聚丙烯酰胺改良甲基丙烯酰化明胶接枝钛合金支架的促成骨性能

Osteogenic properties of polyacrylamide-modified gelatin methacryloyl grafted titanium alloy scaffold

背景:钛及其合金因出色的生物相容性、耐腐蚀性、力学性能被广泛应用于骨科内植物中,但其本身拥有生物惰性不能为成骨细胞提供良好的生长环境,较难形成良好的骨整合。目的:在钛合金支架表面构建甲基丙烯酰化明胶和聚丙烯酰胺复合水凝胶材料,分析其体外促成骨能力。方法:将甲基丙烯酰化明胶与丙烯酰胺混合,加入交联剂与催化剂合成甲基丙烯酰化明胶-聚丙烯酰胺(Gelma-PAAM)水凝胶。将经亲和硅烷表面改性后的钛合金支架分别与甲基丙烯酰化明胶(Gelma)水凝胶、Gelma-PAAM水凝胶混合,完成负载(分别记为Ti-Gelma、Ti-Gelma-PAAM),比较支架表面两种水凝胶的溶胀比、降解率,通过扫描电镜观察水凝胶与钛合金的结合状态。将大鼠骨髓间充质干细胞分别接种于单纯钛合金支架、Ti-Gelma支架、Ti-Gelma-PAAM支架中,检测细胞增殖、黏附及成骨分化能力。结果与结论:①与Gelma水凝胶相比,Gelma-PAAM复合水凝胶具有更高的溶胀比与更低的降解率;②扫描电镜下可见两种水凝胶表面为蜂窝状结构,与多孔钛合金支架结合后呈膜样包裹于支架表面并充斥孔隙,其中Gelma-PAAM复合水凝胶包裹支架得更充分;③CCK-8检测与活-死荧光染色显示,与Ti-Gelma、Ti-Gelma-PAAM支架共培养后的骨髓间充质干细胞增殖良好并保持较高的活性;成骨诱导培养后,单纯钛合金支架组细胞碱性磷酸酶活性、钙沉积量与成骨基因表达量最低,Ti-Gelma-PAAM组细胞碱性磷酸酶活性、钙沉积量与成骨基因表达量最高;④鬼笔环肽骨架染色显示,单纯钛合金支架组、Ti-Gelma组细胞稀疏、伸展不够充分,Ti-Gelma-PAAM组细胞伸展充分、肌动蛋白丝致密;⑤结果表明,Gelma-PAAM水凝胶具有良好的生物相容性与促成骨能力,较Gelma水凝胶更适宜用于钛合金金属表面的促成骨改性。

BACKGROUND:Titanium and its alloys are widely used in orthopedic implants due to their excellent biocompatibility,corrosion resistance,and mechanical properties.However,it has biological inertia itself,cannot provide a good growth environment for osteoblasts,and it is difficult to form good osseointegration.OBJECTIVE:To construct a composite hydrogel material of gelatin methacryloyl and polyacrylamide on the surface of titanium alloy scaffold,and analyze its osteogenic ability in vitro.METHODS:Gelatin methacryloyl was mixed with acrylamide.Crosslinking agent and catalyst were added to synthesize gelatin methacryloyl and acrylamide(Gelma-PAAM)composite hydrogel.The titanium alloy scaffold modified by affinity silane was mixed with the Gelma hydrogel and Gelma-PAAM composite hydrogel to complete the loading(recorded as Ti-Gelma and Ti-Gelma-PAAM,respectively).The swelling ratio and degradation rate of the two hydrogels on the surface of the scaffold were compared.The bonding state between hydrogels and titanium alloy was observed by scanning electron microscope.Rat bone marrow mesenchymal stem cells were inoculated into Ti,Ti-Gelma and Ti-Gelma-PAAM scaffolds,separately.Cell proliferation,adhesion,and osteogenic differentiation were detected.RESULTS AND CONCLUSION:(1)Compared with Gelma hydrogel,Gelma-PAAM hydrogel had higher swelling rate and lower degradation rate.(2)Scanning electron microscope showed that the surface of the two kinds of hydrogels was honeycomb structure.After being combined with porous titanium alloy scaffold,the film was wrapped on the surface of scaffold and filled with pores.Among them,the Gelma-PAAM composite hydrogel coated the scaffold more fully.(3)CCK-8 assay and live/dead fluorescence staining showed that bone marrow mesenchymal stem cells proliferated well after coculture with Ti-Gelma and Ti-Gelma-PAAM scaffolds and maintained high activity.After osteogenic induction culture,alkaline phosphatase activity,calcium deposition,and osteogenic gene expression of cells of titanium alloy scaffold group were the lowest,and alkaline phosphatase activity,calcium deposition,and osteogenic gene expression of cells of Ti-Gelma-PAAM scaffold group were the highest.(4)Phalloidin cytoskeletal staining exhibited that the cells of pure titanium alloy scaffold group and Ti-Gelma scaffold group were sparse and insufficiently extended,while the cells of Ti-Gelatin-PAAM group had the most adequate stretching and the densest filamentous actin.(5)The results show that Gelma-PAAM hydrogel has good biocompatibility and osteogenic ability,and is more suitable for osteogenic modification on the surface of titanium alloy than Gelma hydrogel.