透明质酸-明胶双网络水凝胶促进骨髓间充质干细胞成骨分化的作用

Effect of hyaluronic acid-gelatin double-network hydrogel on osteogenic differentiation of rat bone marrow mesenchymal stem cells

目的·通过双网络(double network,DN)方法构建一种高强度的天然高分子水凝胶,并探讨其对大鼠骨髓间充质干细胞(rat bone mesenchymal stem cells,rBMSCs)成骨分化的促进作用。方法·通过两步光交联法制备透明质酸-明胶双网络(HA-Gel DN)水凝胶,并通过扫描电子显微镜、溶胀、压缩和降解实验评估其物理化学性能。CCK-8和荧光染色检测rBMSCs在水凝胶上的活性和铺展、贴附、增殖能力;定量PCR和Western blotting检测rBMSCs的成骨分化能力。结果·与HA水凝胶和Gel水凝胶相比,HA-Gel DN水凝胶有更适宜于rBMSCs成骨分化的吸水和保水性能[(12.6±0.7)倍,(10.3±0.4)倍],更强的力学性能[(43.7±5.6)k Pa]以及更慢的降解速率[12周,(82.3±3.9)%]。体外实验显示HA-Gel DN水凝胶具有良好的生物相容性,定量PCR显示它能够促进rBMSCs成骨相关基因(Runx2、BSP、OPN、OCN、OSX和ALP)的表达,Western blotting显示它显著提高rBMSCs成骨相关蛋白(OPN、OSX和BSP)的表达。结论·HA-Gel DN水凝胶具有良好的生物相容性和诱导rBMSCs成骨分化的能力,为DN水凝胶成为潜在的骨缺损修复材料提供了新的实验依据。

Objective·To construct a natural polymers-based hydrogels with enhanced mechanical property through double network（DN） strategy under the premise of retaining the good biocompatibility of natural polymers,and then evaluate the beneficial effect of hydrogels on the osteogenic differentiation of rat bone mesenchymal stem cells（rBMSCs）.Methods·Methacrylated hyaluronic acid（HAMA） and methacrylated natural gelatin（Gel MA） were synthesized through reaction of HA and Gel with methacrylic anhydride（MA）,respectively.Thereafter,HA-Gel DN hydrogels were fabricated through two-step photo-crosslinking.The basic physicochemical properties of hydrogels were evaluated by scanning electron microscopy,swelling,compression and degradation analysis.Hydrogels were applied as substrate materials for rBMSCs culture in vitro.Cell viability,attachment,spreading and proliferation were assessed by CCK-8 analysis and fluorescent staining analysis.The osteogenic differentiation of rBMSCs was determined by quantitative PCR and Western blotting analysis.Results·In comparison to HA hydrogels and Gel hydrogels,HA-Gel DN hydrogels showed more suitable physicochemical properties,such as more suitable water absorption and water retention [（12.6± 0.7） fold,（10.3± 0.4） fold],stronger mechanical property [（43.7± 5.6） k Pa] and slower degradation rate [（82.3±3.9）% for 12 weeks] for osteogenic differentiation of rBMSCs.Experiment in vitro revealed that HA-Gel DN hydrogels had good biocompatibility,quantitative PCR revealed that it could promote the expression of osteogenic genes including Runx2,BSP,OPN,OCN,OSX and ALP.Western blotting revealed that the HA-Gel DN hydrogels also increased the levels of osteogenic proteins（OPN,OSX and BSP）.Conclusion·HAGel DN hydrogels have good biocompatibility and promote the osteogenic differentiation of rBMSCs,which provide a new experimental basis for DN hydrogels becoming the potential material for bone defects repair.