Abstract: BACKGROUND: Osteochondral defect of the joint is a difficult problem faced by orthopedic surgeons, and traditional repair methods are difficult to obtain satisfactory curative effects. Hydroxyapatite-polyvinyl alcohol-based composite hydrogel material is a direction of current research. 
OBJECTIVE: To prepare hydroxyapatite-polyvinyl alcohol/collagen-chitosan-gelatin composite hydrogel material and characterize its physical characteristics, to verify its histocompatibility and cell adhesion and proliferation ability after implantation in vivo, and explore its repair effect on rabbit osteochondral defects.
METHODS: The cylindrical porous poly(lactic acid) scaffold was prepared by 3D printing technology (the pore sizes were 1.2, 1.4, 1.6 and 1.8 mm, respectively). The poly(lactic acid) scaffold was injected with polyvinyl alcohol and hydroxyapatite mixed emulsion. After freezing thawing and dichloromethane dissolution, hydroxyapatite-polyvinyl alcohol hydrogel was prepared. Then, the collagen-chitosan-gelatin mixture was injected into the hydroxyapatite-polyvinyl alcohol hydrogel and crosslinked with genipin. Finally, the hydroxyapatite-polyvinyl alcohol/collagen-chitosan-gelatin composite hydrogel was prepared by alcohol cleaning and freeze-drying. The physical characteristics of the four groups of hydrogels were characterized, and the hydrogels with the best performance were screened for follow-up experiments. Hydroxyapatite-polyvinyl alcohol hydrogel and collagen-chitosan-gelatin composite hydrogel were implanted subcutaneously in SD rats. Hematoxylin-eosin staining and Masson staining were used to observe the adhesion growth of cells on the material surface. Osteochondral defect (diameter: 5 mm, depth: 6 mm) models were made in the femoral trochlea of bilateral knee joints of 15 rabbits. The composite hydrogel was implanted on the left side (experimental group), while no material was implanted on the right side (control group). Micro-CT and histology were used to evaluate the repair effect of osteochondral defects.
RESULTS AND CONCLUSION: (1) Based on the results of porosity, water content, mechanical testing and scanning electron microscopy, it was concluded that the hydroxyapatite-polyvinyl alcohol/collagen-chitosan-gelatin composite hydrogel with a pore size of 1.2 mm was more consistent with the general characteristics of natural cartilage, which was used for subsequent experiments. (2) Hematoxylin-eosin staining and Masson staining exhibited that with the extension of subcutaneous implantation time of the materials, the adhesion of cells around the two materials increased significantly, and the proliferation of cells after the implantation of collagen-chitosan-gelatin was better, a large number of cells could be seen growing into the formed network structure, and the network structure was gradually degraded. (3) In the rabbit osteochondral defect experiment, 8 weeks after surgery, Micro-CT examination demonstrated that the material implanted in the experimental group had good integration with the surrounding bone-cartilage, with some bone growth on the surface and inside, while the cartilage and subcartilage in the control group still had obvious defects, without effective repair. Hematoxylin-eosin staining and toluidine blue staining displayed that the composite hydrogel in the experimental group integrated with the surrounding articular cartilage 4-8 weeks after implantation. With the extension of time, new cartilage gradually formed on the surface of the material. At 12 weeks, most of the defect was covered by new cartilage, and good bone growth was also observed in the subcartilage. In the control group, the deep bone defects were mostly repaired and the superficial cartilage and subchondral bone defects were also repaired to a certain extent, but they were mainly replaced by fibrous tissue and part of fibrocartilage 12 weeks after surgery. (4) In conclusion, hydroxyapatite-polyvinyl alcohol/collagen-chitosan-gelatin composite hydrogel material can mimic the structure and function of natural cartilage, and can effectively repair osteochondral defects in animal experiments. 

Key words: hydroxyapatite, polyvinyl alcohol, collagen, chitosan, hydrogel, osteochondral defect