1. The macropores of HA spheres accumulated porous scaffolds were completely interconnected, the pore size could be controlled by the dimension of HA spherulites, and large number of micropores existed on and in HA spheres. No negtive effect on the biocompatibility of the HA porous scaffolds were observed by the process of preparation. A uniform distribution of osteoblasts in HA spherulites accumulating scaffolds could be obtained by the operation of stirring or transferring. By transferring loading method, more osteoblasts were seeded on HA spherulites, which were the main body of HA pourous scaffolds. There were no considerable differences of cells proliferation or differentiation seeded by the three different methods designed.
2. The interpore connectivity and porosity of scaffolds could be improved significantly by treating the wax spheres using dimethyl benzene. There was no significant difference in the shrinkage for the scaffolds with different porosities. Osteoblasts could migrate deeply into the center of the scaffold through the inter-connected pores and showed normal activity. Scaffolds with better interconection pores could supply more space for cell proliferation and differentiation.
3. There was no significant difference of osteoblasts adherent or prolaferation seeded in the two different porous scaffolds in vitro. The two scaffolds with complementary porous structurs can induce new ectopic bone well, and the new bone formed along the edge of the pors. The porous structures of particulate leaching scaffolds were more profitable for ectopic bone formation and vascularize.
实验中以羟基磷灰石为原料,用溶胶-凝胶法制备了HA球状颗粒,将其堆积到多孔管中后组成HA颗粒堆积支架,并对支架的孔隙结构进行了表征；设计了三种不同的成骨细胞接种方法：直接法、混合法和转移法,考察了接种方法不同对成骨细胞生物学特性的影响。用相同的HA浆料添加石蜡球作为造孔剂后制备了颗粒造孔支架,并通过对石蜡球用二甲苯进行处理以改善多孔支架的孔隙贯通性,考察了孔隙贯通性对成骨细胞生物学特性的影响。对孔隙结构互补的这两种HA多孔支架进行体内和体外生物学评价,以考察支架大孔形状对成骨细胞生物学性能的影响和对体内异位成骨的影响。本实验获得的主要结论如下：
1.HA颗粒堆积多孔支架的大孔孔隙完全贯通,并且孔径大小可通过HA颗粒的大小控制,颗粒表面和内部均匀分布着大量微孔；HA颗粒堆积支架具有良好的生物相容性；三种细胞接种方法中,混合法和转移法接种可以使细胞在颗粒堆积支架内部均匀分散,而且转移法可以将更多的细胞接种到颗粒表面,具有更高的接种效率；不同的细胞接种方法对成骨细胞的增殖和分化没有明显影响。
2.通过二甲苯处理用作造孔剂的石蜡球可明显改善支架孔隙的贯通性,并同时提高支架的孔隙率,而支架的抗压强度有所降低；多孔支架的收缩率取决于所配制的HA浆料,与孔隙率无关；支架表面均匀分布着微米和亚微米级的微孔和粗糙结构；HA多孔支架具有良好的细胞相容性,并且成骨细胞可进入多孔支架内部正常生长；贯通性好的支架可以为细胞增殖提供更充分的空间。
3.两种不同的孔隙结构对体外成骨细胞在支架内部粘附和生长的影响没有明显区别；两种支架在动物体内非骨部位都可以很好的诱导成骨发生,并且所生成的新骨沿着孔隙的边缘正常生长,颗粒造孔支架的孔隙结构更有利于体内异位成骨的发生及血管化的产生。
Critical size bone defects due to trauma or disease are very difficult to repair via the natural growth of host tissue. Therefore, there exists a nccessity to replace these defects with bridging materials. Multiple approaches have been attempted by using autografts, allografts, artificial materials and bone tissue engineering scaffolds. The major problem with autografts is the insufficient supply and significant surgical morbidity associated with donor site pain and loss of function. Moreover, patients have to suffer a second procedure. Allografts are associated with infection and inflammation. Therefore, engineered bone tissue has been developed recently. In bone tissue engineering, scaffold served as the matrices of tissue formation plays a pivotal role, and has to fulfill a few basic requirements, that is, high porosity, pore interconnection, proper pore size, required surface properties permitting cell adhesion, and proliferation, desirable mechanical integrity to maintain the predesigned tissue structure, non-cytotoxicity. Large pores in the scaffold can allow effective nutrient supply, gas diffusion and metabolic waste removal. To engineer synthetic bone tissue, cells should be seeded into an appropriate three-dimensional (3D) porous scaffold matrix, and expand the cells in vitro. Recently,3D cultures have been widely adopted for tissue engineering to obtain satisfying artificial bone grafts. Cell seeding is the first step in establishing 3D cultures and achieving the seeding of cells with a uniform distribution throughout the porous scaffold could establish a basis for homogeneous tissue generation. Hydroxyapatite (HA) has a composition and structure very close to natural bone mineral and therefore has been considered to be the ideal material to build bone tissue engineering scaffold due to its osteoconductivity and osteoinductivity.
HA spheres were prepared by the method of sol-gel and water/oil emulsification technology, then accumulated in a porous tube as a porous scaffold. The porosity, macro and micro porous structures of the scaffold were characterized. Three cell seeding methods were designed to distribute osteoblasts into HA spherule scaffolds:direct load, admixture load and transferring load in vitro. The effects on biological property of osteoblasts seeded in the porous scaffold by the three cell seeding methods were investigated. Wax spheres were prepared as porogen to prepare HA porous scaffolds with the same HA slurry, and the wax spheres were treated with dimethyl benzene to improve the interconection of the HA scaffold pores. The effects of interconection on biological property of osteoblasts seeded in the porous scaffold were investigated. The two kinds of HA porous scaffolds with complementary porous structures were evaluated to reseach the influence on osteoblasts in vitro and ectopia osteoinduction in vivo of macropore structures. The main conclusions were obtained as follows:
1. The macropores of HA spheres accumulated porous scaffolds were completely interconnected, the pore size could be controlled by the dimension of HA spherulites, and large number of micropores existed on and in HA spheres. No negtive effect on the biocompatibility of the HA porous scaffolds were observed by the process of preparation. A uniform distribution of osteoblasts in HA spherulites accumulating scaffolds could be obtained by the operation of stirring or transferring. By transferring loading method, more osteoblasts were seeded on HA spherulites, which were the main body of HA pourous scaffolds. There were no considerable differences of cells proliferation or differentiation seeded by the three different methods designed.
2. The interpore connectivity and porosity of scaffolds could be improved significantly by treating the wax spheres using dimethyl benzene. There was no significant difference in the shrinkage for the scaffolds with different porosities. Osteoblasts could migrate deeply into the center of the scaffold through the inter-connected pores and showed normal activity. Scaffolds with better interconection pores could supply more space for cell proliferation and differentiation.
3. There was no significant difference of osteoblasts adherent or prolaferation seeded in the two different porous scaffolds in vitro. The two scaffolds with complementary porous structurs can induce new ectopic bone well, and the new bone formed along the edge of the pors. The porous structures of particulate leaching scaffolds were more profitable for ectopic bone formation and vascularize.
引文
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