Abstract: BACKGROUND: Traditional Chinese medicine-loaded bioscaffolds have been a hot topic and difficulty in recent years. The construction of drug-loaded composite bioscaffold materials provides new ideas and methods for tendon-bone healing.
OBJECTIVE: To explore the role of icariin/hydroxyapatite/methacrylylated silk fibroin hydrogel in promoting tendon-bone healing.
METHODS: (1) Methacrylylated silk fibroin hydrogel (denoted as S hydrogel), hydroxyapatite/methacrylylated silk fibroin hydrogel (denoted as S-H hydrogel) and icariin/hydroxyapatite/methacrylylated silk fibroin hydrogel (denoted as S-H-I hydrogel) were prepared respectively, and the microscopic morphology, physicochemical properties and in vitro drug release properties of the hydrogels were characterized. (2) MC3T3-E1 cells were inoculated on the surfaces of the above three hydrogels, and cell proliferation and activity were detected by CCK-8 assay and live-dead staining. Cell morphology was observed by cytoskeleton staining, and alkaline phosphatase staining and alizarin red staining were performed after osteogenic induction. (3) Eighteen New Zealand rabbits were selected, and the native anterior cruciate ligament was cut along the starting and ending points of the left femur and tibia to construct a tendon-bone injury model. A bone tunnel with a diameter of 3 mm and a length of 10 mm was made along the starting and ending points of the anterior cruciate ligament. The rabbits were randomly divided into three intervention groups: the blank control group (n=6) did not implant any material in the bone tunnel, the control group (n=6) implanted S-H hydrogel in the bone tunnel, and the experimental group (n=6) implanted S-H-I hydrogel in the bone tunnel. The samples were collected 4 and 8 weeks after surgery and stained with hematoxylin-eosin and Msaaon, respectively.
RESULTS AND CONCLUSION: (1) The hydrogels in the three groups all had loose porous structures and similar microscopic morphologies. The pore size of S hydrogel was the largest, and the pore size of S-H-I hydrogel was the smallest. The hydrogels in the three groups all showed the characteristics of swelling and degradation that was fast first and then slow. The compression modulus of S was the largest, and the compression modulus of S-H hydrogel was the lowest. The icariin in S-H-I hydrogel showed the characteristics of initial rapid release and later slow release. (2) CCK-8 assay and live-dead staining showed that S-H-I hydrogel could promote the proliferation of MC3T3-E1 cells and maintain cell activity. Cytoskeleton staining showed that the S-H-I group had the highest cell density, complete and clear skeleton structure, and interconnected cells; alkaline phosphatase staining and alizarin red staining showed that S-H-I hydrogel had the strongest osteogenic ability. (3) Hematoxylin-eosin staining and Msaaon staining showed that compared with S-H hydrogel, S-H-I hydrogel could further promote the orderly arrangement and tissue construction of cells in the tendon-bone injury site, reduce inflammatory cell infiltration, and promote collagen fiber maturation and orderly arrangement. (4) The results showed that icariin/hydroxyapatite/silk fibroin hydrogel had good mechanical properties, biocompatibility and osteogenic induction ability, and could promote tendon-bone healing.

Key words: silk fibroin, hydroxyapatite, icariin, light-cured hydrogel, tendon-bone healing, tendon-bone injury