Role of BCP crystals in regulating the chondrocytic phenotype in osteoarthritis

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Basic calcium phosphate (BCP) calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease. I have found in a previous study that BCP calcification is directly linked to hypertrophic differentiation of chondrocytes. One morphogen regulating chondrocyte differentiation during development is WNT. I found typical signs of OA cartilage changes in severely calcified ttw/ttw mice, which are a natural OA mouse model. OA in ttw/ttw mice was accompanied by an increased pericellular matrix sulfation. The same sulfation pattern was observed in human OA cartilage sections. Immunohistochemical staining showed an accumulation of WNT3a around chondrocytes with increasing OA severity, which could be removed using heparitinase, indicating a binding of Wnt3a to heparansulfate proteoglycans (HSPG). I found an increase in 6-O sulfotransferase expression in human OA cartilage. In line with this finding, β-catenin staining was increased with increasing OA grade in mouse and human cartilage. Explaining this phenomenon I found that BCP crystals were able to bind Wnt3a and induced canonical WNT signalling in monolayer chondrocytes. This effect could be blocked by the extracellular Wnt inhibitor DKK 1. Investigating the effect of BCP crystals on the chondrocyte phenotype, I found a shift towards hypertrophy with increased collagen X and MMP 13, as well as downregulated Sox 9 and aggrecan expression. The calcification of articular cartilage seems to be associated with activation of canonical WNT signalling and subsequent hypertrophic differentiation of chondrocytes. I hypothesize that BCP crystals concentrate Wnt3a in the pericellular matrix, thereby increasing the availability of Wnt3a. The chondrocytes are more prone to induce canonical Wnt signalling with increasing OA severity due to the increased 6-O sulfation of HSPGs. BCP mineralization of extracellular matrix is not an epiphenomenon, but an active step in further perpetuating chondrocyte differentiation in osteoarthritis. The individual questions are:1.) How do BCP crystals interact with chondrocytes at receptor level? Is the LRP receptor or the HSPGs important? Is the negative charge of HSPGs important for the growth of mineralisation? Can the same changes in negative HSPGs be observed during endochondral bone formation?2.) Which intracellular pathways are activated by BCP crystals? Is the non-canonical WNT/Ca2+/ CamKII pathway responsible for the BCP effect? Are other Ca2+ dependent pathways involved in BCP induced chondrocyte differentiation? How do BCP crystals induce changes in the sulfation pattern of HSPGs? Is the induction of HS6ST1 dependent on Ca2+? Are changes in HS6ST1 age dependent?3.) How is the binding of proteins to the surface of BCP crystals mediated? Is it due to lipidation of secreted proteins? Can the dissolution of BCP crystals or the change in HSPG sulfation eliminate the Wnt3a effect?