Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro

Abstract

IntroductionChondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.MethodsHuman articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.ResultsModerately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.ConclusionsPhysiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.

Temporal and Spatial Changes in Cartilage-Matrix-Specific Gene Expression in Mesenchymal Stem Cells in Response to Dynamic Compression

“…Interestingly, PCM staining for sGAG was similar in the core of the construct at day 7 and in the annulus at day 14, suggesting a role for the PCM in determining the response of MSCs to loading. Additionally, the local osmotic environment (tonicity), which is related to both the PCM and ECM, has been shown to regulate chondrocyte cartilage-matrix-specific gene expression and matrix production [39][40][41][42][43] and may be further regulated by the application of dynamic compression.…”

Mesenchymal stem cells secrete factors that inhibit inflammatory processes in short-term osteoarthritic synovium and cartilage explant culture

“…Reverse transcriptase (RT)-PCR primer nucleotide sequences are listed in Table I. Data were normalized to a best keeper index (BKI) of three reference genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), hypoxanthine-guanine phosphoribosyltransferase (HPRT1) 21 . Relative expression levels were calculated using the 2 ÀDDCt method 22 .…”

Voltage-Dependent Calcium Channels in Chondrocytes: Roles in Health and Disease

“…Chondrocytes are surrounded by a pericellular matrix and are situated in cavities called lacunae within the ECM, where they exist in a hypoxic micro-environment [3,4] and control ECM turnover and homeostasis in response to mechanical loading [5] despite having a rather slow (mainly anaerobic) metabolism. Due to the fact that chondrocytes reside within the peculiar milieu of the ECM (which is acidic, hypertonic and hyperosmotic [6][7][8]), and since their function and differentiation is strongly dependent on intracellular calcium homeostasis [9,10], they deploy a whole range of ion channels to enable ion transport across the plasma membrane; the collection of ion channels is referred to as the chondrocyte 'channelome' [11]. Albeit generally considered to be non-excitable cells, chondrocytes and chondroprogenitor cells have been reported to express various voltage-dependent potassium channels (KV), ATP dependent potassium channels (KATP), large and small conductance calcium-activated potassium channels (BK and SK), transient receptor potential channels (primarily TRPV1 and TRPV4), purinergic receptors (both P2X and P2Y subfamily members), voltage gated and epithelial sodium channels, chloride channels, and also voltage-dependent calcium channels (VDCCs) [11][12][13].…”