Abstract

Purpose: Osteoarthritis (OA) is characterized by the gradual degeneration of articular cartilage, bone remodeling, and synovial inflammation. The canonical Wnt/β-catenin pathway has recently been identified as a critical pathway contributing to the pathogenesis of OA by regulating key cellular processes in chondrocytes such as differentiation, inflammation, and production of catabolic proteinases responsible for cartilage matrix degradation in the joint space. In this study, we investigated the effects of micronized dehydrated human amnion-chorion membrane (μdHACM) on normal chondrocytes, the resident cell type of articular cartilage and further evaluated Wnt signaling and matrix metalloproteinase-13 (MMP-13) expression and activity. Methods: Normal human articular chondrocytes (NHACs) isolated from cadaveric knees were obtained from Lonza and formed into 3D pellets in 96-well V-bottom plates coated with 5% Pluronic-F127 to prevent cellular attachment to the surface. This model allowed for NHACs to properly maintain a chondrogenic phenotype and prevent de-differentation to a fibroblast-like phenotype, as measured by quantitative real time PCR (qRT-PCR) for major components of articular cartilage, including collagen type II alpha 1 chain (COL2A1) and aggrecan (ACAN). Cells were cultured in chondrocyte differentiation medium for seven days until stimulation with 10 ng/mL of pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and Oncostatin M (OSM) in basal chondrocyte differentiation medium. Following 24 hours of stimulation, NHACs were treated with varying doses of μdHACM soluble extract (5 mg/mL, 2.5 mg/mL, 0.2 mg/mL) in basal medium for 24 and 48 hours. NHACs were analyzed by qRT-PCR for the expression of target genes critical in the progression of OA. For secreted MMP-13 protein analysis, total protein was first precipitated from conditioned medium (CM) using trichloroacetic acid (TCA)/sodium deoxycholate (DOC), followed by western blotting. For intracellular protein analysis, cell lysate was prepared using RIPA buffer containing protease and phosphatase inhibitors and subjected to sonication to extract total protein. The lysate was subsequently probed for β-catenin and phosphorylated/total glycogen synthase kinase 3β (GSK3β), both key regulators of the canonical Wnt signaling pathway. MMP-13 enzymatic bioactivity in CM was monitored using SensoLyte® Plus 520 MMP-13 Assay (AnaSpec) designed for specifically detecting MMP-13 activity by using a monoclonal anti-human MMP-13 antibody to pull down both pro and active forms of MMP-13. Continuous measurement of MMP-13 activity was recorded using a 5-FAM (fluorophore) and QXL® 520 (quencher) labeled fluorescence resonance energy transfer (FRET) peptide substrate according to the manufacturer’s instructions. Results: TNFα (10 ng/mL) and OSM (10 ng/mL) stimulation of chondrocyte pellets mimicked the key inflammatory and proteolytic signature of OA pathology. In particular, we observed that TNFα/OSM treatment significantly increased the expression of pro-inflammatory marker (MCP-1), three catabolic enzymes (MMP-3, MMP-13 and ADAMTS-4), hypertrophic marker (RUNX2), and the Wnt-related regulatory genes (TCF4, TCF7, LEF1, WISP1, and CCND1). The effectiveness of TNFα/OSM to activate the canonical Wnt/β-catenin pathway in our study is consistent with previously published reports demonstrating TNF as a key inducer of regulatory molecules of the Wnt pathway (Diarra et al, Nature Medicine, 2006). 48 hours of treatment with soluble extracts of μdHACM downregulated expression of MCP-1 (p<0.001), MMP-3 (p=0.004), MMP-13 (p<0.001), ADAMTS-4 (p=0.012), RUNX2 (p=0.048), TCF4 (p<0.001), TCF7 (p=0.001), LEF1 (p<0.001), WISP1 (p=0.008) and CCND1 (p<0.001) genes. We have also identified μdHACM-mediated Wnt inhibition as the major upstream signaling pathway involved in protecting chondrocytes from cytokine-induced inflammation and catabolic degradation. In particular, we have shown that μdHACM not only effectively inhibits the transcriptional regulation of Wnt target genes, but also significantly reduces β-catenin protein levels and Ser9-phosphorylation of GSK3β. We further evaluated MMP-13 due to being the most common downstream target that is upregulated during OA (Goldring et al, Eur Cell Mater, 2014). μdHACM was effective at abolishing MMP-13 protein expression in CM, as measured by western blot analysis. This was consistent with the complete inhibition of MMP-13 enzymatic activity (p<0.001), as demonstrated by the Sensolyte assay. Conclusions: In this study, we have uncovered a novel mechanism for μdHACM-mediated protection of chondrocytes in an in vitro model for OA. μdHACM ameloriated the effects of inflammatory-induced OA in chondrocytes through canonical Wnt inhibition and subsequent downregulation of key inflammatory factors, hypertrophic marker and catabolic enzymes involved in collagen degradation, in particular MMP-13. Collectively, these studies support μdHACM as a promising candidate for a disease modifying osteoarthritic therapy due to its ability to attenuate key degradative pathways mediated by chondrocytes in articular cartilage.

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