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Recreating articular cartilage tri-layered patterning for an engineered in vitro cell construct holds promise for advancing cartilage repair efforts. Our approach involves the development of a mul-tichambered perfusion tissue bioreactor that regulates fluid shear stress levels similar to the gradated hydrodynamic environment in articular cartilage. COMSOL modeling reveals our ta-pered cell chamber design will produce three different shear levels, high in the 22 – 41 mPa range, medium in the 4.5 – 8.4 mPa range, and low in the 2.2 – 3.8 mPa range and distributed across the surface of our mesenchymal stromal cell (MSC) encapsulated construct. In a 14-day bioreactor culture, we assess how fluid shear magnitude and cell vertical location within a 3D construct influence cell chondrogenesis. Notably, Sox9 expression for MSCs cultivated in our reactor shows spatially patterned gene upregulations coding for key chondrogenic marker pro-teins. Beginning with the high shear stress region, lubricin and type II collagen gene increases of 410 and 370-fold indicate cell movement towards a superficial zone architype which is further supported by histological and immunohistochemical stains illustrating the formation of a dense proteoglycan matrix enriched with lubricin, versican, and collagen types I and II molecules. For the medium shear stress region high aggrecan and type II collagen gene expressions of 2.3 and 400-fold, respectively, along with high proteoglycan analyses show movement toward a superfi-cial/mid-zone cartilage architype. For low shear stress regions higher collagen types II and X gene upregulations of 550 and 8,300-fold, the latter being 2x of that for the high shear regime, indicate cell movement with deep zone characteristics. Collectively, biochemical analysis, histol-ogy, and gene expression data demonstrated that our fluid shear bioreactor induced a stratified structure within tissue engineered constructs, demonstrating the feasibility of using this ap-proach to recapitulate the structure of native articular cartilage. 

Abstract Post‐traumatic osteoarthritis develops following an inciting injury to a joint and results in cartilage degeneration. Mechanical loading, including articulation, drives anabolic responses in cartilage clinically, in vivo, and in vitro. Tribological articulation, or sliding of cartilage on a glass counterface, has long been used as an in vitro tool to study cartilage tissue behavior. However, it is unclear if tribological articulation affects chondrocyte fate following injury, and if the timing of articulation impacts the resultant effect. The goal of this study was to investigate the effect of tribological articulation on injured cartilage tissue at two time points: (i) performed immediately after injury and (ii) 24 h after injury. Neonatal bovine femoral cartilage explants were injured using a rapid spring‐loaded impactor and subsequently subjected to tribological articulation. Cell death due to impact injury was highest near the articular surface, suggesting a strain‐dependent mechanism. Immediate articulation following injury mitigated cell death compared to injury alone or delayed articulation; markers for both general cell death and early‐stage apoptosis were markedly decreased in the explants that were immediately slid. Interestingly, mitigation of cell death due to sliding was most predominant at the cartilage surface. Tribological articulation is known to create fluid flow within the tissue, predominantly at the articular surface, which could drive the protective response seen here. Altogether, this work shows that perturbations to the cellular environment immediately following cartilage injury significantly impact chondrocyte fate. 

Abstract The low friction nature of articular cartilage has been attributed to the synergistic interaction between lubricin and hyaluronic acid in the synovial fluid (SF). Lubricin is a mucinous glycoprotein that lowers the boundary mode coefficient of friction of articular cartilage in a dose‐dependent manner. While there have been multiple attempts to produce recombinant lubricin and lubricin mimetic cartilage lubricants over the last two decades, these materials have not found clinical use due to challenges associated with large scale production, manufacturing, and purification. Recently, a novel method using codon scrambling was developed to produce a stable, full‐length bioengineered equine lubricin (eLub) in large reproducible quantities. While preliminary frictional analysis of eLub and other recombinantly produced forms revealed they can lubricate cartilage, a complete tribological characterization is lacking, with previous studies evaluating the friction coefficient only at a single dose or a single speed. The objective of this study was to analyze the dose‐dependent tribological properties of eLub using the Stribeck framework of tribological analysis. Recombinantly produced eLub at doses greater than 1.5 mg/mL exhibits friction coefficients on par with healthy bovine SF, and a maximal 5 mg/mL dose exhibits a nearly 50% lower friction coefficient than healthy SF. eLub also modulates the shift in lubrication mode of the cartilage from the high friction boundary mode to the low friction minimum mode at high concentrations. 

Abstract Intra‐articular injections of hyaluronic acid (HA) are the cornerstone of osteoarthritis (OA) treatments. However, the mechanism of action and efficacy of HA viscosupplementation are debated. As such, there has been recent interest in developing synthetic viscosupplements. Recently, a synthetic 4 wt% polyacrylamide (pAAm) hydrogel was shown to effectively lubricate and bind to the surface of cartilage in vitro. However, its ability to localize to cartilage and alter the tribological properties of the tissue in a live articulating large animal joint is not known. The goal of this study was to quantify the distribution and extent of localization of pAAm in the equine metacarpophalangeal or metatarsophalangeal joint (fetlock joint), and determine whether preferential localization of pAAm influences the tribological properties of the tissue. An established planar fluorescence imaging technique was used to visualize and quantify the distribution of fluorescently labeled pAAm within the joint. While the pAAm hydrogel was present on all surfaces, it was not uniformly distributed, with more material present near the site of the injection. The lubricating ability of the cartilage in the joint was then assessed using a custom tribometer across two orders of magnitude of sliding speed in healthy synovial fluid. Cartilage regions with a greater coverage of pAAm, that is, higher fluorescent intensities, exhibited friction coefficients nearly 2‐fold lower than regions with lesser pAAm (R_rm = −0.59,p < 0.001). Collectively, the findings from this study indicate that intra‐articular viscosupplement injections are not evenly distributed inside a joint, and the tribological outcomes of these materials is strongly determined by the ability of the material to localize to the articulating surfaces in the joint. 

Abstract Articular joints facilitate motion and transfer loads to underlying bone through a combination of cartilage tissue and synovial fluid, which together generate a low‐friction contact surface. Traumatic injury delivered to cartilage and the surrounding joint capsule causes secretion of proinflammatory cytokines by chondrocytes and the synovium, triggering cartilage matrix breakdown and impairing the ability of synovial fluid to lubricate the joint. Once these inflammatory processes become chronic, posttraumatic osteoarthritis (PTOA) development begins. However, the exact mechanism by which negative alterations to synovial fluid leads to PTOA pathogenesis is not fully understood. We hypothesize that removing the lubricating macromolecules from synovial fluid alters the relationship between mechanical loads and subsequent chondrocyte behavior in injured cartilage. To test this hypothesis, we utilized an ex vivo model of PTOA that involves subjecting cartilage explants to a single rapid impact followed by continuous articulation within a lubricating bath of either healthy synovial fluid, phosphate‐buffered saline (PBS), synovial fluid treated with hyaluronidase, or synovial fluid treated with trypsin. These treatments degrade the main macromolecules attributed with providing synovial fluid with its lubricating properties; hyaluronic acid and lubricin. Explants were then bisected and fluorescently stained to assess global and depth‐dependent cell death, caspase activity, and mitochondrial depolarization. Explants were tested via confocal elastography to determine the local shear strain profile generated in each lubricant. These results show that degrading hyaluronic acid or lubricin in synovial fluid significantly increases middle zone chondrocyte damage and shear strain loading magnitudes, while also altering chondrocyte sensitivity to loading. 

Abstract Inflammation of the synovium, known as synovitis, plays an important role in the pathogenesis of osteoarthritis (OA). Synovitis involves the release of a wide variety of pro‐inflammatory mediators in synovial fluid (SF) that damage the articular cartilage extracellular matrix and induce death and apoptosis in chondrocytes. The composition of synovial fluid is dramatically altered by inflammation in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. However, the relationship between key biochemical markers of joint inflammation and mechanical function of SF is not well understood. Here, we demonstrate the application of a novel analytical framework to measure the effective viscosity for SF lubrication of cartilage, which is distinct from conventional rheological viscosity. Notably, in a well‐established equine model of synovitis, this effective lubricating viscosity decreased by up to 10,000‐fold for synovitis SF compared to a ~4 fold change in conventional viscosity measurements. Further, the effective lubricating viscosity was strongly inversely correlated (r = −0.6 to −0.8) to multiple established biochemical markers of SF inflammation, including white blood cell count, prostaglandin E₂(PGE₂), and chemokine ligand (CCLs) concentrations, while conventional measurements of viscosity were poorly correlated to these markers. These findings demonstrate the importance of experimental and analytical approaches to characterize functional lubricating properties of synovial fluid and their relationships to soluble biomarkers to better understand the progression of OA. 

In various biological systems, analyzing how cell behaviors are coordinated over time would enable a deeper understanding of tissue-scale response to physiologic or superphysiologic stimuli. Such data is necessary for establishing both normal tissue function and the sequence of events after injury that lead to chronic disease. However, collecting and analyzing these large datasets presents a challenge—such systems are time-consuming to process, and the overwhelming scale of data makes it difficult to parse overall behaviors. This problem calls for an analysis technique that can quickly provide an overview of the groups present in the entire system and also produce meaningful categorization of cell behaviors. Here, we demonstrate the application of an unsupervised method—the Variational Autoencoder (VAE)—to learn the features of cells in cartilage tissue after impact-induced injury and identify meaningful clusters of chondrocyte behavior. This technique quickly generated new insights into the spatial distribution of specific cell behavior phenotypes and connected specific peracute calcium signaling timeseries with long term cellular outcomes, demonstrating the value of the VAE technique.