Articular cartilage has limited intrinsic repair capacity, and evaluation of chondrogenic differentiation commonly relies on extracellular matrix indicators. Collagen type II immunohistochemistry identifies the fibrillar cartilage framework, whereas Safranin O staining reflects proteoglycan-rich ground substance; both predominantly represent later stages of maturation rather than early cellular commitment. Integrin α10ß1, a collagen type II receptor restricted to cartilage, may instead correspond to earlier phenotypic stages. This study examined the temporal relationship between integrin α10ß1, collagen type II and bone morphogenetic protein-9 (BMP9) during chondrogenesis of human bone-marrow-derived mesenchymal stromal cells (BM-MSCs). BM-MSCs were isolated from bone marrow aspirates obtained during total knee replacement, and characterized by morphology, flow cytometry and gene expression analysis. Chondrogenesis was induced using pellet culture, and samples were analyzed at days 7, 14, 21, 28 and 35 by Safranin O histology and immunohistochemistry for collagen type II, integrin α10ß1 (CD11c) and BMP9.
Cells displayed spindle-shaped morphology and expression of CD105, CD73 and CD90, with minimal hematopoietic markers. Chondrogenic gene expression (SOX9, ACAN and COL2A1) was detected with relatively low hypertrophic marker expression. Integrin α10ß1 staining appeared from early time points in cellular and pericellular regions, preceding prominent extracellular matrix formation. Collagen type II and Safranin O staining became appreciable mainly at later stages, particularly after extended differentiation. BMP9 immunostaining showed diffuse and variable localization without consistent stage association. CD11c corresponds to the cellular phase of chondrogenic commitment, whereas collagen type II identifies fibrillar cartilage formation, and Safranin O reflects proteoglycan accumulation at later stages. Early assessment based solely on matrix markers may underestimate chondrogenic progression.
