纳米级多孔β-磷酸三钙支架材料的制备、表征及细胞相容性研究

发布时间：2018-03-20 04:32

本文选题：纳米　切入点：β-磷酸三钙　出处：《复旦大学》2012年硕士论文　论文类型：学位论文

【摘要】：第一部分纳米级多孔β-磷酸三钙支架材料的制备和表征目的：多孔支架材料是组织工程学的材料基础,它不仅为细胞生长提供支持和保护,而且能够通过与细胞的相互作用调节细胞的形态发生过程,并影响细胞的生长、迁移、增殖和代谢功能。多孔支架材料的形态结构和理化特性,对实现其作用与功能有重要的影响。β-磷酸三钙(β-tricalcium phosphate, β-TCP)材料具有良好的细胞相容性和骨传导性,在骨移植和修复领域发挥了重要作用,致力于提升β-磷酸三钙材料强度及孔隙连通性的研究一直是组织工程学的重点之一。目前,制备纳米级多孔β-TCP支架材料的方法繁多,但是制备方法复杂,对实验室的条件要求较高。本研究的目的在于利用纳米技术,在普通实验室条件下制备纳米级多孔β-TCP支架材料,为进一步的研究奠定基础。方法：以硝酸钙和磷酸氢二铵溶液为原料,液相沉淀法制备纳米级B-TCP粉体,以氯化铵为造孔剂,程序升温法烧结成纳米级多孔β-TCP支架材料。对材料的物相进行X线衍射(X-ray diffraction, XRD)、傅里叶红外线转换光谱分析仪(Fourier transform infrared spectroscopy, FTIR)测试,万能试验机测定材料的抗压强度,液体静力称量法测定材料的孔隙率和吸水率,对材料的微观结构进行扫描电子显微镜(Scanning electron microscope, SEM)观察。结果：材料成分为β-TCP,晶粒尺寸31.5nm,孔隙率73.47±0.21Vol%(n=5),吸水率37.56±0.28Wt%(n=5),抗压强度7.98±0.05MPa(n=5),孔隙均匀丰富,连通性好。结论：实验制备的纳米级β-TCP粉体反应完全,无杂质,晶粒细腻,烧结出的纳米级多孔β-TCP支架材料具有良好的孔隙结构和孔隙连通性,并具备一定的抗压强度,可以作为支架材料为进一步的实验所使用。实验所需原料易于获取,制备工序简单,所得材料性质稳定,可以在普通实验室条件下制备。第二部分纳米级多孔β-磷酸三钙支架材料的细胞相容性研究目的：组织工程学研究中的另一个重要内容是获取良好的种子细胞。脂肪来源干细胞(adipose derived stem cells, ADSCs)是一种具有自我更新能力和一定分化潜能的成体干细胞。近十年来的体外实验已经证实其具有增殖能力和多向分化潜能。由于其来源广、取材便利,因此有希望成为新的骨组织工程种子细胞。理想的支架材料应该能与脂肪干细胞良好复合。有研究表明机械应力对细胞的生长、增殖、分化过程有刺激作用,在细胞与材料的复合过程中,对机械应力刺激作用的研究是不可忽视的。本研究通过与脂肪干细胞的复合了解纳米级多孔β-TCP支架材料的细胞相容性,观察动态接种条件下细胞在支架材料上的生长情况。方法：以兔脂肪来源干细胞为种子细胞,CCK-8法检测细胞毒性。制备细胞-支架材料复合物,分别于第1、4、7天以扫描电子显微镜观察细胞生长情况,评估支架材料的细胞相容性。以动态接种法为实验组,常规接种法为对照组,接种细胞,分别于第1、4、7天取出,CKK-8法检测细胞-支架材料复合物的细胞生长情况。结果：细胞在各浓度支架材料浸提液中生长良好,相对增殖率均≥100%；细胞在材料表面及孔隙内的生长良好,第1天可见细胞在支架材料表面吸附,第4天见细胞向孔隙内生长,第7天观察到细胞外基质。实验组在第4、第7天的OD(optical density)值大于对照组。结论：制备的纳米级多孔B-TCP支架材料无细胞毒性,兔ADSCs可在支架材料的表面及孔隙内生长,支架材料的细胞相容性好。动态接种条件下,纳米级多孔B-TCP支架材料也能表现出良好的细胞相容性,动态接种方式对细胞的短期增殖活性可能有促进作用。
[Abstract]:Preparation and characterization of nanoscale porous beta tricalcium phosphate scaffold
Objective: the porous scaffold material is a material basis of tissue engineering, it not only provides support and protection for cell growth, and can regulate the cell by interacting with cell morphogenesis, and affect cell growth, migration, proliferation and metabolism. The morphological structure of porous scaffolds and physicochemical characteristics of the the role and function has important influence. Beta tricalcium phosphate (beta -tricalcium beta phosphate, -TCP) materials have good biocompatibility and osteoconductivity, play an important role in bone transplantation and repair field, working on improving the strength of beta tricalcium phosphate calcium material and pore connectivity has been one of the key organizations engineering. At present, preparation method of nano porous beta -TCP scaffold material is various, but the preparation method of the complex, requires a sophisticated laboratory. The purpose of this study is to use nano Technology, preparation of nanoscale porous beta -TCP scaffold materials under ordinary laboratory conditions will lay the foundation for further research.
Methods: with calcium nitrate and diammonium hydrogen phosphate solution as raw materials, preparation of nano B-TCP powder by liquid phase precipitation, with ammonium chloride as the pore forming agent, temperature programmed method of sintered nano porous -TCP scaffolds materials. The phase of the composites by X-ray diffraction (X-ray diffraction, XRD), Fourier transformation infrared spectrum analyzer (Fourier transform infrared spectroscopy, FTIR) test, determination of compressive strength of the material universal testing machine, measuring the porosity and water absorption rate of hydrostatic weighing method for scanning electron microscopy on the microstructure of the materials (Scanning electron, microscope, SEM) were observed.
Results: the material composition is beta -TCP, the grain size is 31.5nm, the porosity is 73.47 + 0.21Vol% (n=5), the water absorption rate is 37.56 + 0.28Wt% (n=5), the compressive strength is 7.98 + 0.05MPa (n=5), the pore is even and rich, and the connectivity is good.
Conclusion: nano beta -TCP powder reaction experiment prepared completely, without impurities, grain and sintering of nanometer porous beta -TCP scaffold has good pore structure and pore connectivity, and have certain compressive strength, can be used as scaffold material for the further experiment. Experimental materials required easy to get, the preparation process is simple, the material properties of stability, can be prepared in the laboratory conditions.
Study on the cytocompatibility of second parts of nanoscale porous beta tricalcium phosphate scaffold
Objective: another important research contents in tissue engineering is to obtain good seed cells. Adipose derived stem cells (adipose derived stem cells, ADSCs) is a kind of self renewing ability and differentiation potential of adult stem cells in vitro. In the past ten years has proved its proliferation and multi-directional differentiation potential. Because of its wide source, conveniently so promising new seed cells for bone tissue engineering. The ideal scaffold material should be with adipose derived stem cells. Studies have shown that the composite good mechanical stress on cell growth, proliferation, differentiation has a stimulating effect, in the process of composite materials in the cell and that is not to be ignored in the study of mechanical stress stimulation. Through the study and understanding of adipose derived stem cells composite nano porous -TCP scaffolds materials biocompatibility, dynamic observation of inoculation conditions The growth of the cells on the scaffold material.
Methods: the rabbit adipose derived stem cells as seed cells, the cytotoxicity was measured by CCK-8 method. The preparation of cell scaffold composites, respectively in the first 1,4,7 days by scanning electron microscopy to observe the growth of cells, scaffold materials evaluation of biocompatibility. With dynamic inoculation method as the experimental group, control group, conventional cell inoculation inoculation, were removed in the 1,4,7 days, the growth of cells was detected by CKK-8 cell scaffold complexes.
Results: the cell extracts grew well in various concentrations of scaffold materials, the relative growth rate was more than 100%; the cell growth on the surface and pores of the good, the first day adsorption on the surface of the scaffold cells, fourth days of cell growth to pores were observed seventh days of extracellular matrix in the experimental Group Fourth. Seventh days, the OD (optical density) was higher than that of the control group.
Conclusion: nano porous B-TCP scaffolds prepared by cell toxicity, rabbit ADSCs can grow on the surface of scaffolds and scaffold pores, good biocompatibility. Dynamic inoculation conditions of nano porous B-TCP scaffolds can exhibit good biocompatibility, proliferation of short-term dynamic vaccination cells may play a promoting role.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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