目的 研究基于透明质酸(HA)-己二酸二酰肼(ADH)/RGD寡肽功能化果胶(PAD)的可注射HA-ADH/PAD-RGD水凝胶体系的软骨形成作用.方法 采用腙交联法合成水凝胶.通过分析HA-ADH与PAD-RGD不同质量比(8:2、6:4、4:6)HA-ADH/PAD-RGD水凝胶的形态学、凝胶时间、力学强度、降解时间、细胞增殖和软骨作用等因素,综合评价该水凝胶的特性.结果 质量比从8:2减小到6:4和4:6时,水凝胶时间从(132.8±4.8)s分别增加到(188.7±10.1)s和(328.9±12.6)s(P<0.01),平衡溶胀比(ESR)从32.8±1.5分别依次增大到34.8±2.3和36.4±1.9(P<0.01).经过49 d降解后,8:2、6:4、4:6质量比组水凝胶的剩余质量比依次为85.3％、73.5％和55.9％.另外,随着质量比减小,水凝胶的力学强度不断减小.培养1 d后8:2、6:4、4:6质量比组水凝胶的dsDNA含量分别为(0.7±0.1)μg、(0.6±0.1)μg、(0.6±0.1)μg(P>0.05).培养21 d后,6:4组水凝胶dsDNA含量为(1.9±0.2)μg明显高于8:2组[(1.6±0.1)μg,P<0.05]和4:6组[(1.5±0.1)μg,P<0.05].6:4组水凝胶Ⅱ型胶原基因的表达为2.3±0.2明显高于8:2组(1.3±0.2,P<0.05)和4:6组(1.0±0.1,P<0.05).6:4组糖胺聚糖(GAG)产生量为(16.5±1.4)μg明显高于8:2组[(9.1±1.4)μg,P<0.05]和4:6组[(8.8±1.0)μg,P<0.05].包裹在6:4组水凝胶中软骨细胞胶原Ⅱ型免疫着色的细胞百分比为(83.7±15.7)％,明显高于8:2组[(53.3±9.2)％,P<0.05]和4:6组[(43.9±7.8)％,P<0.05].结论 HA-ADH/PAD-RGD水凝胶具有适宜的凝胶时间,力学强度,良好的组织相容性和细胞相容性,并能增强软骨细胞增殖,维持软骨细胞表型.不同质量比HA-ADH/PAD-RGD水凝胶具有不同性质,6:4质量比水凝胶具有最好的物理化学和生物学性质.%Objective To investigate the chondrogenic effect of injectable hydrogel system based on hyaluronic acid-adipicdihydrazide (HA-ADH)/G4 RGDS-aldehyde pectin (PAD-RGD)in vitro.Methods We synthesized the hydrogel byhydrazine cross-linking method.We evaluated the characteristics of hydrogel by analyzing the different properties such as morphology,gelation time,mechanical strength, degradation time,the proliferation of cells and chondrogenic effect of different weight ratios (8:2,6:4,4:6)of HA-ADH/PAD-RGD. Results When the weight ratio decreased from 8:2 to 4:6,the hydrogel time increased from (132.8±4.8)s to (188.7±10.1)s and (328.9±12.6)s,respectively (P<0.01),and the equilibrium swelling ratio (ESR)increased from 32.8±1.5 to 34.8±2.3 and 36.4±1.9, respectively (P<0.01).After 49 days of degradation,the weight retention rate of the three kinds of hydrogel was 85.3％,73.5％,and 55 .9％,respectively.In addition,as the weight ratio decreased,the mechanical strength of the hydrogel decreased.After 1 day of culture,the dsDNA content of various gels was similar,and the values in the three groups were 0.7±0.1μg,0.6±0.1μg and 0.6±0.1μg,respectively (P>0.05).After 21 days of culture,the dsDNA content in the 6:4 group was significantly higher than that in the 8:2 and 4:6 groups (1.9±0.2,1.6±0.1 and 1.5±0.1)(P<0.05).The expression of type Ⅱ collagen gene in the 6:4 group was 2.3±0.2,which was significantly higher than that of the 8:2 and 4:6 groups (1.3±0.2 and 1±0.1)(P<0.05).The production of Glycosaminoglycan (GAG)in the 6:4 group was 16.5±1.4μg,which was significantly higher than that in the 8:2 and 4:6 groups (9.1±1.4μg,8.8±1.0μg)(P<0.05).The percentage of collagen type Ⅱ immunostaining of chondrocytes encapsulated in the 6:4 hydrogel was 83.7±15.7,which was significantly higher than that of the 8:2 and 4:6 groups (53.3±9.2,43.9±7.8)(P<0.05).Conclusion Our research showed that the hydrogel had the proper gelation time,mechanical strength and good histocompatibility and cytocompatibility.Moreover,it could enhance the proliferation and maintain the phenotype of chondrocytes.Different weight ratios of hydrogel exhibited different properties.Among the three weight ratios,HA-ADH/PAD-RGD at the 6:4 had the best physiochemical and biological properties.
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