DECELLULARIZED EXTRACELLULAR MATRIX PARTICLE-BASED HYDROGEL FOR CARTILAGE REGENERATION

A significant challenge in cartilage tissue engineering is the tendency of chondrocytes to dedifferentiate and lose their original functions during 2D expansion. To address this issue, we propose a biomimetic hydrogel composed of a tyramine hyaluronic acid derivative (THA) and decellularized extracellular matrix (dECM) particles as a novel strategy for modulating chondrocyte phenotype. dECM particles were produced from bovine cartilage tissue. In vitro experiments involved adding different concentrations of dECM particles (0%, 6%, 12%, and 20%) and bovine chondrocytes to THA hydrogels, which were crosslinked enzymatically and photochemically. For ex vivo experiments, a cartilage defect was created on bovine osteochondral explants using a biopsy punch, and the hydrogel containing chondrocytes and 20% dECM particles was implanted into the defect. After 1-week free swelling culture, explants were cultured within a bioreactor under dynamic loading for a 2-week period with dynamic axial compression at 0.1–0.2 mm, and ball oscillation at ±25°, 0.5 Hz for 1 hour/day. In vitro, dECM particles significantly upregulated the expression of ACAN, COL2, and SOX9 in chondrocytes within the hydrogel, with the 20% dECM group showing the highest expression levels by day 14. Compared with the THA group, COL1 expression was significantly downregulated in the 20% dECM group on day 14, and immunohistochemistry confirmed elevated type II collagen expression in this group. Compression tests demonstrated that the 12% and 20% dECM hydrogels had higher initial stiffness compared with THA only, with the 20% group maintaining superior mechanical stability over 14 days. In ex vivo study, under mechanical loading condition, the addition of 20% dECM upregulated ACAN, COL2 and SOX9 gene expression, and downregulated COL1 expression. In the THA-20% dECM group, mechanical loading upregulated ACAN, COL2, and SOX9 gene expression compared with unloaded conditions. THA-20% dECM hydrogels maintained higher GAG and collagen content compared with the THA group, regardless of the loading condition. Mechanical loading enhanced collagen content in the THA-20% dECM group compared with the THA group. Safranin O/Fast Green staining after 21 days revealed consistently high proteoglycan staining in the 20% dECM group, with no significant difference between loaded and unloaded conditions. Embedding dECM particles in cell-loaded hydrogels more closely replicates the natural composition and structure of cartilage, and effectively promotes chondrocyte redifferentiation. Under ex vivo condition, dECM particles demonstrated an enhanced chondrogenic differentiation effect under mechanical loading, evidenced by higher expression of chondrocyte markers and increased collagen content within hydrogels.