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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 (GelMA) 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) kPa] 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.
ABSTRACT
[ ABSTRACT] AIM:To develop the cell model of polymer/liquid crystal and to study the effect of their elasticity on the adhesion of rat bone marrow mesenchymal stem cells (rBM-MSCs).METHODS: Using the method of solvent e-vaporation induced phase separation, the cell model of polymer/liquid crystal was constructed.The surface morphology and phase separation structure were determined by polarized optical microscopy ( POM) , scanning electron microscopy ( SEM) and small angle X-ray scattering ( SAXS ) .rBM-MSCs were separated and expanded by adherent culture.The surface markers of rBM-MSCs were detected by flow cytometry.The cells were induced to osteogenic differentiation and adipogenic differentiation for 2 weeks.After 3 passages, the cells were divided into 4 groups, including total PU control group, 10%membrane group, 30%membrane group and 50%membrane group.The cells were then incubated with rhodamine phalloi-din for cytoskeleton staining and were observed under the confocal laser scanning microscope after cultured for 24 h.RE-SULTS:The cell model of polymer/liquid crystal was constructed successfully using the method of solvent evaporation in-duced phase separation.Flow cytometry results showed that the rBM-MSCs positively expressed CD29, CD44 and CD90, and negatively expressed CD34 and CD45.After stained with alizarin red S and oil red O, the calcium nodule and lipid droplets in rBM-MSCs were observed obviously.The cytoskeleton staining result indicated that the area in total PU control group, 10%membrane group and 30%membrane group were greater, and the actin microfilaments were also clearer than that in 50%membrane group.CONCLUSION:The cell model with suitable content of liquid crystal made a contribution to the rBM-MSCs’ adhesion, but too much liquid crystal inhibits cell adhesion.