High Tough And High Cell Adhesive PVA-HA-HLC Composite Hydrogel For Cartilage Repair

PVA hydrogels have desirable properties for use as soft tissue substitutes,are non-toxic,non-carcinogenic,have strong mechanical strength and good biocompatibility and are widely used in biomedical research.However,the toughness of PVA hydrogels is not sufficient to withstand the requirements of human load-bearing environments.At present,studies have shown that glycosaminoglycan can be used as a plasticizer to increase the toughness and viscoelasticity of PVA hydrogel and greatly increase its compressive modulus.However,the poor biological properties of PVA hydrogels are still not a tissue engineering scaffold.Therefore,by adding human-like collagen(HLC)to PVA-HA hydrogel,the cell affinity is increased to improve biocompatibility.Then,tween 80 was added as a pore-forming agent to increase the overall porosity,thereby preparing a PVA-HA-HLC composite hydrogel with high toughness and high cell adhesion.Afterwards,the experimental conditions were optimized by orthogonal experiment,and the ideal hydrogel was obtained and the relevant performance was detected and analyzed.Physical and chemical properties include macro and microstructure,pore size and porosity determination,swelling ratio,compressive strength and cyclic compression testing.Biological performance tests include cytotoxicity tests,experiments on the growth of hydrogel-adhered cells by live cell staining experiments,and hemolysis experiments,We mixed the hydrogel extract with animal blood to see if hemolysis occurred.Finally,we performed rabbit cartilage defect repair.Experiments to investigate whether PVA composite hydrogel can treat cartilage defects.Research indicates:The optimal preparation process of the novel sponge-like macroporous polyvinyl alcohol(PVA)hydrogel is as follows:hyaluronic acid(HA)dissolved at room temperature(molecular weight1000000 g/mol,concentration 0.6 wt%),and then heated to 90?dissolved(molecular weight89000-98000,alcoholysis degree?99%)of polyvinyl alcohol,the concentration is 7 wt%,and then the temperature is lowered to 37?dissolved concentration of 4 wt%human-like collagen(HLC),dissolved Add 4%tween 80 after completion.After stirring evenly,pour into the 24-well plate of the mold,freeze-thaw once,and then freeze at-20?for 8 h,then take out and melt at room temperature for 2-3 h.Then,it was washed with ultrapure water for 3 days,and water was changed every 4 hours to obtain a PVA~S(PVA-HA-HLC)hydrogel.In the same manner,a PVA~C(PVA-HA)hydrogel obtained without adding HLC was used as a control group.We conducted two experiments of SEM observation and porosity measurement.The results showed that the pore diameter of PVA-HA-HLC hydrogel was between 100-300?m,and dense pores of 10-20?m were formed on the pore walls.The hydrogel is filled with large pores that are staggered and connected,with a porosity of up to 94.5%..The PVA-HA hydrogel has a small pore structure,and the pore wall is smooth and non-porous,and the porosity is only 60%.PVA~S can be quickly absorbed by water after being squeezed in the wet state;in the dry state,the swelling rate can reach about 1200%in 30 s,while the swelling rate of PVA~C hydrogel in the dry state is only about 30 s.150%.In addition,mechanical properties experiments show that the compressive modulus and compressibility of PVA~S hydrogels are 5.56 MPa and 90%,respectively,while the compression modulus and compression ratio of PVA~C hydrogels are 3.34MPa and 80%,respectively.Cytotoxicity experiments showed that both hydrogels have good cytocompatibility,and the toxicity grade is 0,which meets the national biomaterial standards;the experimental results of animal hemolysis indicate that there is no erythrocyte hemolysis in both hydrogels;Adhesion experiments show that mouse chondrocytes can adhere to the pores and pore walls of PVA~S hydrogels and can grow longitudinally into the hydrogel and have good viability.On the PVA~C hydrogel,only a small amount of adhesion to the surface is easy to be eluted by PBS.The results of cartilage repair test showed that after the hydrogel was implanted into rabbits,there were no symptoms such as redness and ulceration,inflammation was small,and inflammation disappeared with the increase of implantation time.After 12 weeks,the cartilage in the blank group did not heal.HE showed that there were many inflammatory cells in the damaged area and could not form new tissue.The cartilage defect in the PVA~C group partially healed,forming new tissue with less inflammation.The defect in the PVA~S group completely healed.HE showed that the new tissue and chondrocytes were filled with damaged sites and the inflammation disappeared.Safranin O-solid green staining experiments showed that there were a large number of chondrocytes in the tissues recovered in the PVA~S group,and only a small amount of chondrocytes in the PVA~C group,and almost no chondrocytes in the blank control group.Therefore,the innovative use of human-like collagen(HLC)and hyaluronic acid(HA)bioactive materials to enhance PVA hydrogels,solve the major defects of PVA hydrogel biological activity,and we use tween 80 to preparate a novel sponge-like macroporous cartilage modified hydrogel with high porosity,fast water absorption rate,excellent mechanical properties and good cell adhesion was prepared by phase separation of the system,and PVA prepared by the common method of freeze-melt physical crosslinking.Compared with the hydrogel,the mechanical strength,toughness and viscoelasticity of the hydrogel are greatly improved,and the pore size and porosity of the hydrogel are imparted,which solves the significant disadvantage of the cell adhesion of the PVA hydrogel.