Vascular calcification is a hallmark of atherosclerosis, which is associated with vascular smooth muscle cell (VSMC) trans-differentiation into chondrocyte-like cells. Crystal formation by hypertrophic chondrocytes relies on the hydrolysis of the mineralization inhibitor inorganic pyrophosphate (PPi) by tissue nonspecific alkaline phosphatase (TNAP). Since PPi depletion in NPP1-/- mice induces chondrogenesis-associated vascular calcification (Johnson, Arterioscler Thromb Vasc Biol 2005), we hypothesized that TNAP participates in VSMC trans-differentiation. We used MOVAS mouse VSMC line and mouse primary articular chondrocytes cultured in DMEM 4.5 g/L glc, 10% FBS, ascorbate, and b-GP, and measured alcian blue staining of sulfated GAGs, TNAP activity, alizarin red staining of calcium and mRNA levels of differentiation markers with real-time PCR. In MOVAS cells, addition of alkaline phosphatase (AP) increased alcian blue staining and a ggrecan levels, and accelerated hypertrophy, as demonstrated by increased expression of type X collagen and osteocalcin, and accelerated mineralization. In support for a role of TNAP in chondrocyte differentiation, treatment of primary chondrocytes with the TNAP inhibitor levamisole reduced alcian blue staining, dropped the levels of type X collagen and osteocalcin and prevented mineralization. Recent observations suggest that microcalcifications form early during atherosclerosis, before the appearance of cartilage metaplasia. We thus speculated that TNAP modulates chondrocyte differentiation through crystals. To address this point, we prepared artificial matrices consisting of type I collagen left unmineralized or mineralized by incubation with AP and phosphate donors. Alizarin red staining and FTIR spectroscopy indicated the presence of apatite crystals in collagen fibrils. These apatite-collagen matrices tended to increase Sox9 and aggrecan expression in MOVAS cells as co mpared with unmineralized ones, suggesting that TNAP stimulates VSMC trans-differentiation into chondrocyte-like cells through apatite crystals. This finding deserves further investigation since microcalcification and subsequent cartilage formation have been respectively associated with atherosclerotic plaque destabilization (Maldonado, Am J Physiol Heart Circ Physiol 2012) and stabilization (Falk, Circulation 1995).