Faculty Bibliography

INTRODUCTION: The synovium of patients with osteoarthritis (OA), presenting with capsular synovial hyperplasia, has cartilage and bone debris bound to the surface or embedded within the tissue [1]. Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with OA. Despite the results of six decades of preliminary animal model and in vitro studies, which have confirmed the deleterious interaction of cartilage wear particles with the synovium, the mechanisms that mediate this interaction remain poorly understood [2-4]. Cartilage wear particles are inherently more complex to study than their non-biologic counterparts, as they can interact with the cells through phagocytosis or integrin binding as well as undergo degradation by cells. In the current study, we hypothesize that the physical interaction between fibroblast-like synoviocytes (FLS) and cartilage wear particles is mediated by phagocytosis and surface binding. METHODS: Tissue Harvest: Synovial tissue and articular cartilage explants were harvested from bovine calf knees (n=3). Synovial tissue was digested with collagenase IV, and the isolated FLS were plated and expanded for two passages. Cartilage explants were maintained in serum free media until use [5]. Particulate Generation: Cartilage particles were generated by manual abrasion with 120 grit sandpaper. The cartilage particle solution was filtered with a 10mum nylon mesh filter to achieve desired particle size. Latex (~ 1mum) and cartilage wear particles (~0.9mum) were counted and sized as in [6]. Imaging: FLS were cultured with FITC labeled latex particles or cartilage particles labeled with dichlorotriazinyl aminofluorescein, fixed and counterstained with TRITC phalloidin (actin) and DAPI (nuclei) after 48 hours. Immunohistochemistry: High density FLS monolayers were cultured for 5 days, fixed and stained for type I collagen and lubricin and counterstained with DAPI. Monolayer Culture: FLS were plated at high density overnight and co-cultured with 250 latex or cartilage particles per cell for 5 days (n=6-8) [7]. Particles alone were also cultured. Conditioned Media: Dense monolayers were cultured +/- 250 cartilage particles per cell for 5 days. In parallel, acellular media only and particle only groups were cultured under the same conditions. Conditioned media was removed from the four original culture groups (media only, particles only, FLS only, FLS + particles). After filtering out the remaining particles, the conditioned media was supplemented with fresh media before application to recipient FLS monolayers for 5 days. Biochemistry: DNA, collagen (COL), nitric oxide (NO) and prostaglandin E2 (PGE2) were determined using the Picogreen, DMMB, OHP, Griess and PGE2 Parameter assays, respectively. Biochemical content of cartilage particles alone was subtracted from the measured sample values to determine the contribution of SF alone. Statistics: Comparisons were analyzed using ANOVA with Tukey's post-hoc test (p<0.05). RESULTS: Similar to the native synovium, FLS organized into a multi-layer cell sheets with positive staining for collagen type I and lubricin (Figure 1A,B,C). Both latex and cartilage particles were capable of being phagocytosed by FLS. Cartilage particles, not only appeared internalized, but attached to the surface of both dense monolayers and individual cells. Staining for the actin cytoskeleton revealed nuclear (internal) and cortical (external) remodeling of the actin cytoskeleton around cartilage wear particles (Figure 1D), compared to only internal remodeling of latex particulates (Figure 1E). Co-culture of FLS with cartilage particulates for 5 days resulted in a significant increase in cell sheet DNA and collagen content compared to control and latex treated groups (Figure 2A,B). Co-culture with cartilage wear particles induced an inflammatory response as measured by the up-regulation of both NO and PGE2 synthesis compared to both control and latex treated samples. Latex particles only had a deleterious effect on collagen synthesis (Figure 2B). No differences in proliferation or extracellular matrix (ECM) synthesis were observed between media only and particle + media groups and FLS + media only and particles + media + FLS. As such, the response of FLS to cartilage particles appears to require particulate surface binding and/or phagocytosis (Figure 3). DISCUSSION: In this study, we developed a 2D model of the synovium to study the interaction of cartilage wear particles with FLS. Based on our findings, we accept our hypothesis that the physical interaction between FLS and cartilage wear particles is mediated by phagocytosis and surface binding. Both latex and cartilage wear particles showed re-arrangement of the actin cytoskeleton around the wear particulate (Figure1 D,E) as has been observed previously with titanium particles [8]. Here, the actin cytoskeleton remodeled at the surface to form a U-shaped organization around the cartilage particle contact site (Figure 1D). Cartilage particles induced more proliferation and secreted more inflammatory factors than latex particles alone. The proliferative response of FLS to cartilage wear particles resulted in an overall increase in ECM content, representative of the thickening of the synovial lining observed in OA patients. Additional insight into the underlying mechanisms that mediate cartilage particulate-FLS interactions may be gleaned from literature studies that have looked at cellular activation through ECM binding to FLS integrins using ECM (e.g. collagen, fibronectin, etc.)-coated beads [9]. These studies have shown that binding to ECM-coated beads increases cellular proliferation and decreases collagenase synthesis, which supports the present results for FLS co-culture with cartilage wear particles (Figure 2). Parallel studies pursued by our laboratory are investigating how the interaction of cartilage wear particles and FLS are affected by physical loading, such as fluid shear that arises in situ from synovial fluid-synovium interactions during joint articulation. (Figure Presented)

Objectives Degeneration of articular cartilage is central to OA pathology; however, the molecular mechanisms leading to these irreversible changes are still poorly understood. Here, we investigated how changes in the chondrocyte translational apparatus may contribute to the pathology of OA. Methods Normal and OA human knee cartilage was used to analyze the activity of different components of the translational machinery. Chondrocytes isolated from lesional and non-lesional areas of OA cartilage were used to estimate relative rate of protein synthesis by metabolic labeling. Experimental OA was induced by transection of the anterior cruciate ligament in rats to investigate changes in the translational apparatus associated with OA. The role of IL-1beta signaling was assessed in vitro using rat articular chondrocytes. Expression of mRNAs was analyzed by qPCR and protein levels by immunohistochemistry and Western blotting. Results We identified several novel traits of OA chondrocytes, including upregulation of the Serine/Threonine kinases AKT2 and AKT3 at the post-transcriptional level and increased rate of total protein synthesis, likely due to inactivation of 4E-BP1, a known repressor of cap-dependent translation. We found that 4E-BP1 inactivation is mTOR-dependent and crucial for upregulation of protein synthesis in general and in particular for MMP13 and ADAMTS5 expression. In addition, IL-1beta treatment led to 4E-BP1 inactivation and upregulation of protein synthesis in articular chondrocytes. Conclusions Precise control of protein synthesis is vital for cartilage homeostasis and its dysregulation contributes to the molecular pathology of OA. Our study therefore identifies a novel set of potential therapeutic targets