Unraveling the Mechanisms of Hypertrophy-Induced Matrix Mineralization and Modifications in Articular Chondrocytes

Chondrocyte hypertrophic differentiation is a main event leading to articular cartilage degradation in osteoarthritis. It is associated with matrix remodeling and mineralization, the dynamics of which is not well characterized during chondrocyte hypertrophic differentiation in articular cartilage. Based on an in vitro model of progressive differentiation of immature murine articular chondrocytes (iMACs) into prehypertrophic (Prehyp) and hypertrophic (Hyp) chondrocytes, we performed kinetics of chondrocyte differentiation from Prehyp to Hyp to follow matrix mineralization and remodeling by immunofluorescence, biochemical, molecular, and physicochemical approaches, including atomic force microscopy, scanning electron microscopy associated with energy-dispersive X-ray spectroscopy (SEM-EDS), attenuated total reflection infrared analyses, and X-ray diffraction. Chondrocyte apoptosis was determined by TUNEL assay. The results show the formation of a mineral phase 7 days after Hyp induction, which spreads within the matrices to form poorly crystalline carbonate-substituted hydroxyapatite after 14 days, then the proportions of crystalline relative to amorphous content increases over time. Hyp differentiation also induced a matrix turnover that occurs over the first 7 days, characterized by a decrease in type II collagen and aggrecan and the concomitant appearance of type X collagen. This is accompanied by an increase in the enzymatic activity of MMP-13, the main collagenase in cartilage. The number of apoptotic chondrocytes slightly increased with Hyp differentiation and SEM-EDS analyses detected phosphorus-rich structures that could correspond to apoptotic bodies. Our findings highlight the mechanisms of matrix remodeling events leading to the mineralization of articular cartilage that may occur in osteoarthritis.