CARTILAGE TGF-BETA · Functional role of endogenous latent TGF-beta activation in the intrinsic repair of mechanically loaded articular cartilage

Functional role of endogenous latent TGF-beta activation in the intrinsic repair of mechanically loaded articular cartilage

Osteoarthritis (OA) is widespread debilitating condition of the synovial joint, initiated by the degeneration of articular cartilage, leading to pain and severe limitations in mobility. Adult cartilage is generally described as having a poor ability for repair as manifested in OA. Yet, healthy human joints are able to function for 8 to 9 decades, a duration that far exceeds the lifecycle of any artificial bearing material. Thus, despite its poorly repairing reputation, healthy articular cartilage is clearly able to maintain itself remarkably well. This Marie Curie fellowship has focused on developing an improved understanding of this maintenance ability in an attempt to better understand the mechanisms that defeat normal tissue maintenance and lead to degeneration and OA. In particular, this fellowship examines the novel hypothesis that articular cartilage maintenance is advanced in part through the action of large stores of latent TGF-beta sequestered in the cartilage extracellular matrix. In response to physiologic mechanical loading, TGF-beta undergoes activation and in turn maintains the structural stability of the cartilage extracellular matrix. In response to excessive loading, TGF-beta activation is unable to counterbalance degradation, leading to the progressive degeneration of the tissue. The long term goal of this research is to develop fundamental insights in the functional role of latent TGF-beta in articular cartilage that can be used for the development of novel OA treatment strategies: 1) molecular inhibition strategies to slow the progression of the disease and 2) tissue engineering strategies for the development of superior replacement articular cartilage. All project objectives were achieved and the project is considered a big success. The significant results obtained during this Marie Curie IIF fellowship can be summarized into the following key projects: Project 1. Identification of functional role of endogenous stores of latent TGF-beta in articular cartilage: The primary proposed aim of the Marie Curie IIF was to elucidate the functional role of latent stores of TGF-beta in articular cartilage. As described in the Midterm Report, an experimental in vitro system was developed to examine the effect of endogenous TGF-beta on cartilage subjected to dynamic mechanical loading. Through this system, we have demonstrated for the first time that, as hypothesized, endogenous TGF-beta plays an important functional role in protecting articular cartilage that is subjected to mechanical loading. This protection comes in the form of maintaining the tensile integrity of the collagen matrix and maintaining chondrocyte viability when the tissue is mechanically loaded. This finding sheds new light on the intrinsic repair capacity of articular cartilage. An abstract of this work was selected as a finalist for the New Investigator Recognition Award at the 2015 Orthopaedic Research Society Annual Meeting. A full-length paper on this work is currently in preparation. Project 2. Biomimetic conjugation of latent TGF-beta to hydrogel scaffolds for cartilage tissue engineering: A long-term goal of the proposed Marie Curie IIF project was to utilize insights into the functional role of TGF-beta in native articular cartilage in order to develop novel strategies in the field of cartilage tissue engineering. To this end, during this Marie Curie IIF fellowship we have adopted a novel biomimetic cartilage tissue engineering strategy whereby latent TGF-beta is conjugated to a cell encapsulated scaffold in order to enhance cellular biosynthetic activity and tissue growth. This strategy mimics the native environment of articular cartilage where chondrocytes are surrounded by large stores of this latent growth factor. In native cartilage, these stores undergo cell-mediated activation and thereby regulate, or enhance, the biosynthetic activity of chondrocytes. This strategy is capable of addressing several of the key challenges in cartilage tissue engineering, including, 1) Reducing abnormal growth heterogeneities through the application of uniform growth factor activity, and 2) Reducing cellular hypertrophy through the delivery of modified growth factor activity. Several novelties have been implemented in order to successfully complete this project. We have developed a novel acrylate modified agarose hydrogel scaffold, which readily accepts growth factors for conjugation while maintaining agarose’s well-characterized exceptional ability to support chondrogenesis and cartilage biosynthesis. Further, we have implemented a novel strategy of nucleophilic catalysis in order to provide efficient conjugation of latent TGF-beta to hydrogel scaffolds. Currently, the final stage of this project is underway, whereby we are monitoring the effect of conjugated latent TGF-beta on the growth of engineered cartilage. An abstract of this project was presented at the 2016 Orthopaedic Research Society Annual Meeting. A full length manuscript of this work will be submitted over the next several months. Project 3. Hyperspectral Raman imaging as a novel tool for quantifying the distribution of ECM in native and engineered cartilage The ability to faithfully characterize the make-up and distribution of the cartilage extracellular matrix is critical for the long-term objectives of the projects initiated during this fellowship: 1) Characterizing the distribution of ECM degeneration, or damage, in mechanically loaded articular cartilage, and 2) Characterizing the distribution of newly deposited ECM in growing tissue engineered cartilage. Quantifications of the distribution of ECM in cartilage tissues has remained a challenge, as routine histological techniques provide only qualitative information and are associated with large artifacts from sample preparation procedures. To address this challenge, we investigated the potential of an exciting new optical technique, hyperspectral Raman imaging, to quantify the distribution of ECM constituents in cartilage tissues. Through this technique, the Raman spectra of a cartilage tissue are acquired at discrete positions throughout a sample. A technique termed multivariate curve resolution (MCR) is applied to deconvolve the spectra of the dominant molecular components in the tissue (glycosaminoglycans, collagen, water), giving rise to the relative concentration distribution of each component in the tissue. We have demonstrated that hyperspectral Raman imaging can faithfully quantify the distributions of cartilage ECM in both native and engineered cartilage tissues. As such, this methodology can now be implemented as a routine technique for producing rapid high-resolution quantifications of the make-up and distribution of ECM in cartilage tissue. This technique can now serve as an invaluable tool to characterize the quality and integrity of engineered cartilage. An abstract on this project was awarded a prestigious podium presentation at the 2016 Orthopaedic Research Society Annual Meeting. A full-length manuscript of this work is currently in preparation and will be submitted to the journal Osteoarthritis and Cartilage. There was no issue on management or project planning; experimental procedures and training activities were planned at least 1-2 months in advance. There was no change on legal status. Planned milestones and deliverables of the project were not affected. In addition to the implementation of improvements to the concept of the project, it evolved into different projects with exploitable results. There were no gender or ethical issues. There was no subcontracting. Regarding management costs, as previously described in part 1, there was no deviation between actual and planned researcher-months.

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