BACKGROUND: Evaluating cartilage repair potential involves subjecting the cellular 3D pellets to chondrogenic differentiation and assessing their differentiation degree using immunohistochemical (IHC) studies. Collagen type II IHC staining stands as a key marker to evaluate mature chondrogenesis, but its non-uniform staining confounded by incomplete differentiation necessitates exploring alternative markers. This study explored the potential of BMP9 and Integrin Alpha 10 as novel early-stage chondrogenic markers in human bone marrow-derived mesenchymal stem cells (BM-MSCs), fibronectin assay derived chondroprogenitors (FAA-CPs), migratory chondroprogenitors (MCPs) and chondrocytes following differentiation.

METHODS: Three human articular cartilage samples and bone marrow aspirate from osteoarthritis patients were collected to isolate the BM-MSCs, FAA-CPs, MCPs and chondrocytes. Following their molecular characterization, differentiation assays were conducted followed by their confirmatory staining and IHC for BMP9 and Integrin alpha10.

RESULT: IHC analysis collagen type II revealed positive with a non-uniform pattern of expression. The same sections subjected to integrin alpha 10 IHC analysis exhibited a greater proportion of uptake and a more uniform distribution throughout the differentiated pellet, addressing a key limitation observed with ECM and collagen staining techniques. The spatial homogeneity observed could be attributed to the functional relevance of integrin alpha 10, a Collagen type II receptor which mediates cell-matrix interactions and is specifically expressed in chondrocytes, further underscoring its potential as an alternative marker. However, BMP9 did not display robust staining.

CONCLUSION: Integrin alpha 10 may serve as a valuable alternative or complementary marker to collagen type II for assessing chondrogenic differentiation, offering a more consistent spatial distribution.