不同交联密度甲基丙烯酰酯明胶/脱细胞半月板细胞外基质复合水凝胶的性能

文题释义：脱细胞半月板细胞外基质：由新鲜半月板组织通过湿法差速离心方法脱细胞制备而来，在去除了异体半月板的免疫原性的同时，仍保留了组织的大部分成分和组织本身的生物学性能，例如影响细胞的活性、调控细胞的增殖和分化、促进组织再生和修复等。甲基丙烯酰酯明胶：是一种明胶的衍生物，由甲基丙烯酸酐与明胶合成而来。在加入光引发剂的基础上经紫外线照射，明胶链上的甲基丙烯酰胺和甲基丙烯酸甲酯侧基聚合形成聚合物链，聚合物链交错纵横，最终形成网状结构。交联密度：通常用来表征交联聚合物里交联键的数量，主要受交联时间和交联光强度的影响，其可明显改变聚合物链的形成情况。背景：交联后的聚合物链对水凝胶的基本性质和细胞相容性存在显著影响，而交联密度可明显改变聚合物链的形成情况，有关交联密度对水凝胶性能影响的针对性研究较少。目的：制备一种细胞相容良好性的复合水凝胶，探究交联密度对该水凝胶性能的影响。方法：配制甲基丙烯酰酯明胶溶液，然后加入脱细胞半月板细胞外基质溶液与LAP溶液，制备预凝胶溶液，采用蓝光对溶液进行交联，交联时间分别为10，30，60 s，检测3种水凝胶的压缩弹性模量、溶胀倍率与降解性能。将半月板纤维软骨细胞加入预凝胶溶液中，采用蓝光对溶液进行交联，交联时间分别为10，30，60 s，检测培养对应时间的细胞活性、形态与聚集。结果与结论：①交联60 s水凝胶的压缩模明显高于交联10，30 s的水凝胶(P < 0.05)；②交联10 s水凝胶的溶胀倍率明显高于交联30，60 s的水凝胶(P < 0.05)，交联30，60 s的水凝胶的溶胀倍率比较差异无显著性意义(P > 0.05)；③随着交联时间的延长，水凝胶的降解时间延长，交联60 s的水凝胶需要80 min完全降解，交联10 s的水凝胶50 min就可以完全降解；④培养24 h后，3组水凝胶中的细胞活性均在95%以上，各组之间无差异(P > 0.05)；⑤培养1 d时，3组水凝胶中的细胞均呈球形且均匀分布；4 d时，3组细胞均开始伸展，交联10 s水凝胶中有较小的细胞团；7 d时，3组细胞树突状伸展更为明显，其中交联10 s水凝胶中形成了较为明显的细胞团；⑥培养1，7，14 d时，交联10 s水凝胶中的细胞活性均在85%以上。培养1 d时，交联10 s水凝胶中的细胞呈球形分布，且分布均匀；培养28 d时，细胞呈树突状伸展，并聚集形成网状结构；⑦结果表明，甲基丙烯酰酯明胶/脱细胞半月板细胞外基质复合水凝胶的性能可通过调整交联密度进行优化。ORCID: 0000-0002-3606-1097(周建)中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Properties of gelatin methacrylate/decellularized meniscus extracellular matrix composite hydrogel with different crosslinking densities

BACKGROUND: Crosslinked polymer chains have a remarkable effect on the fundamental properties and cytocompatibility of hydrogels, and crosslinking density can significantly change the formation of polymer chains. There are few studies on the effect of the properties of hydrogels caused by crosslinking density. OBJECTIVE: To prepare a composite hydrogel with a favorable cytocompatibility and to explore the effect of crosslinking density on the properties of the hydrogel. METHODS: Gelatin methacrylate solution was prepared, and decellularized meniscus extracellular matrix and LAP solution were added to prepare the pre-gel solution, which was crosslinked by blue ray. The crosslinking time was 10, 30 and 60 seconds. The compression elastic modulus, swelling ratio and degradability of hydrogels were detected. Meniscus fibrochondrocytes were added into the pre-gel solution, and crosslinked by blue ray. The cell viability, morphology and gathering were detected at 10, 30 and 60 seconds of crosslinking. RESULTS AND CONCLUSION:(1) The hydrogel with 60 seconds of cross-linking time had higher compression elastic modulus than that of the hydrogel with 10 and 30 seconds of cross-linking time(P < 0.05).(2) The swelling ratio of the hydrogel with 10 seconds of cross-linking time was significantly higher than that of the hydrogel with 30 and 60 seconds of cross-linking time(P < 0.05). The swelling ratio of the hydrogels with 30 versus 60 seconds of cross-linking time had no significant difference(P > 0.05).(3) With the crosslinking time increasing, the degradation time of hydrogels increased. The hydrogels with 60 seconds of cross-linking time degraded completely at 80 minutes, and hydrogels with 10 seconds of cross-linking time degraded completely at 50 minutes.(4) After 24 hours of culture, the cell viability in all groups was over 95%(P > 0.05).(5) At 1 day after culture, the cells were in sphere-shape and distributed evenly in all groups. On day 4, the cells in all groups began to extend, and there were small cell masses in the hydrogels with 10 seconds of cross-linking time. On day 7, the dendritic extension in all groups was obvious, and there were dominant cell masses in the hydrogels with 10 seconds of cross-linking time.(6) After 1, 7 and 14 days of culture, cell viability in all groups was over 85%. At 1 day after culture, the cells in the hydrogels with 10 seconds of cross-linking time were in sphere-shape and distributed evenly. On day 28, the cells extended in dendritic shape, and gathered in reticular formation.(7) In summary, the property of gelatin methacrylate/decellularized meniscus extracellular matrix composite hydrogel can be optimized by adjusting crosslinking density.