【作者】 李星；

【导师】 蒋青； 刘曦；

【作者基本信息】 四川大学 ， 材料工程（专业学位）， 2021， 硕士

【摘要】 近年来,人工骨移植物已经广泛应用于临床上骨缺损的替代和填充。但单一的无机成分不能完全模拟复杂的骨组织。骨组织主要由30%的有机基质和70%的无机矿物质组成,胶原蛋白和磷酸钙占大多数成分。因此,胶原蛋白与羟基磷灰石结合制备仿生的有机-无机杂化生物材料越来越受到人们的关注。但羟基磷灰石在基体材料中容易团聚,且缺乏有效的界面相互作用,导致矿化效果较差、机械性能不稳定和不完全的骨整合,从而导致骨修复失败。通过对羟基磷灰石表面进行功能化修饰是一种可行的策略。透明质酸是一种天然的高分子聚合物,有很好的生物相容性和可降解性,将巯基化透明质酸(HA-SH)作为功能化修饰的羟基磷灰石和I型胶原之间的桥梁,构建结构和组分仿生的复合骨修复支架,有望增强羟基磷灰石和基体材料之间的界面相互作用,促进骨诱导性和仿生矿化的能力。基于上述的背景,本课题通过不同改性方式对纳米羟基磷灰石进行改性来探索合适的改性手段和条件,制备得到了氨基修饰的羟基磷灰石纳米颗粒,进一步通过酰胺化反应将巯基化透明质酸修饰到纳米羟基磷灰石表面,制备得到了功能化修饰的纳米羟基磷灰石粉末。为了探究不同羟基磷灰石的尺寸对于骨诱导性和生物矿化能力的影响,我们将微米级的羟基磷灰石、未改性的纳米羟基磷灰石以及功能化修饰的纳米羟基磷灰石分别与巯基化透明质酸以及I型胶原进行复合,制备了三种可注射杂化交联的水凝胶,并对三种可注射杂化交联水凝胶的理化性能以及细胞相容性进行了考察。分别从体外、体内两个方面考察了复合水凝胶在骨组织工程领域中潜在的应用价值。主要结论如下:(1)合成过程中的原料及最终产物的X射线光电子衍射、红外光谱以及热重分析结果表明,成功制备了氨基修饰的羟基磷灰石以及巯基化透明质酸修饰的羟基磷灰石,正硅酸乙酯作为桥接羟基磷灰石与硅烷偶联剂的媒介,可以提高硅烷偶联剂的接枝量,且经改性后羟基磷灰石晶体结构未发生变化。巯基化透明质酸修饰的羟基磷灰石后,其粒径变大,更加分散均匀,表面电荷变小,且在水溶液中可以保持良好的均匀分散。(2)微米羟基磷灰石和未改性的纳米羟基磷灰石制备的水凝胶中,均有较明显的羟基磷灰石团聚现象,经巯基化透明质酸改性过的纳米羟基磷灰石在基体材料中分散均匀,无明显的大块团聚颗粒出现。相较于其他两种水凝胶,改性纳米羟基磷灰石制备的水凝胶有更高的粘弹性以及更好的抵抗压缩和剪切变形的能力,具有更小的溶胀率以及更温和的降解速率。(3)三种可注射杂化水凝胶均可支持干细胞在其中生长,具有良好的生物相容性,其中改性羟基磷灰石水凝胶能够更好地促进干细胞的粘附和增殖,细胞铺展形态良好。成骨分化相关基因显著上调,且能够促进干细胞分泌更多的成骨相关蛋白。说明干细胞在改性羟基磷灰石水凝胶中有更明显的成骨分化效果(4)经裸鼠皮下植入后,改性羟基磷灰石水凝胶有较高的抗压缩力学性能;其相较其他两种非均匀质水凝胶有更均匀的生物矿化效果和更高的矿化率;包裹在改性羟基磷灰石水凝胶中的干细胞能够保持较好的均匀分布,促进成骨相关基因的表达,分泌大量成骨相关的细胞外基质。综上所述,本研究通过将巯基化透明质酸修饰到纳米羟基磷灰石表面制备得到了功能化修饰的纳米羟基磷灰石,并将其与巯基化透明质酸和I型胶原进行复合制备了一种具有粗糙纤维织构的杂化交联水凝胶,羟基磷灰石在基体材料中分散均匀且与基体材料有效结合,体外细胞实验和裸鼠皮下植入实验表明该种杂化交联水凝胶能够更好的促进干细胞成骨分化,具有更好的骨诱导性和生物矿化能力。更多还原

【Abstract】 Recent years,artificial bone grafts such as hydroxyapatite(HAp),β-tricalciumphosphate(β-TCP)and bioactive glasses had been commonly used in substitution and padding of clinical bone defect.But single inorganic components could not completely simulate complex bone tissue,which was mainly composed of30 % organic matrix and 70 % inorganic mineral including collagen and calcium phosphate.Therefore,highly biomimetic organic-inorganic hybrid biomaterials by combining collagen with HAp were attracting increasingly attention for developing novel artificial bone graft.However,hydroxyapatite agglomerates in the matrix material and lacked effective interface resulted in the poor mineralization effect,unstable mechanical properties and the incomplete osseointegration,thus leading to bone repair failure.Functionally modifying hydroxyapatite could be a feasible strategy.As a hydrophilic natural polymer,hyaluronic acid had good biocompatibility and degradability.Thiolated hyaluronic acid(HA-SH),as a bridge between functionally modified hydroxyapatite and type I collagen,was used to construct structurally and componential biomimetic composite bone repair scaffold,which was expected to enhance the interface between hydroxyapatite and the matrix material.Hence promoting the ability of osteoinductive ability and biomimetic mineralization.Based on the above background,we tried different modification methods of hydroxyapatite to explore a suitable modification method and conditions.Hydroxyapatite nanoparticles modified with amino was prepared,and hydroxyapatite modified with HA-SH by amidation reaction was prepared.To explore the effect of different hydroxyapatite sizes on bone inductivity and biomineralization ability,we blended micron grade hydroxyapatite,unmodified nanometer hydroxyapatite and functionalized nanometer hydroxyapatite respectively with HA-SH and type I collagen.Furthermore,the physical and chemical properties and cell compatibility of three injectable hybrid crosslinked hydrogels were investigated.The potential application of the composite hydrogel in bone tissue engineering was investigated in vitro and in vivo.The main conclusions are as follows:(1)X-ray photoelectron diffraction,infrared spectroscopy,and thermogravimetric analysis results showed that amino-modified hydroxyapatite and HA-SH modified hydroxyapatite were prepared successfully.Ethyl silicate was used as a medium for bridging the hydroxyapatite and the silane coupling agent,which could increase the grafting amount of the silane coupling agent,and the crystal structure of the hydroxyapatite remains unchanged after modification.After modification,hydroxyapatite particle size becomes larger,the dispersion was more uniform,the surface charge became smaller,and it could maintain a good uniform dispersion in the aqueous solution.(2)HA-SH/μHAp/Col and HA-SH/n HAp/Col hybrid hydrogels had obvious hydroxyapatite agglomeration.The hydroxyapatite modified with HA-SH was dispersed in the matrix material uniformly,and no obvious large agglomerated particles appeared.Compared with the other two hydrogels,the modified hydroxyapatite hydrogel had higher viscoelasticity and better resistance to compression and shear deformation.It had a smaller swelling rate and a milder degradation rate.(3)Three injectable hybrid hydrogels could support the growth of stem cells and have good biocompatibility.Among them,the modified hydroxyapatite hydrogel could better promote the adhesion and proliferation of stem cells.Cells spread well in morphology.Osteogenic differentiation-related genes were significantly up-regulated.It could also promote stem cells to secrete more osteogenic-related proteins.These results indicated that stem cells had a more obvious osteogenic differentiation effect in the modified hydroxyapatite hydrogel.(4)After subcutaneous implantation in nude mice,the modified hydroxyapatite hydrogel had higher mechanical properties against compression;it had a more uniform biomineralization effect than the other two heterogeneous hydrogels and a higher mineralization rate;the stem cells encapsulated in the modified hydroxyapatite hydrogel could maintain a better uniform distribution.The expression of osteogenic genes was significantly up-regulated,and a large amount of osteogenic extracellular matrix were secreted.To sum up,this research investigated the surface modification of hydroxyapatite,and prepared a nanometer hydroxyapatite functionalized with HA-SH.In addition,a kind of coarse fiber texture hybrid-crosslinking hydrogels was prepared by combining the modified hydroxyapatite with type I collagen and HA-SH.Hydroxyapatite dispersed uniformly in matrix material and combined with substrate material effectively.Cell experiments in vitro and subcutaneous implantation experiments in nude mice showed that the hybrid cross-linked hydrogel could better promote the osteogenic differentiation of stem cells and had better bone induction and biomineralization ability.更多还原