Abstract: BACKGROUND: Studies have found that crocin can inhibit the apoptosis of osteoarthritis chondrocytes, reduce the inflammatory response of osteoarthritis rats, promote bone formation and inhibit bone loss in osteoporotic rats, and has great application potential in cartilage and bone damage.
OBJECTIVE: To observe the effects of crocin hydrogel on chondrocytes and MC3T3-E1 cells.
METHODS: Crocin hydrogels were prepared by Schiff base crosslinking method by adding different concentrations of crocin solution (0, 15, 20, and 25 mmol/L) into oxidized hyaluronic acid -oxidized chondroitin sulfate -type II collagen mixed solution, and then four kinds of hydrogel extracts were prepared. Human chondrocytes were cultured with four kinds of crocin hydrogel extracts to detect glycosaminoglycan synthesis. Cell proliferation was detected by CCK-8 assay. Cell migration was detected by Transwell chamber assay. RT-qPCR was used to detect the mRNA expression of SOX9, type II collagen, and aggrecan in cells. Four kinds of crocin hydrogel extracts were co-cultured with MC3T3-E1 cells, respectively. Cell proliferation was detected by CCK-8 assay. RT-qPCR was used to detect the mRNA expression of RUNX2, type I collagen, alkaline phosphatase, and osteocalcin in cells. and the alkaline phosphatase activity and mineralized nodule formation after osteogenic differentiation.
RESULTS AND CONCLUSION: (1) CCK-8 assay results showed that 15 and 20 mmol/L crocin hydrogel extract promoted the proliferation of chondrocytes, while 25 mmol/L crocin hydrogel extract inhibited the proliferation of chondrocytes. 15, 20, and 25 mmol/L crocin hydrogel extracts could all promote the migration of chondrocytes, increase the expression of SOX9, type II collagen, aggrecan mRNA and glycosaminoglycan synthesis of chondrocytes in a dose-dependent manner. (2) 15, 20, and 25 mmol/L crocin hydrogel extract could promote the proliferation of MC3T3-E1 cells and increase the expression of RUNX2, type I collagen, alkaline phosphatase, and osteocalcin mRNA in cells after osteogenic differentiation in a dose-dependent manner. 15, 20, and 25 mmol/L crocin hydrogel extract could increase the alkaline phosphatase activity and mineralized nodule formation of MC3T3-E1 cells after osteogenic induction differentiation. Among them, 20 mmol/L crocin hydrogel extract is the most significant in improvement effect of the extract liquid. (3) The results show that crocin hydrogel can promote the proliferation and migration of chondrocytes and the synthesis of cartilage extracellular matrix, and enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. Among them, the comprehensive effect of 20 mmol/L crocin hydrogel is best.

Key words: crocin, hydrogel, chondrocyte, MC3T3-E1 cell, cell proliferation, cell migration, cell differentiation, engineered cartilage material