Preparation Of Photo-crosslinked Hyaluronic Acid-polyethylene Glycol Hydrogel Scaffold Andits Effects On Proliferation And Osteogenic Differentiation Of BMSCs

Objective:（1）The photo crosslinked hyaluronic acid polyethylene glycol hydrogel scaffolds with different concentrations were prepared,the physical and chemical properties of the hydrogels were characterized,and the biocompatibility was tested.（2）Isolation,culture and identification of rabbit bone marrow mesenchymal stem cells in vitro;（3）Rabbit BMSCs/ hyaluronic acid-polyethylene glycol hydrogel complexes were constructed;The effect of hydrogel on proliferation and differentiation of bone marrow mesenchymal stem cells was studied.Methods:（1）Preparation of photo crosslinked hyaluronic acid polyethylene glycol hydrogel scaffold: EDC,NHS,N-（3-aminopropyl）methyl acrylamide was successively added to hyaluronic acid aqueous solution.Adjust the p H of the reaction mixture to 6.5,and react at room temperature for 12 h.After 12 h,equal amounts of EDC,NHS and N-（3-aminopropyl）methyl acrylamide are added,and the reaction continues for 12 h.Then freeze-drying 3d;Photo-crosslinked hyaluronic acid-polyethylene glycol diacrylate hydrogels with different concentrations were prepared by mixing a certain amount of methacrylated hyaluronic acid and polyethylene glycol diacrylate with photo-crosslinked agent under UV irradiation for a certain time.The internal structure of hydrogels with different concentrations was observed by electron microscopy,the swelling rate of hydrogels with different concentrations was detected,and the degradation performance of hydrogels was simulated in vitro.The compression modulus was measured by universal mechanical testing machine,and the cytotoxicity of hydrogels with different concentrations of photo-crosslinked hyaluronic acid and polyethylene glycol was compared by CCK8 method.（2）Isolation,culture and identification of rabbit BMSCs: BMSCs were isolated,cultured and purified by whole bone marrow adherent method.The cell morphology was observed by light microscope and the surface markers were detected by flow cytometry to identify the cells.CCK8 method was used to compare the proliferation of stem cells with different concentrations of hydrogel.The group with the best proliferation effect and BMSCs were selected for osteogenic induction and differentiation culture for 21 days.Compared with the simple petri dish group,the effect of hydrogel on BMSCs osteogenic differentiation was studied.ARS,ALP qualitative and quantitative analysis and fluorescence quantitative PCR were used to measure the expression of marker genes related to osteogenic differentiation.Results:（1）It was confirmed that hyaluronic acid had been methacrylated by Fourier transform infrared spectrometer and nuclear magnetic resonance hydrogen spectrometer,and methacrylated hyaluronic acid was successfully synthesized.By dissolving a certain amount of methacrylated hyaluronic acid,polyethylene glycol diacrylate and LAP in aqueous solution and irradiating 30 seconds with UV light,photo-crosslinked hyaluronic acid-polyethylene glycol composite hydrogel scaffolds with different concentrations can be successfully prepared.Compared with H3P1 and H2P1 groups,the pores in H4P1 concentration group have compact overall structure,complete pore wall circumference,smaller pore diameter and higher density.Through swelling experiment,the swelling property of H4P1 is lower than that of H3P1 and H2P1 groups.By simulating the degradation of human hyaluronic acid,it was confirmed that the degradation rate of H4P1 group was lower than that of H3P1 and H2P1 concentration groups.According to the mechanical properties of hydrogel,the compression modulus of H4P1 group was higher than that of H3P1 and H2P1 concentration groups.By using crosslinking agent LAP and I2959 respectively,it was concluded that the gel promoting time of crosslinking agent LAP was faster than that of I2959.CCK8 experiment proved that the absorbance of H4P1 concentration group was higher than that of H3P1 and H2P1 concentration group,and it had good biocompatibility.（2）Bone marrow mesenchymal stem cells were successfully extracted by identifying the adherent cells.CCK8 experiment showed that hydrogel H4P1 concentration group had higher absorbance and better proliferation effect than H3P1 and H2P1 concentration groups.Real-time quantitative PCR showed that the expression of osteogenic marker genes Runx2 and OPN of bone marrow mesenchymal stem cells combined with hydrogel was significantly increased compared with blank control group（P<0.05）,indicating that the photo-crosslinked hyaluronic acid-polyethylene glycol hydrogel could promote the osteogenic differentiation of bone marrow mesenchymal stem cells.Conclusion:（1）After the addition of photo-crosslinked agent,HA-Ac and PEGDA can quickly synthesize photo-crosslinked hyaluronic acid-polyethylene glycol composite hydrogel scaffold,which has good porosity,compression modulus,swelling rate and biocompatibility;In different concentrations of photo-crosslinked hyaluronic acid-polyethylene glycol hydrogels,H4P1 group had more dense pores,stronger compression modulus,lower swelling rate,less degradation and better promoting cell proliferation and differentiation than H2P1 and H3P1 groups.（2）Bone marrow mesenchymal stem cells with good activity can be isolated and cultured by whole bone marrow method;Photo-crosslinked hyaluronic acid-polyethylene glycol hydrogel can promote the proliferation and osteoblast differentiation of the cells.