Abstract
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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