材料性质调控干细胞成骨分化及壳聚糖基骨修复材料

发布时间：2018-02-23 03:35

本文关键词： 骨组织工程间充质干细胞等离子体聚合纳米银壳聚糖　出处：《清华大学》2015年博士论文　论文类型：学位论文

【摘要】：本论文研究工作围绕骨组织工程中材料与细胞、材料与生长因子之间的相互关系开展,重点研究了材料性质对间充质干细胞成骨分化的影响以及载BMP-2多肽的壳聚糖基骨修复材料。采用等离子体聚合的方法,分别以烯丙基胺(AAm)、丙烯酸(AAc)、1,7-辛二烯(OD)和乙醇(ET)为单体,制备了分别富含氨基( NH2)、羧基( COOH)、甲基( CH3)和羟基( OH)的表面。研究了体外培养在这些表面上的脂肪干细胞(hASCs)的行为,尤其是干细胞的成骨分化。在此基础上,通过控制等离子体聚合过程中1,7-辛二烯(OD)和烯丙基胺(AAm)的用量,制备并表征了氨基含量逐渐变化的化学梯度表面,探索等离子体聚合改性表面影响脂肪干细胞分化的相关机理。结果表明,氨基表面能促进hASCs的贴附、铺展、增殖以及成骨分化。其可能的机理是表面化学性质通过与血清蛋白作用来影响hASCs的行为,而不是直接与细胞相互作用,ERK1/2可能是涉及表面化学性质影响干细胞分化的信号通路。制备了直径约为30 nm的单分散纳米银粒子,并研究了不同浓度纳米银(0、10、50和100μg/m L)对人间充质干细胞(hMSCs)成骨分化的影响。存在于hMSCs中的纳米银不影响其细胞周期、碱性磷酸酶(ALP)活性、骨钙素(OC)基因表达、骨桥蛋白(OPN)表达以及体外矿化钙结节的形成,表明纳米银不影响hMSCs的成骨分化能力。这些结果为纳米银在骨组织工程中的应用,尤其是作为抗菌剂加入骨组织工程支架/植入物的应用提供积极的参考意义。在壳聚糖基骨修复材料载BMP-2多肽研究中,先通过2-亚氨基硫烷盐酸盐对壳聚糖进行巯基改性。然后将巯基化壳聚糖与末端为半胱氨酸的BMP-2多肽P24反应,制备接枝多肽的壳聚糖(CS-P24),再与羟基磷灰石粉(HA)复合,制备CS-P24/HA复合支架。同时将制备所得水溶性巯基化壳聚糖(CS-TBA)与β-甘油磷酸钠(β-GP)和羟基磷灰石(HA)复合,制备CS-TBA/β-GP/HA温敏性凝胶体系。巯基化壳聚糖凝胶体系具有多孔结构,羟基磷灰石均匀分布其中;具有合适的降解速率以及较低的细胞毒性;由于巯基化凝胶体系中的巯基与BMP-2多肽P24中的巯基发生反应,可以减缓BMP-2多肽的释放。
[Abstract]:This paper focuses on the relationship between materials and cells, materials and growth factors in bone tissue engineering. The effects of material properties on osteogenic differentiation of mesenchymal stem cells and chitosan based bone repair materials loaded with BMP-2 polypeptide were studied. The surfaces rich in amino groups (NH _ 2H _ 2, C _ 2O _ 3, methyl (Ch _ 3) and hydroxyl) were prepared. The behavior of adipose stem cells cultured on these surfaces in vitro, especially the osteogenic differentiation of stem cells, was studied. A chemically gradient surface with a gradual change in amino content was prepared and characterized by controlling the amount of 1-octylenedienediol (ODO) and allylamine (Aam) in plasma polymerization. The effects of plasma polymerization on the differentiation of adipose stem cells were investigated. The results showed that the surface of amino group could promote the adhesion and spread of hASCs. Proliferation and osteogenic differentiation. The possible mechanism is that surface chemical properties affect the behavior of hASCs by interacting with serum proteins. Instead of interacting directly with cells, ERK1 / 2 may be a signaling pathway involving surface chemical properties affecting stem cell differentiation. Monodisperse silver nanoparticles, about 30 nm in diameter, have been prepared. The effects of different concentrations of nano-silver on osteogenic differentiation of human mesenchymal stem cells (hMSCs) were studied. The cell cycle, alkaline phosphatase (ALP) activity and osteocalcin (OC) gene expression were not affected by the presence of nano-silver in hMSCs. The expression of osteopontin (OPN) and the formation of mineralized calcium nodules in vitro indicate that nano-silver does not affect the osteogenic differentiation ability of hMSCs. These results are the application of nano-silver in bone tissue engineering. Especially the application of bone tissue engineering scaffolds / implants as antimicrobial agents provides a positive reference for the study of chitosan based bone repair materials loaded with BMP-2 peptides. The chitosan was modified by 2-iminositane hydrochloride, and then the thioglycolated chitosan reacted with the BMP-2 polypeptide P24 with cysteine terminus to prepare the graft polypeptide, CS-P24N, which was then compounded with hydroxyapatite powder. CS-P24/HA composite scaffolds were prepared. CS-TBA / 尾 -GPHA thermosensitive gel system was prepared by compounding CS-TBA with 尾 -glycerophosphate (尾 -GPN) and hydroxyapatite (HA). The mercaptochitosan gel system had porous structure. Hydroxyapatite is homogeneously distributed among them; it has suitable degradation rate and low cytotoxicity. The release of BMP-2 peptide can be slowed down by the reaction between thiol group and sulfhydryl group in BMP-2 polypeptide P24.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R68

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