Type II collagen levels correlate with mineralization by articular cartilage vesicles.

Pathologic calcification commonly occurs in the extracellular matrix of articular cartilage affected by severe osteoarthritis (OA). Two types of calcium-containing crystals dominate: calcium pyrophosphate dihydrate (CPPD) crystals, which are relatively unique to articular cartilage, and basic calcium phosphate (BCP) crystals, which are similar to the hydroxyapatite mineral of bone and other calcified tissues. While the mechanisms of BCP and CPPD crystal formation have not been fully elucidated, small extracellular organelles known as articular cartilage matrix vesicles (ACVs) have been implicated in this process (1). These organelles can be enzymatically isolated from both porcine and human articular cartilage; and when provided with calcium and a source of phosphate or pyrophosphate, they generate BCP and CPPD crystals identical to those found in human osteoarthritic joints (2) (3). ACVs are easily isolatable from both normal and osteoarthritic cartilage (1). When removed from their extracellular milieu, normal porcine ACVs mineralize more effectively than ACVs derived from human osteoarthritic cartilage (1). As ACVs rarely mineralize in normal cartilage, this observation suggests a potential role for the normal extracellular matrix in suppressing ACV mineralization. Certainly, in other models, changes in extracellular matrix with osteoarthritis and aging facilitate pathologic crystal formation. For example, increased extracellular activity of the matrix-modifying transglutaminase enzymes promotes CPPD crystal formation in chondrocyte monolayers (4). While there is ample evidence that factors such as levels of ATP or phosphate and levesl of enzymes regulating pyrophosphate/phosphate ratio affect mineralization (2), we recently showed that the ability of ACVs to generate pathologic calcium crystals was also affected by their extracellular milieu (5). In these studies, isolated ACVs were embedded in agarose gels containing various extracellular matrix components and their mineralization behavior was measured. We showed that the type II collagen found in normal cartilage suppressed ACV-induced CPPD crystal formation, while the combination of type I and II collagens seen in osteoarthritis stimulated both CPPD and BCP crystal formation. Collagens did not affect activity of mineralization-regulating enzymes on ACVs. Thus, how collagens regulate ACV mineralization remains unknown. The behavior of growth plate matrix vesicles (GPMV) during normal bone growth has served as a paradigm for exploring the role of ACVs in pathologic articular cartilage mineralization. In contrast to type II collagen's effect on ACVs, Kirsch and Wuthier showed that mineral formation by GPMVs was stimulated by type II collagen, likely through annexin V-type II collagen interactions (6). GPMVs tightly bound type II and X collagens via annexins I, II and V, alkaline phosphatase, and link protein (7). In contrast, we observed very little type II collagen in isolated porcine ACVs, while annexin V, CD44 and link protein were easily detectable (5). Collagens and collagen receptors on ACVs remain otherwise uncharacterized. Articular chondrocytes, from which ACVs are derived, contain multiple collagen receptors and binding proteins including integrins, annexins, and NG2, a proteoglycan which binds type VI collagen (8), a key collagen in the pericellular matrix. Recently, the discoidin domain receptors (DDRs) have joined this group. DDR2 preferentially binds type II collagen and is up-regulated in osteoarthritis, where receptor activation triggers catabolic protease release (9). The purpose of the current work was to further characterize collagens and collagen receptors on porcine and human ACVs released enzymatically from whole cartilage and to determine if this might explain the differences in biomineralization of isolated ACVs. We show here that the persistence of type II collagen on isolated human ACVs could explain the seemingly paradoxical mineralization behavior of human ACVs ex vivo.