Purpose: Joint injuries, specifically destabilizing injuries that involve the meniscus and ligaments, are well-known risk factors for osteoarthritis (OA). OA is characterized by chronic progressive joint tissue degeneration and debilitating pain that significantly effects an individual’s quality of life. Currently, no treatment can prevent progression of OA after injury, and limited options for early and intermediate stages of disease are only provide partial symptomatic relief. The goal of this study was to identify novel chondro-protective compounds using a high-throughput cartilage injury system that functions as a model of post-traumatic osteoarthritis (PTOA). We utilized an in vitro high throughput mechanical injury (HiTMI) platform to screen over 500 small molecule compound libraries to identify potential early modulators of the cartilage injury response. We have previously established a unique injury response model that mimics that of native cartilage, and enables fast and reliable screening of chemical libraries for therapeutic discovery in a “high” throughput manner. Here we describe identification of top performing compounds and the mechanisms or pathways they may potentially improve in cell response to joint injury. In total, we screened 6 libraries containing 523 natural and synthetic compounds involved in a variety of cell responses, including matrix degradation, apoptosis, and inflammation. The libraries consisted of natural compounds, redox compounds, and variety of inhibitors critical for cell function, including protease, kinase and phosphatase inhibitors. Six compounds rose to the top from a series of primary and secondary screenings that had either effects on reducing matrix degradation or apoptosis, or both. Our platform rapidly can identify compounds that affect cell behavior and health in the acute phase post injury. Here, we explore the effect of the top compounds on cell function and inflammatory cytokines to elucidate the pathways they affect. As more is learned about their mechanism of action these or other similar or same family compounds could of significant clinical importance to this growing form of OA.
Methods: We use cartilage tissue analogs (CTA) that mimics native tissue. In this model, bovine chondrocytes are cultured at high density cultures above a hydrogel coating (poly 2-hydroxyethyl methacrylate) preventing cell attachment. Within 24 hours, chondrocytes coalesce to form a stable construct that possess phenotypic matrix and mechanical characteristics that are similar to native cartilage. CTAs are well-suited for high-throughput testing, since they can be generated in large numbers and each bear an identical profile. We use an Instron to deliver a single compressive injury similar to that which occurs in a clinical setting. Immediately following injury, CTAs were treated with library compounds at 10 μM for 2 and 4 days. For primary screenings medium was collected to assess matrix loss by measuring released glycosaminoglycans by DMMB assay and indirectly measured for cell stress/death (LDH) CytoToxONE, Roche), two hallmarks of acute cartilage injury. Following our primary screens, additional secondary screens were performed on positive hits, in which we selected molecules which reversed the inflammatory and catabolic events (MMP activity) induced my IL-1β. In our secondary screen we measured apoptosis (TUNEL assay) and MMP activity in 4-day media using the SensoLyte ® 520 Generic MMP assay. Inflammatory biomarkers were assessed in 4-day media by using a human-bovine cross-reacting multiplex immunoassay system (BioRad®) where 6 biomarkers were effectively assayed using the ProTM inflammatory 37-plex panel.
Results: Using this HiTMI platform and CTAs we screened a total of 523 small molecules from 6 libraries. The selection of most active compounds was achieved by successive primary screenings (38 hits) using the GAG assay as a surrogate to proteoglycan degradation (32 hits) and the LDH assay (6 hits) a measure of cell stress. Of these initial 38 hits, we then identified 6 high performing compounds based on secondary screens using apoptosis and MMP assays (Fig. 1A). The final 6 chondroprotective compounds with therapeutic potential are: triptolide, (S)-10-hydroxycamptothecin, RWJ-60475, Z-VAD-FMK, trphostin 9, and SB-20219. Such drugs that can prevent early events of matrix degeneration following injury, can ultimately reduce structural disruption, inhibit catabolic processes, and maintain and or improve function of cartilage. To further explore the mechanism/pathways these compounds are having a positive effect on we examined a panel of inflammation biomarkers. Of the biomarkers tested IL- 11,19, 32, and 35, and MMP2 were affected by at least one of the 6 compounds. In fig. 1B-D we highlight those biomarkers (MMP2, IL-11, and IL-35) most strongly influenced by treatment. MMP2, a key factor involved in the pathogenesis of OA, was decreased in response to several compound (fig. 1b). Interestingly, both IL-11 and IL-35 have been studied in the clinical setting and are considered biomarkers for several inflammatory diseases including rheumatoid arthritis.
Conclusions: Collectively, Results of this study present a novel strategy for the identifying PTOA related compounds with therapeutic potential. Our system can rapidly identify novel compounds that act to interfere with the cartilage injury response, and affect inflammation and matrix degradation. With the successful follow up of these top compounds with additional molecular pathway characterization and in vivo animal injury model, this new testing platform has the potential to identify new and early therapy for this common clinical problem of PTOA.
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