Objective: Chondrogenic differentiation of mesenchymal stem cells (MSCs) into hyaline cartilage is complicated by terminal hypertrophic differentiation. In growth plate, parathyroid hormone-related peptide (1-34) (PTHrP) plays a crucial role in maintaining chondrocytes in their proliferation state by counteracting the hypertrophic differentiation. This study aims to test the effect of PTHrP supplementation at different time points on chondrogenic differentiation of MSCs and assess the final quality of differentiated chondrocytes.
Methods: Human periosteum and bone marrow MSCs isolated from 3 patient samples (donor unmatched) were characterized by flow cytometry and multilineage differentiation. The cells were differentiated into chondrocytes in the presence of transforming growth factor-β (TGF-β) and the PTHrP (1-34) was added from 4th or 14th day of culture. The outcome was analyzed by histology, immunohistochemistry, and gene expression.
Results: Flow cytometry and multilineage differentiation confirmed that the cells isolated from periosteum and bone marrow exhibited the phenotype of MSCs. During chondrogenic differentiation, pellets that received PTHrP from the 4th day of culture showed a significant reduction in hypertrophic markers (COL10A1 and RUNX) than the addition of PTHrP from the 14th day and TGF-β alone treated samples. Furthermore, 4th day supplementation of PTHrP significantly improved the expression of cartilage-specific markers (COL2A1, SOX9, ACAN) in both periosteum and bone marrow-derived MSCs. Histology and immunostaining with collagen type X data corroborated the gene expression outcomes.
Conclusion: The outcome showed that supplementing PTHrP from the 4th day of chondrogenic differentiation produced better chondrocytes with less hypertrophic markers in both bone marrow and periosteal-derived MSCs.
Keywords: bone marrow MSCs; chondrocyte hypertrophy; chondrogenic differentiation; parathyroid hormone–related peptide; periosteum MSCs.