Alan J. Grodzinsky

Osteoarthritis is a debilitating disease of synovial joints affecting millions of people worldwide. Despite this, there are currently no disease modifying osteoarthritis drugs (DMOADs), mainly due to poor retention within the joint space. Previously, our group has shown that partially PEGylated cationic poly(amido amine) (PAMAM) dendrimers electrostatically bind to articular cartilage and improve therapy retention times, resulting in reduced severity of osteoarthritis symptoms. Here, we develop a greater understanding of how PEGylation influences dendrimer-cartilage interactions through systematic modulation of PEG chain length and accessible charged amines, defined as PAMAM primary amines freely accessible to the physiological environment and void of any interactions with PEG. Utilizing various models, we found that cartilage binding strength and binding kinetics increased with increasing accessible charged amines, while biocompatibility decreased, independent of PEG chain length. Conversely, decreasing accessible charged amines or increasing PEG chain length enhanced diffusion through cartilage explants. We found accessible charged amines controlled electrostatic binding strength while PEG chain length controlled reversibility of binding. When PEG-PAMAM conjugates were compared to other cationic molecules studied for their cartilage binding properties, all dendrimers tested exhibited significantly greater binding affinities and binding site densities but reduced Donnan partitioning coefficients. Finally, we found controlling binding strength and cartilage diffusion is critical to achieving extended retention within healthy joints in vivo. Future studies can use the enhanced mechanistic understanding of dendrimer-cartilage interactions established here to optimize PEG-PAMAM conjugates as a cartilage drug delivery platform.

Collagen damage in articular cartilage plays a key role in post-traumatic osteoarthritis, but the underlying mechanobiological pathways leading to collagen fibril degeneration after injury remain incompletely understood. We hypothesized that mechanical injurious loading induces localized cellular damage in cartilage, which in turn triggers the release of collagen-degrading matrix metalloproteinases (MMPs) and depth-wise collagen loss. To investigate this, we developed a computational mechano-signaling model for injured bovine cartilage, in which injury-induced cell damage is caused by excessive localized shear strains, leading to downstream MMP release, and spatially heterogeneous collagen degradation. The model predictions were compared to ex vivo cartilage explant experiments over 12 days post-injury. By day 12, the simulated bulk and depth-wise collagen loss aligned with our experimental findings quantified via Fourier-transform infrared microspectroscopy imaging (~30% average loss in the model vs. ~ 35% in the experiment). The results suggest that injury-induced cell damage and the downstream MMP activity can partly explain the depth-wise collagen content loss observed in the early days after cartilage injury. Ultimately, combining the current mechanistic approach with joint-level computational models could enhance the prediction of the onset and progression of cartilage degeneration following joint trauma.

Abstract Purpose Osteoarthritis (OA) is a debilitating disease characterized by degeneration of articular cartilage with no clinically approved treatment available to date despite the multitude of approaches that have been proposed. Two fundamentally different therapeutic strategies are pro-anabolic and anti-catabolic treatments. However, one of the major challenges to developing a successful intervention for OA is the fast clearance of the therapeutics from the joint space, which makes evaluating these proposed strategies difficult. In this work, we utilize a modular cationic dendrimer nanoformulation conjugated with therapeutic proteins which grants them longer retention in joint space and makes comparison of the approaches possible. Methods Dendrimer conjugated with insulin-like growth factor-1 (dend-IGF1) and interleukin-1 receptor antagonist (dend-IL1RA) were used as representative pro-anabolic and anti-catabolic therapeutics, respectively. Preservation of bioactivity of the final formulations was tested in vitro and ex vivo , and the therapeutic efficacy was tested in vivo in a post-traumatic OA model in rats. Results Studies in a rat model revealed that both therapeutics when conjugated to the dendrimers show improved pharmacokinetics and inhibited OA progression in vivo . Furthermore, dend-IL1RA showed significant efficacy in pain mitigation as well. This work supports the concept that dendrimer-protein conjugates provided an extended half-life within cartilage tissue that can greatly enhance the treatment efficacy of different types of biologic treatments. Conclusion Therefore, not only do the formulations studied in this work present promising avenues for OA treatment, but they also open up the possibility of exploring other therapeutics to treat anionic avascular tissues, such as meniscus or cornea, using this platform technology. Lay Summary More than 650 million people worldwide suffer from osteoarthritis (OA), but unfortunately, there is no clinically approved treatment, and the only standard of care is pain management. Although there are two leading mechanisms of treatment that can be used, one being using pro-anabolic drugs to repair cartilages and the other one being using anti-catabolic drugs that prevents cartilage degradation, these drugs get rapidly cleared from the knee joints after injection. Therefore, in this study, we attached insulin-like growth factor-1 (IGF-1) which is a pro-anabolic drug and interleukin-1 receptor antagonist (IL-1RA) which is an anti-catabolic drug to a positively charged polymer that can stick to the cartilage via charge-charge interaction to prevent these drugs from being rapidly cleared. Our findings reveal that by doing so, the drugs were able to stay within the joint space longer and was able to delay OA progression. Especially, IL-1RA – polymer conjugate was able to significantly reduce pain as well.

Degenerative musculoskeletal diseases like osteoarthritis can be initiated by joint injury. Injurious overloading-induced mechanical straining of articular cartilage and subsequent biological responses may trigger cartilage degradation. One early sign of degradation is loss of aggrecan content which is potentially accelerated near chondral lesions under physiological loading. Yet, the mechanoinflammatory mechanisms explaining time-dependent degradation in regions with disparate mechanical loading are unclear and challenging to assess with experiments alone. Here, we developed computational models unraveling potential mechanisms behind aggrecan content adaptation in fibril-reinforced porohyperelastic cartilage after single injurious overloading (50% compressive strain magnitude, 100%/s strain rate) followed by physiological cyclic loading (15% strain, 1 Hz, haversine waveform). The simulated adaptation of aggrecan content was compared spatially and at several time points to tissue composition found in Safranin-O-stained sections of young bovine knee cartilage subjected to the same loading protocols. Incorporating mechanical strain-driven cell damage and downstream proteolytic enzyme release, fluid flow-driven aggrecan depletion, and fluid pressure-stimulated regulation of aggrecan biosynthesis, the models agreed with experiments and exhibited 14%-points greater near-lesion aggrecan loss after 12 days of physiological loading compared to without loading. The near-lesion aggrecan loss was driven by fluid flow and proteolytic aggrecanase activity, while chondroprotective pro-anabolic responses (increased aggrecan biosynthesis) were prominent in the deeper tissue despite damaged superficial layer. This significant advancement in mechanistic understanding incorporated into cartilage adaptation model can help in development and guidance of personalized therapies, such as rehabilitation protocols and tissue-engineered constructs.

Objectives: Meniscal integrity is crucial for knee joint stability and the prevention of osteoarthritis (OA) development. Recent studies suggested that mechanical overload and interleukin (IL)-17A may be important intertwined players in meniscal degeneration, but a direct impact of IL-17A on the meniscus has not been investigated. Therefore, the aim of this study was to analyze the effect of IL-17A on meniscal tissue with and without combined mechanical injury (MI). Methods: Meniscal explant disks (1 mm height, 3 mm diameter) were isolated from bovine menisci (preserving the native tibial superficial zone) and exposed to IL-17A [0–100 ng/mL] and/or MI (single compression, 50% strain, strain rate 1 mm/sec). After three days of incubation in a serum-free medium, the proteoglycan release (sGAG; DMMB assay), mRNA level of matrix-degrading enzymes (qRT-PCR), aggrecan degradation (NITEGE immunostaining), and cell death (histomorphometry of nuclear blebbing/apoptosis and condensed nuclei/unspecified cell death) were determined. Statistics: one- and two-way ANOVA with Tukey’s multiple comparisons or Kruskal–Wallis with post hoc testing. Results: IL-17A increased sGAG release in a dose-dependent significant manner. MI also induced the release of sGAG significantly, but the combination with IL-17A showed the highest levels. Both IL-17A and MI individually affected the mRNA levels for ADAMTS4 and MMP-13 slightly, but the combination of both particularly induced a significant increase in mRNA levels. Signals for the ADAMTS4-related aggrecan neoepitope NITEGE were elevated by IL-17A in superficial areas of the excised tissue and by MI in superficial and deeper areas. The combination of both stimuli intensified this signal further. MI increased the number of cells with condensed nuclei significantly and induced apoptosis in a small proportion of cells. IL-17A had no significant impact on the amount of condensed or apoptotic nuclei. Conclusions: Our findings emphasize an interaction between inflammatory cytokine IL-17A signaling and mechanical stress since IL-17A induced matrix degeneration in meniscal tissue, which intensified in combination with a trauma. The latter might create a post-traumatic environment that promotes meniscal degeneration and subsequently osteoarthritis progression.

Abstract Delivering therapeutics directly to synovial joints to treat osteoarthritis (OA) is challenging due to the dense negatively charged cartilage matrix and rapid turnover of synovial fluid, leading to high clearance rates. Our lab has identified polyamidoamine (PAMAM) dendrimers as optimal nanocarriers to overcome delivery challenges to cartilage due to their positive charge and small size, which enables them to bind to cartilage and diffuse through tissue to deliver therapeutics to chondrocytes. Previously, we have developed and characterized dendrimers functionalized with polyethylene glycol (PEG), demonstrating improved biocompatibility and enhanced transport through cartilage matrix. To improve the design of our therapeutic system, in this next phase of work, we characterized dendrimer-protein interactions or protein coronas that form on dendrimers after being immersed in synovial fluid. We also analyzed how synovial fluid protein coronas affect biological outcomes of dendrimers in synovial joints, specifically uptake in cartilage and internalization by chondrocytes. We identified that protein coronas can reduce dendrimer uptake in cartilage and chondrocytes; however, uptake reduction is mitigated by varying PEG chain length and density. Although protein coronas can be perceived as “biological barriers” to uptake, we demonstrate that dendrimers conjugated with insulin-like growth factor 1 (IGF-1) have better engagement with IGF-1 receptors after being pre-coated with a synovial fluid protein corona. Overall, these studies offer further insight into the mechanisms of how positively charged dendrimers target and transport through cartilage, bridging knowledge gaps between ex vivo and in vivo work. Abstract Figure

Abstract Damage of collagen fibril network in articular cartilage plays a key role in post-traumatic osteoarthritis but the main underlying mechanobiological mechanisms of fibrils degeneration early after injury are not fully understood. This study explores the hypothesis that injurious loading leads to cellular damage that triggers the release of matrix metalloproteinases (MMPs), resulting in loss of collagen content in cartilage. To investigate this, we developed a computational mechano-signaling model simulating spatial collagen loss in bovine cartilage. In the model, the injurious loading causes excessive shear strains in tissue matrix, leading to cell damage and subsequent release of MMPs. The model was compared to ex vivo cartilage explant experiments over 12 days post-injury where collagen content was assessed via Fourier-transform infrared microspectroscpy. By day 12, the simulated collagen loss aligned with our experimental findings along most of tissue depth (∼30% bulk average loss in the model vs. ∼35% in the experiment). The results suggest that injury-related cell damage and the downstream MMP activity could partly explain the depth-wise collagen content loss in early days after ex vivo cartilage injury. Ultimately, combining the current approach with joint-level computational models could enhance the prediction of the onset and progression of cartilage degeneration. Author Summary Knee injuries can initiate an irreversible degeneration of articular cartilage which can later lead to the development of post-traumatic osteoarthritis over the years. Currently, there is no cure or effective intervention to repair degenerated cartilage or halt disease progression. Yet, opportunities for intervention depend on a thorough understanding of mechanisms that govern the very early changes in cartilage tissue composition such as the collagen fibrils. In this work, we developed a finite element computational modeling framework to simulate cartilage following injurious loading, focusing on the early loss of collagen content. The model aims to capture the role of injury-induced cellular damage in elevating proteolytic activity within the tissue, which could rapidly degrade collagen fibrils and result in significant collagen loss. This model framework offers a tool for studying the degradation of the collagen fibril network, testing hypotheses involving cell-driven mechano-signalling pathways and evaluating potential treatment interventions aimed at preventing collagen loss.

Joint trauma often leads to articular cartilage degeneration and post-traumatic osteoarthritis (PTOA). Pivotal determinants include trauma-induced excessive tissue strains that damage cartilage cells. As a downstream effect, these damaged cells can trigger cartilage degeneration via oxidative stress, cell death, and proteolytic tissue degeneration. N-acetylcysteine (NAC) has emerged as an antioxidant capable of inhibiting oxidative stress, cell death, and cartilage degeneration post-impact. However, the temporal effects of NAC are not fully understood and remain difficult to assess solely by physical experiments. Thus, we developed a computational finite element analysis framework to simulate a drop-tower impact of cartilage in Abaqus, and subsequent oxidative stress-related cell damage, and NAC treatment upon cartilage proteoglycan content in Comsol Multiphysics, based on prior ex vivo experiments. Model results provide evidence that immediate NAC treatment can reduce proteoglycan loss by mitigating oxidative stress, cell death (improved proteoglycan biosynthesis), and enzymatic proteoglycan depletion. Our simulations also indicate that delayed NAC treatment may not inhibit cartilage proteoglycan loss despite reduced cell death after impact. These results enhance understanding of the temporal effects of impact-related cell damage and treatment that are critical for the development of effective treatments for PTOA. In the future, our modeling framework could increase understanding of time-dependent mechanisms of oxidative stress and downstream effects in injured cartilage and aid in developing better treatments to mitigate PTOA progression.

Abstract Degenerative musculoskeletal diseases like osteoarthritis can be initiated by joint injury. Injurious overloading-induced mechanical straining of articular cartilage and subsequent biological responses may trigger cartilage degradation. One early sign of degradation is loss of aggrecan content which is potentially accelerated near chondral lesions under physiological loading. Yet, the mechanoinflammatory mechanisms explaining time-dependent degradation in regions with disparate mechanical loading are unclear and challenging to assess with experiments alone. Here, we developed computational models unraveling potential mechanisms behind aggrecan content adaptation. Incorporating mechanical strain-driven cell damage and downstream proteolytic enzyme release, fluid flow-driven aggrecan depletion, and fluid pressure-stimulated regulation of aggrecan biosynthesis, the models agreed with experiments and exhibited 14%-points greater near-lesion aggrecan loss after 12 days of physiological loading compared to without loading. This significant advancement in mechanistic understanding incorporated into cartilage adaptation model can help in development and guidance of personalized therapies, such as rehabilitation protocols and tissue- engineered constructs.

Abstract Joint injury can lead to articular cartilage damage, excessive inflammation, and post‐traumatic osteoarthritis (PTOA). Collagen is an essential component for cartilage function, yet current literature has limited understanding of how biochemical and biomechanical factors contribute to collagen loss in injured cartilage. Our aim was to investigate spatially dependent changes in collagen content and collagen integrity of injured cartilage, with an explant model of early‐stage PTOA. We subjected calf knee cartilage explants to combinations of injurious loading (INJ), interleukin‐1α‐challenge (IL) and physiological cyclic loading (CL). Using Fourier transform infrared microspectroscopy, collagen content (Amide I band) and collagen integrity (Amide II/1338 cm −1 ratio) were estimated on days 0 and 12 post‐injury. We found that INJ led to lower collagen content near lesions compared to intact regions on day 0 ( p < 0.001). On day 12, near‐lesion collagen content was lower compared to day 0 ( p < 0.05). Additionally, on day 12, INJ, IL, and INJ + IL groups exhibited lower collagen content along most of tissue depth compared to free‐swelling control group ( p < 0.05). CL groups showed higher collagen content along most of tissue depth compared to corresponding groups without CL ( p < 0.05). Immunohistochemical analysis revealed higher MMP‐1 and MMP‐3 staining intensities localized within cell lacunae in INJ group compared to CTRL group on day 0. Our results suggest that INJ causes rapid loss of collagen content near lesions, which is intensified on day 12. Additionally, CL could mitigate the loss of collagen content at intact regions after 12 days.

Abstract Joint trauma often leads to articular cartilage degeneration and post-traumatic osteoarthritis (PTOA). Pivotal determinants include trauma-induced excessive tissue strains that damage cartilage cells. As a downstream effect, these damaged cells can trigger cartilage degeneration via oxidative stress, cell death, and proteolytic tissue degeneration. N-acetylcysteine (NAC) has emerged as antioxidant capable of inhibiting oxidative stress, cell death, and cartilage degeneration post-impact. However, temporal effects of NAC are not fully understood and remain difficult to assess solely by physical experiments. Thus, we developed a computational framework to simulate a drop tower impact of cartilage with finite element analysis in ABAQUS, and model subsequent oxidative stress-related cell damage, and NAC treatment upon cartilage proteoglycan content in COMSOL Multiphysics, based on prior ex vivo experiments. Model results provide evidence that by inhibiting further cell damage by mechanically induced oxidative stress, immediate NAC treatment can reduce proteoglycan loss by mitigating cell death (loss of proteoglycan biosynthesis) and enzymatic proteoglycan depletion. Our simulations also indicated that delayed NAC treatment may not inhibit cartilage proteoglycan loss despite reduced cell death after impact. These results enhance understanding of temporal effects of impact-related cell damage and treatment that are critical for the development of effective treatments for PTOA. In the future, our modeling framework could increase understanding of time-dependent mechanisms of oxidative stress and downstream effects in injured cartilage and aid in developing better treatments to mitigate PTOA progression. Author summary Post-traumatic osteoarthritis is a debilitating disease which is often initiated by trauma and characterized by cartilage degeneration. The degeneration is partly driven by trauma-induced damage, including oxidative stress, to cartilage cells. Multiple drugs have been studied to counteract the cell damage, but it remains difficult to inhibit the disease progression since temporal effects of such treatments are not fully understood. Here, we developed a computational framework to study effects of antioxidant treatment in mechanically impacted cartilage and compared our computational simulations with previously published experiments. Our results showed that the high strain induced cell damage occurring in the mechanically impacted region could be mitigated by N-acetylcysteine treatment. This mechanism could partly explain reduced cartilage proteoglycan loss compared to untreated samples. Our modeling framework could help enhance development of treatments to better inhibit osteoarthritis progression.

Post-traumatic Osteoarthritis (PTOA) results from traumatic joint injuries (such as an ACL rupture). Mechanical impact and an immediate synovial inflammatory response can result in joint tissue degradation and longer-term progression to PTOA. Astronauts are susceptible to increased exercise-related joint injuries leading to altered musculoskeletal physiology, further escalated due to microgravity and increased exposure to ionizing radiation. We applied a human Cartilage-Bone-Synovium (CBS) coculture model to test the potential of low-dose dexamethasone (Dex) and IGF-1 in ameliorating PTOA-like degeneration on Earth and the International Space Station-National Laboratory (ISS-NL, ISS for short). CBS cocultures were established using osteochondral plugs (CB) subjected to compressive impact injury (INJ) followed by coculture with synovium (S) explants. Study groups consisted of control (CB); disease [CBS + INJ]; treatment [CBS + INJ + Dex + IGF-1]; and drug-safety [CB + Dex + IGF-1]. Outcome measures included cell viability, altered matrix glycosaminoglycans (GAG) and collagens, multiplex-ELISA quantification of released cytokines, histopathology, and metabolomic and proteomic analyses of spent media. A 21-day study on ISS-NL explored PTOA-like pathogenesis and treatment in microgravity. Tissue cards for study groups were cultured in custom-built culture chambers within multi-use variable-g platforms (MVPs). A marked upregulation in the release of inflammatory cytokines and tissue-GAG loss was observed in CBS + INJ groups in space and ground controls utilizing tissues from the same donors, similar to that reported in a previous multi-donor study on Earth; these changes were partly ameliorated by Dex + IGF-1, but with donor variability. Metabolomic and proteomic analyses revealed an array of distinct differences between metabolites/proteins released to the medium in Space versus on Earth.

<title>Abstract</title> Objective Meniscal integrity is crucial for knee joint stability and prevention of osteoarthritis (OA) development. Recent studies suggested that mechanical overload and interleukin (IL)-17A may be important intertwined players in meniscal degeneration, but a direct impact of IL-17A on the meniscus has not been investigated. Therefore, the aim of this study was to analyze the effect of IL-17A on meniscal tissue with and without combined mechanical injury (MI). Methods Meniscal explant disks (1 mm height, 3 mm diameter) were isolated from the lower surface of bovine menisci and exposed to IL-17A [0-100 ng/ml] and/or MI (single compression, 50% strain, strain rate 1 mm/sec). After three days of incubation in a serum-free medium the proteoglycan release (sGAG; DMMB assay), mRNA level of matrix-degrading enzymes (qRT-PCR), aggrecan degradation (NITEGE immunostaining), and cell death (histomorphometry of nuclear blebbing/apoptosis and condensed nuclei/unspecified cell death) was determined. Statistics: one-way ANOVA with Tukeys multiple comparisons or Kruskal-Wallis-post hoc test. Results IL-17A increased sGAG release in a dose-dependent manner. MI also induced release of sGAG, but the combination with IL-17A raised this effect to a significant level even for an IL-17A concentration which showed no significant effect on its own. Both, IL-17A and MI individually affected the mRNA levels for ADAMTS-4 and MMP-13 slightly, but the combination of both induced a significant increase in mRNA levels. Signals for the ADAMTS-4-related aggrecan neoepitope NITEGE were elevated by IL-17A in superficial explant tissue and by MI in superficial and deeper areas. The combination of both stimuli intensified this signal further. MI increased the number of cells with condensed nuclei significantly and induced apoptosis in a small proportion of cells. IL-17A alone had no significant impact on the amount of condensed nuclei but increased the number of apoptotic cells slightly and even more in combination with MI. Conclusion IL-17A induces matrix degeneration in meniscal tissue which in combination with a trauma intensifies significantly. The latter might be responsible for a post-traumatic joint environment that promotes meniscal degeneration and subsequently OA, and highlights IL-17A as a potential target in PTOA therapy.

Topical skin care products and hydrating compositions (moisturizers or injectable fillers) have been used for years to improve the appearance of, for example facial wrinkles, or to increase "plumpness". Most of the studies have addressed these changes based on the overall mechanical changes associated with an increase in hydration state. However, little is known about the water mobility contribution to these changes as well as the consequences to the specific skin layers. This is important as the biophysical properties and the biochemical composition of normal stratum corneum, epithelium, and dermis vary tremendously from one another. Our current studies and results reported here have focused on a novel approach (dynamic atomic force microscopy-based nanoindentation) to quantify biophysical characteristics of individual layers of ex vivo human skin. We have discovered that our new methods are highly sensitive to the mechanical properties of individual skin layers, as well as their hydration properties. Furthermore, our methods can assess the ability of these individual layers to respond to both compressive and shear deformations. In addition, since human skin is mechanically loaded over a wide range of deformation rates (frequencies), we studied the biophysical properties of skin over a wide frequency range. The poroelasticity model used helps to quantify the hydraulic permeability of the skin layers, providing an innovative method to evaluate and interpret the impact of hydrating compositions on water mobility of these different skin layers.

INTRODUCTION: Mechanical forces are emerging as key drivers of fibrosis. Elucidating this pathophysiology is relevant in degenerative spinal disease (DSD) where mechanical stress on the spine is associated with fibrotic changes in ligament and bone, with resultant compression of the neural elements. Though DSD affects most people as they age and costs $3.6 billion annually, the crucial bridge between macroscale mechanical forces driving pathologic degenerative mechanosensing remains unknown. METHODS: Validated lumbo-pelvic finite element models (FEMs) were used to calculate LF macroscale stress; LF was collected from 120 patients undergoing surgery and studied via histology, Western blot and novel Atomic Force Microscopy (AFM) stiffness mapping. A novel device to apply strain to LF was engineered to further investigate the real-time effect of resulting stress on myofibroblast accumulation and transcriptome-level gene expression. RESULTS: Spinopelvic FEM predicted LF adjacent to a fused level experiences increased stress during spine flexion. Supporting this model, LF resected adjacent to a fused level was observed to have significant increases in pro-fibrotic myofibroblasts (4.3-fold) and downstream ROCK effectors compared with LF from non-DSD and DSD patients without prior fusion. AFM and mathematical modeling revealed myofibroblasts localize to islands of elevated stiffness and microstress within LF. Through application of strain on LF explants, we observed stress-dependent myofibroblast accumulation (3.66-fold, P &lt; .0002) and blocked this accumulation through ROCK inhibition. Finally, through transcriptomic analysis on human LF, we identify a stress-driven hyperacute transcriptional response and find this response is also altered by ROCK inhibition. CONCLUSIONS: These findings implicate stress-driven myofibroblast accumulation as a mechanosensitive pro-fibrotic pathway in DSD. These observations warrant detailed future investigation into ROCK inhibitors as a potentially surgery-sparing adjunct to non-operative therapy in ASD and DSD-mediated spinal stenosis.

Post-traumatic osteoarthritis (PTOA), characterized by articular cartilage degradation initiated in an inflammatory environment after traumatic joint injury, can lead to alterations in cartilage biomechanical properties. Low dose dexamethasone (Dex) shows chondroprotection in cartilage challenged with inflammatory cytokines, but little is known about the structural biomechanical response of human cartilage to Dex in such a diseased state. This study examined changes in the biomechanical properties and biochemical composition of the cartilage within human osteochondral explants in response to treatment with exogenous cytokines, Dex, and a regimen of cyclic loading at the start and end of culture. Osteochondral explants were harvested from five pairs of human ankle talocrural joints (Collins grade 0-1) and cultured for 10 days with/without exogenous cytokines (100 ng/mL TNFα, 50 ng/mL IL-6, 250 ng/mL sIL-6R) ± Dex (100 nM). Biomechanical testing on day-0 and day-10 enabled estimation of the unconfined compression equilibrium modulus (Ey), dynamic stiffness (Ed) and hydraulic permeability (kp) of cartilage excised from bone, accompanied by biochemical assessment of media and cartilage tissue. Dex preserved chondrocyte cell viability and decreased sulfated glycosaminoglycan (sGAG) loss and nitric oxide release, but did not alter Ey, Ed and kp (before or after loading) on day-10. In the cytokine/cytokine+Dex treated groups, sGAG content exhibited a weaker correlation with Ey and Ed than at baseline, suggesting an important role for structural rather than biochemical changes in producing biomechanical alterations in response to cytokines and Dex. These findings aid in forming a more complete profile of potential clinical effects of Dex for use in OA/PTOA treatment regimens.

Osteoarthritis (OA) is a common musculoskeletal disease that leads to deterioration of articular cartilage, joint pain, and decreased quality of life. When OA develops after a joint injury, it is designated as post-traumatic OA (PTOA). The etiology of PTOA remains poorly understood, but it is known that proteoglycan (PG) loss, cell dysfunction, and cell death in cartilage are among the first signs of the disease. These processes, influenced by biomechanical and inflammatory stimuli, disturb the normal cell-regulated balance between tissue synthesis and degeneration. Previous computational mechanobiological models have not explicitly incorporated the cell-mediated degradation mechanisms triggered by an injury that eventually can lead to tissue-level compositional changes. Here, we developed a 2-D mechanobiological finite element model to predict necrosis, apoptosis following excessive production of reactive oxygen species (ROS), and inflammatory cytokine (interleukin-1)-driven apoptosis in cartilage explant. The resulting PG loss over 30 days was simulated. Biomechanically triggered PG degeneration, associated with cell necrosis, excessive ROS production, and cell apoptosis, was predicted to be localized near a lesion, while interleukin-1 diffusion-driven PG degeneration was manifested more globally. Interestingly, the model also showed proteolytic activity and PG biosynthesis closer to the levels of healthy tissue when pro-inflammatory cytokines were rapidly inhibited or cleared from the culture medium, leading to partial recovery of PG content. The numerical predictions of cell death and PG loss were supported by previous experimental findings. Furthermore, the simulated ROS and inflammation mechanisms had longer-lasting effects (over 3 days) on the PG content than localized necrosis. The mechanobiological model presented here may serve as a numerical tool for assessing early cartilage degeneration mechanisms and the efficacy of interventions to mitigate PTOA progression.

H-NMR, allows us to study more closely the non-covalent interactions between PEG and PAMAM. We find that increasing PEG chain length increases the number of non-covalent interactions. Additionally, at low grafting densities, increasing the number of PEG chains on the PAMAM surface also increases the non-covalent interactions. At higher grafting densities, however, PEG chains sterically repel one another, forcing chains to elongate away from the surface and reducing the number of interactions between PAMAM and individual PEG chains. The data presented here provides a framework for a more precise mechanistic understanding of how the length and density of tethered PEG chains on PAMAM dendrimers influence drug delivery properties.