Abstract: BACKGROUND: The outcome of large osteochondral defects is poor. Double-layer or multi-layer scaffolds have become a new treatment strategy because they are more consistent with cartilage anatomy and mimicry. 
OBJECTIVE: To investigate the repair effect of nerve growth factor/type II collagen-silk fibroin-chitosan/type I collagen-silk fibroin-chitosan-nano hydroxyapatite cartilage and subchondral bone bionic scaffold in the repair of cartilage defects.  
METHODS: Double-layer scaffolds loaded with type II collagen-silk fibroin-chitosan/type I collagen-silk fibroin-chitosan-nano hydroxyapatite were prepared by freeze-drying, emulsification, and solvent evaporation. The physical and chemical properties of the scaffolds were tested and the appropriate proportion of scaffold components was selected for animal experiments. A double-layer scaffold containing nerve growth factor was prepared by immersion in nerve growth factor sustained-release microsphere solution. Thirty New Zealand white rabbits were randomly divided into three groups (n=10). An osteochondral defect model was established. The blank control group did not receive any treatment. The control group was implanted with a double-layer scaffold without nerve growth factor. The experimental group was implanted with a double-layer scaffold loaded with nerve growth factor. The gross observation of cartilage repair and the morphological observation of cartilage tissue were performed 4, 8 and 12 weeks after operation.
RESULTS AND CONCLUSION: (1) The relevant results of porosity, water absorption expansion rate, and hot water dissolution rate concluded that when the mass ratio of type II collagen:silk fibroin:chitosan in the upper scaffold was 1:1:1, and the mass ratio of type I collagen:silk fibroin:chitosan:nano-hydroxyapatite in the lower scaffold was 1:1:1:1. The double-layer scaffold had good physical and chemical properties and was suitable for animal experiments. (2) The average particle size of the sustained release of nerve growth factor microspheres was (25.87±6.54) μm, the drug loading capacity was 0.516 μg/mg, and the encapsulation rate was 40.5%. (3) In animal experiments, the gross observation results showed that the cartilage repair of the experimental group was faster than that of the control group and the blank control group, and the repair effect was the best. Hematoxylin-eosin staining and alxin blue staining showed that fibrous tissue and a small number of cartilage lacunae were visible in the defect area of the blank control group at 12 weeks after surgery; most of the repaired tissue in the damaged area of the control group was fibrocartilage, and the subchondral bone was not completely rebuilt, and the boundary between the osteochondral layer was not clear. In the experimental group, the repair between the cartilage layer and the subchondral bone layer was complete, the boundary was obvious, and the tide line was visible. Immunohistochemical staining showed that type II collagen in the repaired cartilage of the experimental group was more than that in the control group and the blank control group at 12 weeks after surgery. (4) The double-layer bionic scaffold of cartilage and subchondral bone with good physical and chemical properties was successfully prepared in the experiment. The scaffold loaded with nerve growth factor could better promote the repair of rabbit cartilage defects.

Key words: double-layer scaffold, nerve growth factor, sustained-release microsphere, cartilage defect, tissue engineering, collagen, biocompatibility