RGD和透明质酸双功能聚乙烯醇水凝胶的制备、表征及成骨相容性评价

Yan Li, Lijie Sun, Lingli Zhou, Yonggang Ma, Mingyuan Liu
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After three days of culture, the proliferation\n activity ranks in order as PVA-HA-SH/PVAPA-RGD group > PVA-RGD group > PVA-HA group > PVA group > Control group. After 4 hours of culture, cell adhesion is sequenced as PVA-HA-SH/PVAPA-RGD group > bicomponent group > PVA group > Control group. After 48 hours of culture,\n immunofluorescence staining demonstrates more sufficient cellular spreading and adherence, clearer actin fibers, more vinculin numbers in PVA-HA-SH/PVAPA-RGD group. In addition, significantly higher ALP activity (after 3 days’ culturing) and osteogenic gene expression level (after 7\n days’ culturing) have been witnessed in PVA-HA-SH/PVAPA-RGD group than those of other groups. 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引用次数: 0

摘要

近年来,聚乙烯醇水凝胶在骨组织工程领域一再面临提高生物活性的迫切需求。采用巯基炔烃点击化学反应制备了含有RGD肽和透明质酸的双功能聚乙烯醇水凝胶。聚乙烯醇与透明质酸接枝可以降低降解率,改善力学性能。RGD肽可以改善细胞在材料表面的粘附和迁移。通过点击化学反应制备了双功能聚乙烯醇水凝胶,并通过扫描电子显微镜、核磁共振氢谱、埃尔曼试验和凯氏氮测定对其进行了表征。通过拉伸性能和水接触角测试对双功能聚乙烯醇水凝胶的物理性能进行了评价。根据水凝胶组成的不同,设计了五组进行实验:对照组、PVA组、PVA-HA组、PVAPA-RGD组和PVA-HA-SH/PVAPA-RGD组,每组与MC3T3-E1细胞共培养,用激光扫描共聚焦显微镜观察细胞形态。然后分别用CCK-8法和细胞计数法检测细胞增殖活性和细胞粘附率。用ALP活性和qRT-PCR检测成骨分化和成骨标志基因ALP、OCN、Runx2、Col I和OPN的表达。实验结果表明,双功能聚乙烯醇水凝胶的拉伸强度为1.42±0.61MPa,拉伸模量为0.58±0.03MPa,断裂伸长率为297.53±42.36%,水接触角为18.40±2.2°,增殖活性依次为PVA-HA-SH/PVAPA-RGD组>PVA-RGD组>PVA-HA组>PVA组>对照组。培养4小时后,细胞粘附顺序为PVA-HA-SH/PVAPA-RGD组>双组分组>PVA组>对照组。培养48小时后,免疫荧光染色显示PVA-HA-SH/PVAPA-RGD组有更充分的细胞扩散和粘附,肌动蛋白纤维更清晰,长春花蛋白数量更多。此外,PVA-HA-SH/PVAPA-RGD组的ALP活性(培养3天后)和成骨基因表达水平(培养7天后)显著高于其他组。总体实验结果证实了双功能聚乙烯醇水凝胶体系具有优异的亲水性和良好的力学性能,对MC3T3-E1细胞的增殖、粘附和成骨分化具有显著影响。
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Preparation, Characterization and Osteoblastic Compatibility Evaluation of Bifunctional Polyvinyl Alcohol Hydrogels Containing RGD and Hyaluronic Acid
In recent years, polyvinyl alcohol hydrogel has been repeatedly confronted with urgent needs for more enhanced bio-activity in the field of bone tissue engineering. We used sulfydryl-alkyne click chemical reaction to prepare a bifunctional polyvinyl alcohol hydrogel containing RGD peptide and hyaluronic acid. Polyvinyl alcohol grafted with hyaluronic acid can reduce degradation rate and improve mechanical properties. RGD peptide can improve cell adhesion and migration on material surface. The bifunctional polyvinyl alcohol hydrogel is prepared by click chemistry reaction and characterized by scanning electron microscopy, 1H NMR, Ellman’s test and Kjeldahl nitrogen determination. The physical properties of bifunctional polyvinyl alcohol hydrogel are evaluated in terms of tensile properties and water contact angle test. According to varying hydrogel composition, five groups are designed to conduct the experiment as follows: Control group, PVA group, PVA-HA group, PVAPA-RGD group, and PVA-HA-SH/PVAPA-RGD group, each of which is to be co-cultured with MC3T3-E1 cells to observe cell morphology by laser scanning confocal microscope. Then, CCK-8 method and cell counting method were used to detect the proliferation activity and cell adhesion rate, respectively. ALP activity and qRT-PCR were used to detect osteogenic differentiation and expression of osteogenic marker genes ALP, OCN, Runx2, Col I and OPN. The experimental results show that the tensile strength of the bifunctional polyvinyl alcohol hydrogel is 1.42±0.61 MPa, with tensile modulus as 0.58±0.03 MPa, and elongation at break as 297.53±42.36%. The water contact angle is 18.40±2.2°. After three days of culture, the proliferation activity ranks in order as PVA-HA-SH/PVAPA-RGD group > PVA-RGD group > PVA-HA group > PVA group > Control group. After 4 hours of culture, cell adhesion is sequenced as PVA-HA-SH/PVAPA-RGD group > bicomponent group > PVA group > Control group. After 48 hours of culture, immunofluorescence staining demonstrates more sufficient cellular spreading and adherence, clearer actin fibers, more vinculin numbers in PVA-HA-SH/PVAPA-RGD group. In addition, significantly higher ALP activity (after 3 days’ culturing) and osteogenic gene expression level (after 7 days’ culturing) have been witnessed in PVA-HA-SH/PVAPA-RGD group than those of other groups. The overall experimental results have confirmed its excellent hydrophilicity and fine mechanical properties of the bifunctional polyvinyl alcohol hydrogel system, which would exert a remarkable effect on proliferation, adhesion and osteogenic differentiation of MC3T3-E1 cells.
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