Superficial zone chondrocytes secrete a factor that regulates deep zone cartilage mineralization by modulating polyphosphate levels

Abstract

Purpose: Articular cartilage is an avascular tissue with a complex depth-dependent zonal architecture with unique matrix composition and organization, vital for maintaining joint function. Osteoarthritis (OA) is a degenerative disease that disrupts this zonal arrangement and manifests initially as loss of the superficial zone (SZ) followed by progressive matrix loss and advancement of the calcified zone into the remaining articular cartilage region. We have shown previously that polyphosphate, a naturally occurring linear polymer of phosphates, can regulate mineralization in articular cartilage. The hypothesis of this study is that superficial zone cells modulate calcification of deep zone cartilage (DZ) by modulating levels of cartilage polyphosphate levels through a soluble factor. Methods: Articular chondrocytes were isolated from either the SZ or DZ of bovine cartilage obtained from the metacarpal-phalangeal joints (6-9 months). The cells were seeded individually on 3D membrane inserts in conditions that favour formation of cartilage tissue and grown in side-by-side co-culture for up to 7 days in mineralization inducing media (MIM, β glycerophosphate). As a control DZ cells were grown in co-culture with DZ cells. To determine if SZ cells were producing a soluble factor, SZ cells were grown alone and the resulting conditioned media (SZ-CM) was collected daily and added to cultures of DZ cells to determine its effect on mineralization. To examine the role of FGF signalling in regulating mineralization, DZ cells were treated with FGF-18 (10ng/mL), or CM supplemented with an FGF-receptor inhibitor (PD173074 60nM) in MIM. Calcium and phosphate contents were determined biochemically. Representative tissues were fixed in formalin and embedded in paraffin. Sections were stained with Von kossa. Polyphosphate (PP) was visualized by staining un-waxed histological sections with DAPI, which shifts the emission spectra to the yellow-green range when it binds to PP. PP was quantified following extraction from the tissues by measurement of phosphate levels after alkaline phosphatase digestion. A minimum of three independent experiments were performed and each condition was done in duplicate or triplicate. Results are expressed as mean ± standard error of the mean and analysed using a one-way analysis of variance followed by Tukey’s post hoc test. Statistical significance was assigned at p < 0.05. Results: Side by side co-culture of DZ cells with SZ cells inhibited DZ mineralization, compared to co-culture with DZ, as demonstrated by significantly decreased calcium and phosphate content and decreased mineral deposits observed by Von Kossa staining. Treatment with SZ-CM inhibited DZ mineralization, resulting in significantly less calcium and phosphate content compared to treatment with DZ-CM suggesting that the cells were producing a soluble factor. SZ-CM was unable to inhibit mineralization after heat treatment or trypsin digestion, suggesting that the factor is a protein. Polyphosphate was present within the matrix of tissue grown in non-mineralizing conditions, and staining diminished in mineralized tissue. Polyphosphate levels were significantly higher in cartilage co-cultured with SZ cells, compared to DZ cells. FGF-18 has been shown to attenuate hypertrophic differentiation and we investigated whether this might be the factor responsible for influencing mineralization. Treatment of DZ cells with FGF-18 mimicked the effects of the SZ-CM, with significantly less mineralization and more polyphosphate accumulation within the tissue. Furthermore, treatment of DZ cells with SZ-CM in the presence of the FGF-receptor inhibitor rescued mineralization, and significantly reduced polyphosphate accumulation within cartilage formed by DZC. Conclusions: SZ cells release a soluble factor, likely a protein as it is sensitive to heat treatment and trypsin digestion, that inhibits mineralization of cartilage formed by DZ cells by up-regulating PP. Further investigation is required to determine if SZ cells secrete a factor that directly or indirectly, by up-regulating FGF-18 expression, inhibits mineralization of cartilage formed by DZC, and whether a single factor or combination of factors is required.