Evaluation of chondrogenesis and osteogenesis in human mesenchymal stem cells, chondrocytes, and chondroprogenitors using molecular markers, cellular markers and polarized microscopy

Fibronectin adhesion assay progenitors (FAA-CPs) and migratory assay progenitors (MCPs), subsets of mesenchymal-like stromal cells (MSCs), exhibit superior in-vitro chondrogenic potential compared to bone marrow (BM)-MSCs and chondrocytes. To assess this potential, differentiation studies followed by confirmatory staining for collagen deposition are utilized. Polarized light microscopy (PLM), based on birefringence principles, is a valuable tool for visualizing organized collagen fibers. Its use as a predictive tool for assessing chondrogenesis and osteogenesis has not been reported.

This study involved FAA-CPs, MCPs, chondrocytes, and BM-MSCs derived from osteoarthritic knee joints (n = 3). After phenotypic characterization, the cells underwent chondrogenic and osteogenic differentiation, followed by Picrosirius red staining and PLM analysis, including immunohistochemical analysis for collagen types I, II, and X.

Birefringence assessment revealed greater collagen fibril alignment and significant remodeling in the BM-MSC group, which exhibited an arcade-like pattern. The MCP group displayed well-organized collagen fibrils in pericellular zones and as a peripheral band, while chondrocytes and FAA-CPs exhibited lower intensity birefringence, indicating random alignment. Areas with higher collagen type II deposition corresponded to reduced collagen type I and the absence of collagen type X, highlighting the unique fibrillar network seen with PLM was indicative of collagen type II.

While its application for osteogenesis was limited, probably due to the non-fibrillar nature of collagen type X, its value for chondrogenesis is notable. Although not directly reflecting chondrogenesis, PLM can serve as a valuable tool for gaining insights into collagen remodeling, particularly concerning collagen type II during chondrogenic differentiation.