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Objective: To study mechanical overload of mature meniscal tissue under normal and pro-inflammatory conditions in vitro. Method: Three days after a single unconfined compression (strain: 25-75%, strain rate 1/s) of meniscal explants from 16 to 24 months-old cattle combined with interleukin-1-treatment (IL-1, 10 ng/ml) release of glycosaminoglycans (GAGs; dimethylmethylene blue (DMMB) assay), lactate dehydrogenase (LDH; cytotoxicity detection kit), and nitric oxide (NO; Griess assay), as well as gene transcription (quantitative reverse transcription polymerase chain reaction (RT-PCR)) and numbers of cells with condensed nuclei (CN; histomorphometry) were determined. Results: Mean peak stresses during compression were about five (25%), 11 (50%), and 30 MPa (75%), respectively. GAG and LDH release and numbers of CN increased whereas NO production and mRNA levels of matrix metalloproteinase (MMP)-2, -3 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 decreased strain-dependently after compression. IL-1 induced an increase in GAG and NO release as well as MMP-2, -3 and ADAMTS-4 levels, but had no impact on the LDH release and slightly increased numbers of CN. However, in combination with compression the tissue responses were reduced and LDH and CN levels were increased compared to IL-1 alone. Conclusion: Our data suggest that a single impact compression induces cell damage and release of GAG and reduces the NO production and transcription of certain matrix-degrading enzymes. It also reduces the capacity of meniscal tissue to respond to IL-1, which might be related to the cell damage and suggests that the compression-related GAG release might rather be the result of immediate extracellular matrix-damage than a cell-mediated event. This, however, needs to be confirmed in future studies. [ABSTRACT FROM AUTHOR]

Objective: Our goal was to test the hypothesis that specific integrin receptors regulate chondrocyte biosynthetic response to dynamic compression at early times in 3D gel culture, during initial evolution of the pericellular matrix, but prior to significant accumulation of further-removed matrix. The study was motivated by increased use of dynamic loading, in vitro, for early stimulation of tissue engineered cartilage, and the need to understand the effects of loading, in vivo, at early times after implantation of constructs.Methods: Bovine articular chondrocytes were seeded in 2% agarose gels (15x10(6)cells/mL) and incubated for 18 h with and without the presence of specific integrin blockers (small-molecule peptidomimetics, function-blocking antibodies, and RGD-containing disintegrins). Samples were then subjected to a 24-h dynamic compression regime found previously to stimulate chondrocyte biosynthesis in 3D gel as well as cartilage explant culture (1 Hz, 2.5% dynamic strain amplitude, 7% static offset strain). At the end of loading, proteoglycan (PG) synthesis ((35)S-sulfate incorporation), protein synthesis ((3)H-proline incorporation), DNA content (Hoechst dye 33258) and total glycosaminoglycan (GAG) content (dimethyl methylene blue (DMMB) dye binding) were assessed.Results: Consistent with previous studies, dynamic compression increased PG synthesis and total GAG accumulation compared to free-swelling controls. Blocking alphavbeta3 abolished this response, independent of effects on controls, while blocking beta1 abolished the relative changes in synthesis when changes in free-swelling synthesis rates were observed.Conclusions: This study suggests that both alphavbeta3 and beta1 play a role in pathways that regulate stimulation of PG synthesis and accumulation by dynamic compression, but through distinct complementary mechanisms. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS: Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS: In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION: Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to determine the role of nitric oxide (NO) in tumor necrosis factor alpha (TNF-alpha)-induced matrix damage, compared to interleukin 1 beta (IL-1beta), in bovine cartilage explant cultures.Methods: Cartilage explants were subjected to treatment with TNF-alpha (100ng/ml), IL-1beta (10 ng/ml) and to the nitric oxide synthase inhibitor, N-methyl-arginine (L-NMA; 1.25 mM) for 26, 50 or 120 h (5 days). The collected medium was analyzed for sulfated glycosaminoglycan (sGAG), nitrate and nitrite, matrix metalloproteinase (MMP) activity by zymography, and aggrecan degradation by immunoblotting of aggrecan-G1 and aggrecan-G1-NITEGE fragments. RNA was extracted from the 26 and 50 h treated explants for real time quantitative PCR analyses.Results: TNF-alpha and IL-1beta treatment caused a 3-5 fold increase in sGAG release with an increase in aggrecanase-specific aggrecan breakdown and an increase in nitrate and nitrite production. L-NMA treatment inhibited almost 50% of the sGAG release caused by TNF-alpha treatment, with concomitant decrease in the aggrecanase-specific-NITEGE neo-epitope of aggrecan released into the medium. No L-NMA effect was identified with IL-1beta. TNF-alpha and IL-1beta both increased a disintegrin and matrix metalloproteinase with thrombospondin motif (ADAMTS)4 and ADAMTS5 transcription with no effect by L-NMA, suggesting that NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-alpha. TNF-alpha and IL-1beta both caused an increase in protease transcription (MMP-3, MMP-13, ADAMTS4 and ADAMTS5) and in pro-inflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase (COX)-2, as well as a decrease in matrix protein transcription, including collagen II, aggrecan, fibromodulin and link protein (IL-1beta only), and an increase in MMP-3 and MMP-9 secretion. L-NMA had no effect on gene transcription or MMP secretion.Conclusion: NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-alpha treatment while having no effect on IL-1beta treated cartilage explants. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Arthritis is one of the hallmarks of late-stage Lyme disease. Previous studies have shown that infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in degradation of proteoglycans and collagen in cartilage. B burgdorferi do not appear to produce any exported proteases capable of digesting proteoglycans and collagen, but instead, induce and activate host proteases, such as matrix metalloproteinases (MMPs), which results in cartilage degradation. The role of aggrecanases in Lyme arthritis has not yet been determined. We therefore sought to delineate the contribution of aggrecanases to joint destruction in Lyme arthritis. METHODS: We examined the expression patterns of aggrecanases 1 and 2 (ADAMTS 4 and 5, respectively) in B burgdorferi-infected primary human chondrocyte cell cultures, in synovial fluid samples from patients with active Lyme arthritis, and in the joints of mice by real-time quantitative reverse transcription-polymerase chain reaction and immunoblotting techniques. Bovine cartilage explants were used to determine the role of aggrecanases in B burgdorferi-induced cartilage degradation. RESULTS: ADAMTS-4, but not ADAMTS-5, was induced in human chondrocytes infected with B burgdorferi. The active forms of ADAMTS-4 were increased in synovial fluid samples from patients with active Lyme arthritis and were elevated in the joints of mice infected with B burgdorferi. Using cartilage explant models of Lyme arthritis, it appeared that the cleavage of aggrecan was predominantly mediated by 'aggrecanases' rather than MMPs. CONCLUSION: The induction of ADAMTS-4 by B burgdorferi results in the cleavage of aggrecan, which may be an important first step that leads to permanent degradation of cartilage. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Joint injury in young adults leads to an increased risk of developing osteoarthritis (OA) later in life. This study was undertaken to determine if injurious mechanical compression of cartilage explants results in changes at the level of gene transcription that may lead to subsequent degradation of the cartilage. METHODS: Cartilage was explanted from the femoropatellar groove of newborn calves. Levels of messenger RNA encoding matrix molecules, proteases, their natural inhibitors, transcription factors, and cytokines were assessed in free swelling control cultures as compared with cartilage cultures at 1, 2, 4, 6, 12, and 24 hours after application of a single injurious compression. RESULTS: Gene-expression levels measured in noninjured, free swelling cartilage varied over 5 orders of magnitude. Matrix molecules were the most highly expressed of the genes tested, while cytokines, matrix metalloproteinases (MMPs), aggrecanases (ADAMTS-5), and transcription factors showed lower expression levels. Matrix molecules showed little change in expression after injurious compression, whereas MMP-3 increased approximately 250-fold, ADAMTS-5 increased approximately 40-fold, and tissue inhibitor of metalloproteinases 1 increased approximately 12-fold above the levels in free swelling cultures. Genes typically used as internal controls, GAPDH and beta-actin, increased expression levels approximately 4-fold after injury, making them unsuitable for use as normalization genes in this study. The expression levels of tumor necrosis factor alpha and interleukin-1beta, cytokines known to be involved in the progression of OA, did not change in the chondrocytes after injury. CONCLUSION: Changes in the level of gene expression after mechanical injury are gene specific and time dependent. The quantity of specific proteins may be altered as a result of these changes in gene expression, which may eventually lead to degradation at the tissue level and cause a compromise in cartilage structure and function. [ABSTRACT FROM AUTHOR]

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., (© 2024 The Author(s). Journal of Orthopaedic Research ® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Cationic poly(amido amine) (PAMAM) dendrimers exhibit great potential for use in drug delivery, but their high charge density leads to an inherent cytotoxicity. To increase biocompatibility, many studies have attached poly(ethylene glycol) (PEG) chains to the dendrimer surface. It is unclear how these tethered PEG chains influence the physicochemical properties of the dendrimer. Here, we develop a fluorescence-based assay utilizing anionic biological tissue to quantify the electrostatic binding affinity of a library of PEG-PAMAM conjugates with various PEG chain lengths and grafting densities. We find that covalently bound PEG chains reduce the electrostatic binding affinity more significantly than what can be achieved through covalent bonds only. Contrary to previous thought, this reduction is not explained by the steric hindrance effects of PEG chains, suggesting that other, non-covalent interactions between PEG and PAMAM are present. Using acetylated PAMAM conjugates, we convert electrostatic binding affinity to the number of charged amines accessible to the physiological environment. These data, coupled with 1 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.

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 (E y ), dynamic stiffness (E d ) and hydraulic permeability (k p ) 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 E y , E d and k p (before or after loading) on day-10. In the cytokine/cytokine+Dex treated groups, sGAG content exhibited a weaker correlation with E y and E d 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kosonen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium., (© 2022. The Author(s).)

There are no drugs or treatment methods known to prevent the development of post-traumatic osteoarthritis (PTOA), a type of osteoarthritis (OA) that is triggered by traumatic joint injuries and accounts for ∼12% of the nearly 600 million OA cases worldwide. Lack of effective drug delivery techniques remains a major challenge in developing clinically effective treatments, but cationic delivery carriers can help overcome this challenge. Scaling up treatments that are effective in in vitro models to achieve success in preclinical in vivo models and clinical trials is also a challenging problem in the field. Here we use a cationic green fluorescent protein (GFP) as a carrier to deliver Insulin-Like Growth Factor 1 (IGF-1), a drug considered as a potential therapeutic for PTOA. GFP-IGF-1 conjugates were first synthesized as fusion proteins with different polypeptide linkers, and their transport properties were characterized in human cartilage explants. In vitro experimental data were used to develop a predictive mathematical transport model that was validated using an independent in vitro experimental data set. The model was used to predict the transport of these fusion proteins upon intra-articular injection into human knee joints. The predictions included results for the rate and extent of fusion protein penetration into cartilage, and the maximum levels of fusion proteins that would escape into systemic circulation through the joint capsule. Together, our transport measurements and model set the stage for translation of such explant culture studies to in vivo preclinical studies and potentially clinical application. STATEMENT OF SIGNIFICANCE: The lack of blood supply in cartilage and rapid clearance of drugs injected into human knees presents a major challenge in developing clinically effective treatments for osteoarthritis. Cationic delivery carriers can target negatively charged cartilage and help overcome this problem. Scaling up treatments that are effective in vitro to achieve success in vivo is also challenging. Here, we use a cationic green fluorescent protein (GFP) to deliver Insulin-Like Growth Factor-1 (IGF-1) into cartilage. Experiments measuring transport of GFP-IGF-1 fusion proteins in human cartilage explants were used to develop and validate a mathematical model to predict fusion protein transport upon injection into human knee joints. This work translates such explant culture studies to in vivo preclinical studies and potentially clinical application., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Patent Application: Grodzinsky, A.J. and Krishnan, Y.; ”Multi-Targeted, Tunable, Sustained Delivery of Payloads to Charged Avascular Tissues.,” US Patent Application No. 16/883,419, January 28th, 2021 (Publication No. US-2021-0023236-A1). Grants: NIH-NIBIB Grant R01-EB026344, NIH-NCATS Grant UH3 TR002186, DOE Grant DE-SC0007106., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease., Methods: Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin's grade 0-2, 23-83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile., Results: Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified., Conclusion: CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression., (© 2022. The Author(s).)

Injurious overloading and inflammation perturbate homeostasis of articular cartilage, leading to abnormal tissue-level loading during post-traumatic osteoarthritis. Our objective was to gain time- and cartilage depth-dependent insights into the early-stage disease progression with an in vitro model incorporating for the first time the coaction of (1) mechanical injury, (2) pro-inflammatory interleukin-1 challenge, and (3) cyclic loading mimicking walking and considered beneficial for cartilage health. Cartilage plugs (n = 406) were harvested from the patellofemoral grooves of young calves (N = 6) and subjected to injurious compression (50% strain, rate 100%/s; INJ), interleukin-1α-challenge (1 ng/ml; IL), and cyclic loading (intermittent 1 h loading periods, 15% strain, 1 Hz; CL). Plugs were assigned to six groups (control, INJ, IL, INJ-IL, IL-CL, INJ-IL-CL). Bulk and localized glycosaminoglycan (GAG) content (DMMB assay, digital densitometry), aggrecan biosynthesis ( 35 S-sulfate incorporation), and chondrocyte viability (fluorescence microscopy) were assessed on days 3-12. The INJ, IL, and INJ-IL groups exhibited rapid early (days 2-4) GAG loss in contrast to CL groups. On day 3, deep cartilage of INJ-IL-CL group had higher GAG content than INJ group (p < 0.05). On day 12, INJ-IL-CL group showed more accumulated GAG loss (normalized with control) than INJ-IL group (average fold changes 1.97 [95% CI: 1.23-2.70]; 1.66 [1.42-1.89]; p = 0.007). Aggrecan biosynthesis increased in CL groups on day 12 compared to day 0. Despite promoting aggrecan biosynthesis, this cyclic loading protocol seems to be beneficial early-on to deep cartilage, but later becoming incapable of restricting further degradation triggered by marked but non-destructive injury and cytokine transport., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Excessive tissue deformation near cartilage lesions and acute inflammation within the knee joint after anterior cruciate ligament (ACL) rupture and reconstruction surgery accelerate the loss of fixed charge density (FCD) and subsequent cartilage tissue degeneration. Here, we show how biomechanical and biochemical degradation pathways can predict FCD loss using a patient-specific finite element model of an ACL reconstructed knee joint exhibiting a chondral lesion. Biomechanical degradation was based on the excessive maximum shear strains that may result in cell apoptosis, while biochemical degradation was driven by the diffusion of pro-inflammatory cytokines. We found that the biomechanical model was able to predict substantial localized FCD loss near the lesion and on the medial areas of the lateral tibial cartilage. In turn, the biochemical model predicted FCD loss all around the lesion and at intact areas; the highest FCD loss was at the cartilage-synovial fluid-interface and decreased toward the deeper zones. Interestingly, simulating a downturn of an acute inflammatory response by reducing the cytokine concentration exponentially over time in synovial fluid led to a partial recovery of FCD content in the cartilage. Our novel numerical approach suggests that in vivo FCD loss can be estimated in injured cartilage following ACL injury and reconstruction. Our novel modeling platform can benefit the prediction of PTOA progression and the development of treatment interventions such as disease-modifying drug testing and rehabilitation strategies., (© 2021 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Post-traumatic osteoarthritis (PTOA) does not currently have clinical prognostic biomarkers or disease-modifying drugs, though promising candidates such as dexamethasone (Dex) exist. Many challenges in studying and treating this disease stem from tissue interactions that complicate understanding of drug effects. We present an ex vivo human osteochondral model of PTOA to investigate disease effects on cartilage and bone homeostasis and discover biomarkers for disease progression and drug efficacy., Methods: Human osteochondral explants were harvested from normal (Collins grade 0-1) ankle talocrural joints of human donors (2 female, 5 male, ages 23-70). After pre-equilibration, osteochondral explants were treated with a single-impact mechanical injury and TNF-α, IL-6, and sIL-6R ± 100 nM Dex for 21 days and media collected every 2-3 days. Chondrocyte viability, tissue DNA content, and glycosaminoglycan (sGAG) percent loss to the media were assayed and compared to untreated controls using a linear mixed effects model. Mass spectrometry analysis was performed for both cartilage tissue and pooled culture medium, and the statistical significance of protein abundance changes was determined with the R package limma and empirical Bayes statistics. Partial least squares regression analyses of sGAG loss and Dex attenuation of sGAG loss against proteomic data were performed., Results: Injury and cytokine treatment caused an increase in the release of matrix components, proteases, pro-inflammatory factors, and intracellular proteins, while tissue lost intracellular metabolic proteins, which was mitigated with the addition of Dex. Dex maintained chondrocyte viability and reduced sGAG loss caused by injury and cytokine treatment by 2/3 overall, with donor-specific differences in the sGAG attenuation effect. Biomarkers of bone metabolism had mixed effects, and collagen II synthesis was suppressed with both disease and Dex treatment by 2- to 5-fold. Semitryptic peptides associated with increased sGAG loss were identified. Pro-inflammatory humoral proteins and apolipoproteins were associated with lower Dex responses., Conclusions: Catabolic effects on cartilage tissue caused by injury and cytokine treatment were reduced with the addition of Dex in this osteochondral PTOA model. This study presents potential peptide biomarkers of early PTOA progression and Dex efficacy that can help identify and treat patients at risk of PTOA., (© 2022. The Author(s).)

Mucus is a selectively permeable hydrogel that protects wet epithelia from pathogen invasion and poses a barrier to drug delivery. Determining the parameters of a particle that promote or prevent passage through mucus is critical, as it will enable predictions about the mucosal passage of pathogens and inform the design of therapeutics. The effect of particle net charge and size on mucosal transport has been characterized using simple model particles; however, predictions of mucosal passage remain challenging. Here, we utilize rationally designed peptides to examine the integrated contributions of charge, hydrophobicity, and spatial configuration on mucosal transport. We find that net charge does not entirely predict transport. Specifically, for cationic peptides, the inclusion of hydrophobic residues and the position of charged and hydrophobic residues within the peptide impact mucosal transport. We have developed a simple model of mucosal transport that predicts how previously unexplored amino acid sequences achieve slow versus fast passage through mucus. This model may be used as a basis to predict transport behavior of natural peptide-based particles, such as antimicrobial peptides or viruses, and assist in the engineering of synthetic sequences with desired transport properties., (Copyright © 2021. Published by Elsevier Inc.)

Research in low Earth orbit (LEO) has become more accessible. The 2020 Biomanufacturing in Space Symposium reviewed space-based regenerative medicine research and discussed leveraging LEO to advance biomanufacturing for regenerative medicine applications. The symposium identified areas where financial investments could stimulate advancements overcoming technical barriers. Opportunities in disease modeling, stem-cell-derived products, and biofabrication were highlighted. The symposium will initiate a roadmap to a sustainable market for regenerative medicine biomanufacturing in space. This perspective summarizes the 2020 Biomanufacturing in Space Symposium, highlights key biomanufacturing opportunities in LEO, and lays the framework for a roadmap to regenerative medicine biomanufacturing in space., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Adjuvant therapy in autologous chondrocyte implantation (ACI) can control the post-traumatic environment and guide graft maturation to support cartilage repair. To investigate both aspects, we examined potential chondro-regenerative effects of lysed platelet concentrate (PC) and supplementary interleukin 10 (IL-10) on mechanically injured cartilage and on clinically used ACI scaffolds. ACI remnants and human cartilage explants, which were applied to an uniaxial unconfined compression as injury model, were treated with human IL-10 and/or PC from thrombocyte concentrates. We analyzed nuclear blebbing/TUNEL, sGAG content, immunohistochemistry, and the expression of COL1A1, COL2A1, COL10A1, SOX9, and ACAN. Post-injuriously, PC was associated with less cell death, increased COL2A1 expression, and decreased COL10A1 expression and, interestingly, the combination with Il-10 or Il-10 alone had no additional effects, except on COL10A1, which was most effectively decreased by the combination of PC and Il-10. The expression of COL2A1 or SOX9 was statistically not modulated by these substances. In contrast, in chondrocytes in ACI grafts the combination of PC and IL-10 had the most pronounced effects on all parameters except ACAN. Thus, using adjuvants such as PC and IL-10, preferably in combination, is a promising strategy for enhancing repair and graft maturation of autologous transplanted chondrocytes after cartilage injury.

Objectives: In this study, we apply a clustering method to proteomic data sets from bovine and human models of post-traumatic osteoarthritis (PTOA) to distinguish clusters of proteins based on their kinetics of release from cartilage and examined these groups for PTOA biomarker candidates. We then quantified the effects of dexamethasone (Dex) on the kinetics of release of the cartilage media proteome., Design: Mass spectrometry was performed on sample medium collected from two separate experiments using juvenile bovine and human cartilage explants (3 samples/treatment condition) during 20- or 21-day treatment with inflammatory cytokines (TNF-α, IL-6, sIL-6R) with or without a single compressive mechanical injury. All samples were incubated with or without 100 nM Dex. Clustering was performed on the correlation between normalized averaged release vectors for each protein., Results: Our proteomic method identified the presence of distinct clusters of proteins based on the kinetics of their release over three weeks of culture. Clusters of proteins with peak release after one to two weeks had biomarker candidates with increased release compared to control. Dex rescued some of the changes in protein release kinetics the level of control, and in all conditions except control, there was late release of immune-related proteins., Conclusions: We demonstrate a clustering method applied to proteomic data sets to identify and validate biomarkers of early PTOA progression and explore the relationships between the release of spatially related matrix components. Dex restored the kinetics of release to many matrix components, but not all factors that contribute to cartilage homeostasis., Competing Interests: Conflict of Interest The authors do not have any conflict of interest.

Background: Microfracture augmentation can be a cost-effective single-step alternative to current cartilage repair techniques. Trypsin pretreatment combined with a growth factor-functionalized self-assembling KLD hydrogel ("functionalized hydrogel") has been shown to improve overall cartilage repair and integration to surrounding tissue in small animal models of osteochondral defects., Hypothesis: Microfracture combined with trypsin treatment and a functionalized hydrogel will improve reparative tissue quality and integration as compared with microfracture alone in an equine model., Study Design: Controlled laboratory study., Methods: Bilateral cartilage defects (15-mm diameter) were created on the medial trochlear ridge of the femoropatellar joints in 8 adult horses (16 defects total). One defect was randomly selected to receive the treatment, and the contralateral defect served as the control (microfracture only). Treatment consisted of 2-minute trypsin pretreatment of the surrounding cartilage, subchondral bone microfracture, and functionalized hydrogel premixed with growth factors (platelet-derived growth factor and heparin-binding insulin-like growth factor 1). After surgery, all horses were subjected to standardized controlled exercise on a high-speed treadmill. Clinical evaluation was conducted monthly, and radiographic examinations were performed at 2, 16, 24, 32, 40, and 52 weeks after defect creation. After 12 months, all animals were euthanized. Magnetic resonance imaging, arthroscopy, gross pathologic evaluation of the joint, histology, immunohistochemistry, and biomechanical analyses were performed. Generalized linear mixed models (with horse as random effect) were utilized to assess outcome parameters. When P values were <.05, pairwise comparisons were made using least squares means., Results: Improved functional outcome parameters were observed for the treatment group, even though mildly increased joint effusion and subchondral bone sclerosis were noted on imaging. Microscopically, treatment resulted in improvement of several histologic parameters and overall quality of repaired tissue. Proteoglycan content based on safranin O-fast green staining was also significantly higher in the treated defects., Conclusion: Trypsin treatment combined with functionalized hydrogel resulted in improved microfracture augmentation., Clinical Relevance: Therapeutic strategies for microfracture augmentation, such as those presented in this study, can be cost-effective ways to improve cartilage healing outcomes, especially in more active patients.

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-β. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage.

The periodontal ligament (PDL) plays a critical role in providing immediate response to abrupt high loads during mastication while also facilitating slow remodeling of the alveolar bone. The PDL exceptional functionality is permitted by the unique nonuniform structure of the tissue. Two distinct areas that are critical to PDL function were previously identified: the furcation and the dense collar. Despite their hypothesized functions in tooth movement and maintenance, these 2 regions have not yet been compared within the context of their native environment. Therefore, the objective of this study is to elucidate the extracellular matrix (ECM) structure, composition, and biomechanical function of the furcation and the collar regions while maintaining the 3-dimensional (3D) structure in the murine PDL. We identify significant difference between the collar and furcation regions in both structure and mechanical properties. Specifically, we observed unique longitudinal structures in the dense collar that correlate with type VI collagen and LOX, both of which are associated with increased type I collagen density and tissue stiffness and are therefore proposed to function as scaffolds for tooth stabilization. We also found that the collar region is stiffer than the furcation region and therefore suggest that the dense collar acts as a suspense structure of the tooth within the bone during physiological loading. The furcation region of the PDL contained more proteins associated with reduced stiffness and higher tissue remodeling, as well as a dual mechanical behavior, suggesting a critical function in loads transfer and remodeling of the alveolar bone. In summary, this work unravels the nonuniform nature of the PDL within the 3D structural context and establishes understanding of regional PDL function, which opens new avenues for future studies of remodeling, regeneration, and disease.

Objectives: In this exploratory study, we used discovery proteomics to follow the release of proteins from bovine knee articular cartilage in response to mechanical injury and cytokine treatment. We also studied the effect of the glucocorticoid Dexamethasone (Dex) on these responses., Design: Bovine cartilage explants were treated with either cytokines alone (10 ng/ml TNFα, 20 ng/ml IL-6, 100 ng/ml sIL-6R), a single compressive mechanical injury, cytokines and injury, or no treatment, and cultured in serum-free DMEM supplemented with 1% ITS for 22 days. All samples were incubated with or without addition of 100 nM Dex. Mass spectrometry and western blot analyses were performed on medium samples for the identification and quantification of released proteins., Results: We identified 500 unique proteins present in all three biological replicates. Many proteins involved in the catabolic response of cartilage degradation had increased release after inflammatory stress. Dex rescued many of these catabolic effects. The release of some proteins involved in anabolic and chondroprotective processes was inconsistent, indicating differential effects on processes that may protect cartilage from injury. Dex restored only a small fraction of these to the control state, while others had their effects exacerbated by Dex exposure., Conclusions: We identified proteins that were released upon cytokine treatment which could be potential biomarkers of the inflammatory contribution to cartilage degradation. We also demonstrated the imperfect rescue of Dex on the effects of cartilage degradation, with many catabolic factors being reduced, while other anabolic or chondroprotective processes were not., Competing Interests: Conflict of Interest The authors do not have any conflict of interest.

Secondary joint damage is the process by which a single injury can lead to detrimental changes in adjacent tissue structures, typically through the spread of inflammatory responses. We recently developed an in vitro model of secondary joint damage using a murine rotator cuff explant system, in which injuries to muscle and bone cause massive cell death in otherwise uninjured tendon. The purpose of the present study was to test the ability cytokine-targeted and broad-spectrum therapeutics to prevent cell death and tissue degeneration associated with secondary joint damage. We treated injured bone-tendon-muscle explants with either interleukin-1 receptor antagonist, etanercept, or dexamethasone (DEX) for up to 7 days in culture. Only the low-dose DEX treatment was able to prevent cell death and tissue degeneration. We then identified a critical window between 24 and 72 h following injury for maximal benefit of DEX treatment through timed administration experiments. Finally, we performed two tendon-only explant studies to identify mechanistic effects on tendon health. Interestingly, DEX did not prevent cell death and degeneration in a model of cytokine-induced damage, suggesting other targets of DEX activity. Future studies will aim to identify factors in joint inflammation that may be targeted by DEX treatment, as well as to investigate novel delivery strategies. Statement of clinical significance: Overall, this work demonstrates beneficial effects of DEX administration on preventing tenocyte death and extracellular matrix degeneration in an explant model of secondary joint damage, supporting the clinical use of low-dose glucocorticoids for short-term treatment of joint inflammation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:139-149, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Purpose of the Study:  The incidence of tendon injuries increases dramatically with age, which presents a major clinical burden. While previous studies have sought to identify age-related changes in extracellular matrix structure and function, few have been able to explain fully why aged tissues are more prone to degeneration and injury. In addition, recent studies have also demonstrated that age-related processes in humans may be sex-dependent, which could be responsible for muddled conclusions in changes with age. In this study, we investigate short-term responses through an ex vivo explant culture model of stress deprivation that specifically questions how age and sex differentially affect the ability of tendons to respond to altered mechanical stimulus. Materials and Methods:  We subjected murine flexor explants from young (4 months of age) and aged (22-24 months of age) male and female mice to stress-deprived culture conditions for up to 1 week and investigated changes in viability, cell metabolism and proliferation, matrix biosynthesis and composition, gene expression, and inflammatory responses throughout the culture period. Results and Conclusions : We found that aging did have a significant influence on the response to stress deprivation, demonstrating that aged explants have a less robust response overall with reduced metabolic activity, viability, proliferation, and biosynthesis. However, age-related changes appeared to be sex-dependent. Together, this work demonstrates that the aging process and the subsequent effect of age on the ability of tendons to respond to stress-deprivation are inherently different based on sex, where male explants favor increased activity, apoptosis, and matrix remodeling while female explants favor reduced activity and tissue preservation.

While glucocorticoids have been used for over 50 years to treat rheumatoid and osteoarthritis pain, the prescription of glucocorticoids remains controversial because of potentially harmful side effects at the molecular, cellular and tissue levels. One member of the glucocorticoid family, dexamethasone (DEX) has recently been demonstrated to rescue cartilage matrix loss and chondrocyte viability in animal studies and cartilage explant models of tissue injury and post-traumatic osteoarthritis, suggesting the possibility of DEX as a disease-modifying drug if used appropriately. However, the literature on the effects of DEX on cartilage reveals conflicting results on the drug's safety, depending on the dose and duration of DEX exposure as well as the model system used. Overall, DEX has been shown to protect against arthritis-related changes in cartilage structure and function, including matrix loss, inflammation and cartilage viability. These beneficial effects are not always observed in model systems using initially healthy cartilage or isolated chondrocytes, where many studies have reported significant increases in chondrocyte apoptosis. It is crucially important to understand under what conditions DEX may be beneficial or harmful to cartilage and other joint tissues and to determine potential for safe use of this glucocorticoid in the clinic as a disease-modifying drug.

Objective: To investigate whether and how a sedentary lifestyle contributes to knee osteoarthritis (OA) incidence and severity., Design: An experiment was conducted using Hartley guinea pigs, an established idiopathic knee OA model. To simulate a sedentary lifestyle, growing animals (n = 18) were housed for 22 weeks in small cages that restricted their mobility, while another group of animals (n = 17) received daily treadmill exercise to simulate moderate physical activity. After the experiment, histological assessments, biochemical assays, and mechanical testing were conducted to compare tibial articular cartilage structure, strength, and degree of OA degeneration between sedentary and physically active animals. Groups were also compared based on body weight and composition, as well as gut microbial community composition assessed using fecal 16S rRNA gene sequencing., Results: Prevalence of knee OA was similar between sedentary and physically active animals, but severity of the disease (cartilage lesion depth) was substantially greater in the sedentary group (P = 0.02). In addition, during the experiment, sedentary animals developed cartilage with lower aggrecan quantity (P = 0.03) and accumulated more body weight (P = 0.005) and visceral adiposity (P = 0.007). Groups did not differ greatly, however, in terms of cartilage thickness, collagen quantity, or stiffness, nor in terms of muscle weight, subcutaneous adiposity, or gut microbial community composition., Conclusions: Our findings indicate that a sedentary lifestyle promotes the development of knee OA, particularly by enhancing disease severity rather than risk of onset, and this potentially occurs through multiple pathways including by engendering growth of functionally deficient joint tissues and the accumulation of excess body weight and adiposity., (Copyright © 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Impact Statement: A critical attribute for the long-term success of cartilage defect repair is the strong integration between the repair tissue and the surrounding native tissue. Current approaches utilized by physicians fail to achieve this attribute, leading to eventual relapse of the defect. This article demonstrates the concept of a simple, clinically viable approach for enhancing tissue integration via the combination of a safe, transient enzymatic treatment with a locally delivered, retained growth factor through an in vitro hydrogel/cartilage explant model.

The compression of brain tissue by a tumour mass is believed to be a major cause of the clinical symptoms seen in patients with brain cancer. However, the biological consequences of these physical stresses on brain tissue are unknown. Here, via imaging studies in patients and by using mouse models of human brain tumours, we show that a subgroup of primary and metastatic brain tumours, classified as nodular on the basis of their growth pattern, exert solid stress on the surrounding brain tissue, causing a decrease in local vascular perfusion as well as neuronal death and impaired function. We demonstrate a causal link between solid stress and neurological dysfunction by applying and removing cerebral compression, which respectively mimic the mechanics of tumour growth and of surgical resection. We also show that, in mice, treatment with lithium reduces solid-stress-induced neuronal death and improves motor coordination. Our findings indicate that brain-tumour-generated solid stress impairs neurological function in patients, and that lithium as a therapeutic intervention could counter these effects.

Stereociliary imprints in the tectorial membrane (TM) have been taken as evidence that outer hair cells are sensitive to shearing displacements of the TM, which plays a key role in shaping cochlear sensitivity and frequency selectivity via resonance and traveling wave mechanisms. However, the TM is highly hydrated (97% water by weight), suggesting that the TM may be flexible even at the level of single hair cells. Here we show that nanoscale oscillatory displacements of microscale spherical probes in contact with the TM are resisted by frequency-dependent forces that are in phase with TM displacement at low and high frequencies, but are in phase with TM velocity at transition frequencies. The phase lead can be as much as a quarter of a cycle, thereby contributing to frequency selectivity and stability of cochlear amplification.

Osteoarthritis is a debilitating joint disease affecting nearly 30 million people for which there are no disease-modifying therapies. Several drugs that have failed clinical trials have shown inefficient and inadequate delivery to target cells. Anabolic growth factors are one class of such drugs that could be disease-modifying if delivered directly to chondrocytes, which reside deep within dense, anionic cartilage tissue. To overcome this biological barrier, we conjugated a growth factor to a cationic nanocarrier for targeted delivery to chondrocytes and retention within joint cartilage after direct intra-articular injection. The nanocarrier uses reversible electrostatic interactions with anionic cartilage tissue to improve tissue binding, penetration, and residence time. Amine terminal polyamidoamine (PAMAM) dendrimers were end functionalized with variable molar ratios of poly(ethylene glycol) (PEG) to control surface charge. From this small family of variably PEGylated dendrimers, an optimal formulation showing 70% uptake into cartilage tissue and 100% cell viability was selected. When conjugated to insulin-like growth factor 1 (IGF-1), the dendrimer penetrated bovine cartilage of human thickness within 2 days and enhanced therapeutic IGF-1 joint residence time in rat knees by 10-fold for up to 30 days. In a surgical model of rat osteoarthritis, a single injection of dendrimer-IGF-1 rescued cartilage and bone more effectively than free IGF-1. Dendrimer-IGF-1 reduced width of cartilage degeneration by 60% and volumetric osteophyte burden by 80% relative to untreated rats at 4 weeks after surgery. These results suggest that PEGylated PAMAM dendrimer nanocarriers could improve pharmacokinetics and efficacy of disease-modifying osteoarthritis drugs in the clinic., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Osteoarthritis (OA), the most common form of arthritis, is a multi-factorial disease that primarily affects cartilage as well as other joint tissues such as subchondral bone. The lack of effective drug delivery, due to the avascular nature of cartilage and the rapid clearance of intra-articularly delivered drugs via the synovium, remains a major challenge in the development of disease modifying drugs for OA. Cationic delivery carriers can significantly enhance the uptake, penetration and retention of drugs in cartilage by interacting with negatively charged matrix proteoglycans. In this study, we used "supercharged" green fluorescent proteins (GFPs), engineered to have a wide range of net positive charge and surface charge distributions, to characterize the effects of carrier charge on transport into cartilage in isolation of other factors such as carrier size and shape. We quantified the uptake, extent of cartilage penetration and cellular uptake of the GFP variants into living human knee cartilage and bovine cartilage explants. Based on these results, we identified optimal net charges of GFP carriers for potential drug targets located within cartilage extracellular matrix as well as the resident live chondrocytes. These cationic GFPs did not have adverse effects on cartilage in terms of measured cell viability and metabolism, cartilage cell biosynthesis and matrix degradation at doses needed for drug delivery. In addition to quantifying the kinetics of GFP uptake, we developed a predictive mathematical model for transport of the GFP variants that exhibited the highest uptake and penetration into cartilage. This model was further used to predict the transport behavior of GFPs during scale-up to in vivo applications such as intra-articular injection into human knees. The insights gained from this study set the stage for development of cartilage-targeted delivery systems to prevent cartilage degeneration, improve tissue regeneration and reduce inflammation that may cause degradation of other joint tissues affected by OA., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cartilage provides low-friction properties and plays an essential role in diarthrodial joints. A hydrated ground substance composed mainly of proteoglycans (PGs) and a fibrillar collagen network are the main constituents of cartilage. Unfortunately, traumatic joint loading can destroy this complex structure and produce lesions in tissue, leading later to changes in tissue composition and, ultimately, to post-traumatic osteoarthritis (PTOA). Consequently, the fixed charge density (FCD) of PGs may decrease near the lesion. However, the underlying mechanisms leading to these tissue changes are unknown. Here, knee cartilage disks from bovine calves were injuriously compressed, followed by a physiologically relevant dynamic compression for twelve days. FCD content at different follow-up time points was assessed using digital densitometry. A novel cartilage degeneration model was developed by implementing deviatoric and maximum shear strain, as well as fluid velocity controlled algorithms to simulate the FCD loss as a function of time. Predicted loss of FCD was quite uniform around the cartilage lesions when the degeneration algorithm was driven by the fluid velocity, while the deviatoric and shear strain driven mechanisms exhibited slightly discontinuous FCD loss around cracks. Our degeneration algorithm predictions fitted well with the FCD content measured from the experiments. The developed model could subsequently be applied for prediction of FCD depletion around different cartilage lesions and for suggesting optimal rehabilitation protocols.

Hyaline cartilages, fibrocartilages and elastic cartilages play multiple roles in the human body including bearing loads in articular joints and intervertebral discs, providing joint lubrication, forming the external ears and nose, supporting the trachea, and forming the long bones during development and growth. The structure and organization of cartilage's extracellular matrix (ECM) are the primary determinants of normal function. Most diseases involving cartilage lead to dramatic changes in the ECM which can govern disease progression (e.g., in osteoarthritis), cause the main symptoms of the disease (e.g., dwarfism caused by genetically inherited mutations) or occur as collateral damage in pathological processes occurring in other nearby tissues (e.g., osteochondritis dissecans and inflammatory arthropathies). Challenges associated with cartilage diseases include poor understanding of the etiology and pathogenesis, delayed diagnoses due to the aneural nature of the tissue and drug delivery challenges due to the avascular nature of adult cartilages. This narrative review provides an overview of the clinical and pathological features as well as current treatment options available for various cartilage diseases. Late breaking advances are also described in the quest for development and delivery of effective disease modifying drugs for cartilage diseases including osteoarthritis, the most common form of arthritis that affects hundreds of millions of people worldwide., (Copyright © 2018. Published by Elsevier B.V.)

Purpose: Tendinopathy is a significant clinical problem thought to be associated with altered mechanical loading. Explant culture models allow researchers to alter mechanical loading in a controlled in vitro environment while maintaining tenocytes in their native matrix. However, current models do not accurately represent commonly injured tendons, ignoring contributions of associated musculature and bone, as well as regional collagen structure. This study details the characterization of amouse rotator cuff explant culture model, including bone, tendon, and muscle (BTM)., Materials and Methods: Following harvest, BTM explants were maintained in stress-deprived culture for one week and tendon was then assessed for changes in cell viability, metabolism, matrix structure and content., Results: Matrix turnover occurred throughout culture as manifested in both gene expression and biosynthesis, but this did not translate to net changes in total collagen or sulfated glycosaminoglycan content. Furthermore, tendon structure was not significantly altered throughout culture. However, we found significant cell death in BTM tendons after 3 days in culture, which we hypothesize is cytokine-induced. Using a targeted multiplex assay, we found high levels of pro-inflammatory cytokines released to the culture medium from muscle and bone, levels that did cause cell deathin tendon-alone controls., Conclusions: Overall, this model presents an innovative approach to understandingrotator cuff injury and tenocyte mechanobiology in a clinically-relevant tendon structure. Our model can be a powerful tool to investigate how mechanical and biological stimuli can alter normal tendon health and lead to tendon degeneration, and may provide a testbed for therapeutics for tendon repair.

Active matrix metalloproteases (MMPs) play a significant role in the pathogenesis of many diseases including osteoarthritis (OA), which involves progressive proteolytic degradation of cartilage. Clinical success of OA interventions that target MMPs has been limited by a lack of information about the presence and activity of specific disease-related proteases. We therefore developed a chemoproteomics approach based on MS to characterize the release and activity of MMPs in an in vitro model of the early inflammatory phase of posttraumatic OA (PTOA). We designed and synthesized chemical activity-based probes (ABPs) to identify active MMPs in bovine cartilage explants cultured for 30 days with the proinflammatory cytokine, interleukin-1α. Using these probes in an activity-based protein profiling-multidimensional identification technology (ABPP-MudPIT) approach, we identified active MMP-1, -2, -3, -7, -9, -12, and -13 in the medium after 10 days of culture, the time at which irreversible proteolysis of the collagen network in the explant was detected using proteolytic activation of FRET-quenched MMP substrates. Total MMP levels were quantified by shotgun proteomics, which, taken with ABPP-MudPIT data, indicated the presence of predominantly inactive MMPs in the culture medium. The selectivity of the ABPP-MudPIT approach was further validated by detection of specific endogenous MMPs activated de novo with 4-aminophenylmurcuric acetate. The utility of the new ABPP-MudPIT approach for detecting molecular biomarkers of PTOA disease initiation and potential targets for therapeutics motivates possible application in other diseases involving MMP activity., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rotator cuff disorders are one of the most common causes of shoulder pain and disability in the aging population but, unfortunately, the etiology is still unknown. One factor thought to contribute to the progression of disease is the external compression of the rotator cuff tendons, which can be significantly increased by age-related changes such as muscle weakness and poor posture. The objective of this study was to investigate the baseline compressive response of tendon and determine how this response is altered during maturation and aging. We did this by characterizing the compressive mechanical, viscoelastic, and poroelastic properties of young, mature, and aged mouse supraspinatus tendons using macroscale indentation testing and nanoscale high-frequency AFM-based rheology testing. Using these multiscale techniques, we found that aged tendons were stiffer than their mature counterparts and that both young and aged tendons exhibited increased hydraulic permeability and energy dissipation. We hypothesize that regional and age-related variations in collagen morphology and organization are likely responsible for changes in the multiscale compressive response as these structural parameters may affect fluid flow. Importantly, these results suggest a role for age-related changes in the progression of tendon degeneration, and we hypothesize that decreased ability to resist compressive loading via fluid pressurization may result in damage to the extracellular matrix (ECM) and ultimately tendon degeneration. These studies provide insight into the regional multiscale compressive response of tendons and indicate that altered compressive properties in aging tendons may be a major contributor to overall tendon degeneration.

In this study, a poroviscoelastic finite element model (FEM) was developed and used in conjunction with an AFM-based wide-bandwidth nanorheology system to predict the frequency-dependent mechanical behavior of tendon and dermis subjected to compression via nanoindentation. The aim was to distinguish between loading rates that are dominated by either poroelasticity, viscoelasticity, or the superposition of these processes. Using spherical probe tips having different radii, the force and tip displacement were measured and the magnitude, E ∗ , and phase angle, ϕ, of the dynamic complex modulus were evaluated for mouse supraspinatus tendon and mouse dermis. The peak frequencies of the phase angle were associated with the characteristic time constants of poroelastic and viscoelastic material behavior. The developed FE model could predict the separate poroelastic and viscoelastic responses of these soft tissues over a 4 decade frequency range, showing good agreement with experimental results. We observed that poroelasticity was the dominant energy dissipation mechanism for mouse dermis and supraspinatus tendon at higher indentation frequencies (10 2 to 10 4  Hz) whereas viscoelasticity was typically dominant at lower frequencies (<10 2  Hz). These findings show the underlying mechanical behavior of biological connective tissues and give insight into the role played by these different energy dissipation mechanisms in governing the function of these tissues at nanoscale., Statement of Significance: Soft biological tissues exhibit complex, load- and time-dependent mechanical behavior. Evaluating their mechanical behavior requires sophisticated experimental tools and numerical models that can capture the fundamental mechanisms governing tissue function. Using an Atomic-force-microscopy-based rheology system and finite element models, the roles of the two most dominant time-dependent mechanisms (poroelasticity and viscoelasticity) that govern the dynamic loading behavior of mouse skin and tendon have been investigated. FE models were able to predict and quantify the contribution of each mechanism to the overall dynamic response and confirming the presence of these two distinct mechanisms in the mechanical response. Overall, these results provide novel insight into the viscoelastic and poroelastic properties of mouse skin and tendon and promote better understanding of the underlying origins of each mechanism., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)