Human osteoarthritic joint explant model reflects diverse disease-relevant changes in inflammation and degradation

Abstract

Purpose: Immune cell-mediated inflammation has been implicated in the pathogenesis and progression of osteoarthritis (OA). However, our current understanding of the phenotype and interactions between joint resident and infiltrating immune cells is not comprehensive enough to appropriately guide the design of new inflammation-targeting OA therapeutics. We have developed a human OA joint explant model containing osteoarthritic synovium and cartilage that more accurately represents the human immune condition. The explants contain joint cell types within their endogenous matrix, including resident synovial macrophages and fibroblasts that are drivers of inflammation and fibrosis. The purpose of this study is to optimize a human OA joint explant model to be representative of the human condition by maintaining in vivo morphology, viability, and responsiveness to inflammation-mediated changes in gene expression and extracellular matrix (ECM) dynamics. Methods: Cartilage and synovium are obtained from late-stage OA knee replacement. Full-depth 1.5 mm diameter punch-cut cartilage and minced (fat removed) synovial tissue are randomly distributed by wet weight into 24-wells or transwells inserts for 48 hours before co-culture. Pro-inflammatory cytokine conditions are used as positive controls (oncostatin M + interleukin 1B). Treatment conditions or other cell types can be co-cultured with the joint explants for 2-day or 7-day time points. Explant conditions are evaluated by cartilage, synovium, and conditioned medium harvest: viability (resazurin), tissue gene expression (RT-qPCR), secreted cytokine/protease (immunoassay) and proteoglycan (GAG) content (dimethylmethylene blue), and morphological (H&E) and ECM (Safranin-O) histology. Synovium tissue is digested for flow cytometry of fibroblast and polarized monocyte/macrophage surface markers. Conditions are compared by principal component analysis for gene panels and analysis of variance for single dependent variable assays. Results: 48-hour cartilage gene expression demonstrates distinct clusters between the pro-inflammatory cytokine control and cartilage alone control conditions within a 26-gene panel capturing catabolic, anabolic, and inflammatory/chemotactic genes (N=12; ΔΔCT values relative to cartilage alone control in PCA plot). The pro-inflammatory control upregulates genes such as IL6, CCL2, MMP1, MMP13, ADAMTS4 while downregulating ACAN, PRG4, COL2A1, TIMP1. The synovium-cartilage co-culture cluster overlaps with the control group, but a subset of this condition is shifted towards the pro-inflammatory control cluster. GAG loss into the medium was significantly increased with the pro-inflammatory positive control (N=18, P<0.0001). The mean GAG loss is higher in the synovium-cartilage co-culture but is not statistically significant. These changes are observable by GAG staining of histological sections. Synovium digest reveals presence of CD90+ fibroblasts and CD45+CD14+ monocytes/macrophage before and after explant co-culture. Conclusions: Our human OA joint explant model is viable and demonstrates changes relevant to inflammation and cartilage degradation in response to pro-inflammatory insult. Co-culture of synovium and cartilage as our baseline OA joint condition shows a shift towards the pro-inflammatory response. Increasing the sample size will allow us to annotate patient subsets based on their clinical outcomes to identify subsets with different levels of synovium-derived inflammatory involvement in their disease, and therefore understand the scope of our model. We position our explant model as a useful complement to in vivo animal models in understanding immune contribution to OA progression and for developing new OA treatment strategies ranging from small molecule immunomodulators to cell-based therapies.