Study On The Formation Of Temperature-sensitive F127-DOPA-HRP Hydrogel And Its Treatment Effect Of Articular Cartilage Defects

Objectives:The main aim of this study is to prepare temperature-sensitive F127-DOPA-HRP hydrogel,evaluate its related performance and explore its effect on mesenchymal stem cells(MSC)in vitro and in vivo,and its ability to repair cartilage defects,providing a possible new method for cartilage defect repair.Methods:1.Synthesize F127-DOPA-HRP hydrogel on the basis of F127 poloxamer monomer using 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide(EDC)chemical branch method and oxidation using HRP and hydrogen peroxide;2.Evaluate the infrared spectroscopy and nuclear magnetic spectroscopy-based molecular structure,phase transition temperature,electron microscopy-based microscopic morphology,rheological properties,adhesive properties one by one;3.MSC-based experiments in vitro: plant mesenchymal stem cells on F127-DOPA-HRP hydrogel and use electron microscopy,calcein AM and safranin o and fast green staining to observe the morphology,adhesion,aggregation and chondrogenesis of mesenchymal stem cells;4.Cartilage defect repair in vivo: a 2mm x 2mm cartilage defect was constructed using a dental drill in the sulcus of the rat femur,and F127-DOPA-HRP was injected to the defect site.Results:1.Infrared spectroscopy and nuclear magnetic spectroscopy results:on the basis of F127 poloxamer monomer,the DOPA group was successfully introduced by the chemical branch method to form a temperature-sensitive F127-DOPA-HRP hydrogel,which is fast at 20 degrees Celsius for complete phase change;2.Electron microscopy results: F127-DOPA and F127-DOPA-HRP have clear and obvious three-dimensional network and microscopic pore structure,and both are denser than F127,proving that F127-DOPA and F127-DOPA-HRP hydrogels have a good 3D structure;3.Rheological test results: Compared with F127 hydrogel(6.28±0.305),F127-DOPA hydrogel has a higher shear elastic modulus(10.53±0.586,p<0.001);after adding HRP,compared with F127-DOPA hydrogel,the shear elastic modulus of F127-DOPA-HRP is further enhanced(16.03±0.585,p<0.0001);4.Adhesion test results: Compared with F127 hydrogel(0.68±0.153 N,p<0.0001),F127-DOPA hydrogel(0.27±0.103 N,p<0.0001)F127-DOPA-HRP 0.5% hydrogel(0.20±0.042 N,p<0.0001),and F127-DOPA-HRP 2% hydrogel(2.42±0.049 N,p<0.0001),F127-DOPA-HRP1% hydrogel has the highest adhesive force(3.26±0.028 N);5.In vitro cell experiment results: 24 h CCK8 experiment showed compared with the control group,F127(p=0.999),F127-DOPA-HRP0.5%(p=0.999),F127-DOPA-HRP 1%(p=0.149)and F127-DOPA-HRP2%(p=0.631)had no significant difference;48h CCK8 experiment showed compared with the control group,F127(p=0.577),F127-DOPA-HRP 0.5%(p=0.740),F127-DOPA-HRP 1%(p=0.326)and F127-DOPA-HRP 2%(p=0.363)had no significant difference;electron microscopy,calcein AM and safranin o and fast green staining show that F127-DOPA-HRP hydrogel can effectively support the adhesion,aggregation and chondrogenesis of mesenchymal stem cells group;6.In vivo cartilage defect repair experiment results: After adding F127-DOPA-HRP hydrogel to the defect site for 4 weeks,compared with the operation group(5.00±0.707),the F127-DOPA-HRP hydrogel filling group achieved better repair score(9.40±0.894,p<0.0001).Conclusion:F127-DOPA-HRP hydrogel has good temperature responsiveness and rheological properties,a certain degree of mechanical strength,adhesive capacity,and biocompatibility.It can also effectively support the growth of mesenchymal stem cells and repair articular cartilage defects with possibility to be a potential method of cartilage repair.