Discovery of potential therapeutics for post-traumatic osteoarthritis using a high throughput mechanical injury platform

Abstract

Purpose: The aim of this study was to utilize a high throughput cartilage injury platform that can be used as a model of post-traumatic osteoarthritis (PTOA) and rapidly assess a large number of compounds that are relevant to many cell functions after compressive injury. We developed and validated an in vitro high throughput mechanical injury (HiTMI) platform for investigating PTOA using engineered cartilage. The injury response in this model mimics that of native cartilage, and enables screening of chemical libraries for therapeutic discovery in a “high” throughput manner. Here we describe the screening of a number of small molecule compound libraries targeting various pathways using this HiTMI platform. Our goal was to identify ‘hits’ (i.e. significant regulators) that might serve as novel therapeutics for early intervention in PTOA. Six libraries were evaluated, including natural and synthetic compounds with potential to regulate a variety of cell responses, such as apoptosis, cell signaling, and cell proliferation. We probed compounds libraries that contained protease, kinase, and phosphatase inhibitors, all critical to cell function. An additional natural compound library and one focused on anti-apoptosic molecules was also utilized. Rapidly identifying compounds that may affect cell behavior and health in the acute phase post injury could be timely and of significant clinical importance to this growing form of osteoarthritis. Methods: Our model consists of engineered cartilage tissue analogs (CTA), which have been extensively characterized in our laboratory. In this model, chondrocytes are cultured in high density cultures above a hydrogel coating (poly 2-hydroxyethyl methacrylate) to prevent cell attachment. Within 24 hours, chondrocytes coalesce to form a stable construct that remains in suspension and increases in mass with time. Chondrocytes in CTAs possess phenotypic characteristics and deposit ECM that is similar to native cartilage. Since CTA can be generated in large numbers (100’s), each bearing an identical profile, they were used in our screening studies using a multi-platen mechanical injury device we recently fabricated. This device uses an Instron to deliver a single compressive injury, similar to that which occurs in a clinical setting. We have previously shown this to mimic the injury and cell responses in cartilage explants. CTA (4-5 months) were compressed to 75% strain at a rate of 50% strain/sec. Immediately following injury, CTA were treated with library compounds at 10 υM. Since this is a HTS platform, all primary screenings were carried out for 48 hours post injury/treatment. Medium was collected to assess proteoglycan loss (by measuring released or soluble glycosaminoglycans (GAG) using the DMMB assay) and indirectly measure cell stress / death (by measuring LDH using the CytoToxONE Homogenous Membrane Integrity Assay), two hallmarks of acute cartilage injury. Additional secondary screening was performed on positive hits using early (48 h) and late (96 h) time points and extended dose ranges. Results: Using this cartilage analog injury platform, we screened a total of 428 molecules. Primary screening resulted in 5 positive hits in the protease inhibitor, 9 in the apoptosis, 8 in the phosphatase, 10 in the kinase, and lastly 12 in the natural compound library (Figure 1). For example, results from the kinase inhibitor library revealed that there were 10 compounds that met the ‘hit’ criteria based on the released sGAG. These molecules included included PDGFR tyrosine kinase inhibitors. Conversely, for example, one compound, AG-370 resulted in worsening of the response (increased sGAG and LDH). The third phosphatase library screening resulted in identification of potential hits including compounds inhibitors of calcineurin. From 23 compounds screened through secondary screening, and based on a 20% or better threshold, more than 3 of these compounds remain significantly positive with respect to GAG release, relative to the injury-control. The compounds, with activities specific to pan-caspase inhibition, tyrosine phosphatase inhibition, and JNK-pathway signaling activation, resulted in a released-GAG reduction of 43%, 24% and 25%, respectively (Figure 2). Conclusions: Collectively, the results of this study present a promising strategy for identification of potential PTOA related therapeutic targets. This system permits the rapid identification of compounds that may act to interfere with the chondrocyte’s response to injury, both in regard to the early cell death and the later matrix degradation that occurs. With the successful follow up of positive hits with additional molecular pathway assays and an in vivo injury animal model, this new testing platform has the potential to identify new and early therapy for this common clinical problem of trauma-related osteoarthritis,View Large Image Figure ViewerDownload Hi-res image Download (PPT)