In Vitro Induction of Hypertrophic Chondrocyte Differentiation of Naïve MSCs by Strain

In the context of bone fractures, the influence of the mechanical environment on the healing outcome is widely accepted, while its influence at the cellular level is still poorly understood. This study explores the influence of mechanical load on naïve mesenchymal stem cell (MSC) differentiation, focusing on hypertrophic chondrocyte differentiation. Unlike primary bone healing, which involves the direct differentiation of MSCs into bone-forming cells, endochondral ossification uses an intermediate cartilage template that remodels into bone. A high-throughput uniaxial bioreactor system (StrainBot) was used to apply varying percentages of strain on naïve MSCs encapsulated in GelMa hydrogels. This research shows that cyclic uniaxial compression alone directs naïve MSCs towards a hypertrophic chondrocyte phenotype. This was demonstrated by increased cell volumes and reduced glycosaminoglycan (GAG) production, along with an elevated expression of hypertrophic markers such as MMP13 and Type X collagen. In contrast, Type II collagen, typically associated with resting chondrocytes, was poorly detected under mechanical loading alone conditions. The addition of chondrogenic factor TGFβ1 in the culture medium altered these outcomes. TGFβ1 induced chondrogenic differentiation, as indicated by higher GAG/DNA production and Type II collagen expression, overshadowing the effect of mechanical loading. This suggests that, under mechanical strain, hypertrophic differentiation is hindered by TGFβ1, while chondrogenesis is promoted. Biochemical analyses further confirmed these findings. Mechanical deformation alone led to a larger cell size and a more rounded cell morphology characteristic of hypertrophic chondrocytes, while lower GAG and proteoglycan production was observed. Immunohistology staining corroborated the gene expression data, showing increased Type X collagen with mechanical strain. Overall, this study indicates that mechanical loading alone drives naïve MSCs towards a hypertrophic chondrocyte differentiation path. These insights underscore the critical role of mechanical forces in MSC differentiation and have significant implications for bone healing, regenerative medicine strategies and rehabilitation protocols.