Surgery of the Equine Urinary Tract.

Urinary surgery in the horse may be challenging. More straightforward procedures, such as urinary bladder or urachal defects, do not usually require specialized equipment or imaging, although laboratory work is helpful. Congenital or acquired conditions of the ureters or kidneys may necessitate advanced diagnostic work-ups including advanced imaging /or and minimally invasive procedures. Some surgery of the lower urinary tract is done in the sedated, standing adult horse. Surgery involving the kidney typically requires general anesthesia. Laparoscopy and associated tools are frequently used. Although many of the surgical procedures discussed are quite involved, they are becoming more commonplace.

Application of a variational autoencoder for clustering and analyzing in situ articular cartilage cellular response to mechanical stimuli.

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

Connexin 43 Regulates Intercellular Mitochondrial Transfer from Human Mesenchymal Stromal Cells to Chondrocytes.

Background: The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin43 (Cx43, encoded by GJA1 ) and the truncated internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. Methods: Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1+ and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 hrs in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. Results: Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. Conclusions: Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA.

STRAINS: A big data method for classifying cellular response to stimuli at the tissue scale.

Cellular response to stimulation governs tissue scale processes ranging from growth and development to maintaining tissue health and initiating disease. To determine how cells coordinate their response to such stimuli, it is necessary to simultaneously track and measure the spatiotemporal distribution of their behaviors throughout the tissue. Here, we report on a novel SpatioTemporal Response Analysis IN Situ (STRAINS) tool that uses fluorescent micrographs, cell tracking, and machine learning to measure such behavioral distributions. STRAINS is broadly applicable to any tissue where fluorescence can be used to indicate changes in cell behavior. For illustration, we use STRAINS to simultaneously analyze the mechanotransduction response of 5000 chondrocytes-over 20 million data points-in cartilage during the 50 ms to 4 hours after the tissue was subjected to local mechanical injury, known to initiate osteoarthritis. We find that chondrocytes exhibit a range of mechanobiological responses indicating activation of distinct biochemical pathways with clear spatial patterns related to the induced local strains during impact. These results illustrate the power of this approach.

Mesenchymal stromal cells donate mitochondria to articular chondrocytes exposed to mitochondrial, environmental, and mechanical stress.

Articular cartilage has limited healing capacity and no drugs are available that can prevent or slow the development of osteoarthritis (OA) after joint injury. Mesenchymal stromal cell (MSC)-based regenerative therapies for OA are increasingly common, but questions regarding their mechanisms of action remain. Our group recently reported that although cartilage is avascular and relatively metabolically quiescent, injury induces chondrocyte mitochondrial dysfunction, driving cartilage degradation and OA. MSCs are known to rescue injured cells and improve healing by donating healthy mitochondria in highly metabolic tissues, but mitochondrial transfer has not been investigated in cartilage. Here, we demonstrate that MSCs transfer mitochondria to stressed chondrocytes in cell culture and in injured cartilage tissue. Conditions known to induce chondrocyte mitochondrial dysfunction, including stimulation with rotenone/antimycin and hyperoxia, increased transfer. MSC-chondrocyte mitochondrial transfer was blocked by non-specific and specific (connexin-43) gap-junction inhibition. When exposed to mechanically injured cartilage, MSCs localized to areas of matrix damage and extended cellular processes deep into microcracks, delivering mitochondria to chondrocytes. This work provides insights into the chemical, environmental, and mechanical conditions that can elicit MSC-chondrocyte mitochondrial transfer in vitro and in situ, and our findings suggest a new potential role for MSC-based therapeutics after cartilage injury.

Human mesenchymal stromal cells release functional mitochondria in extracellular vesicles.

Cartilage and other skeletal soft tissues heal poorly after injury, in part due to their lack of vascularity and low metabolic rate. No pharmacologic approaches have proven effective in preventing chronic degenerative disease after joint injury. Mesenchymal stromal cells (MSCs) have been investigated for their ability to treat pain associated with osteoarthritis (OA) and preserve articular cartilage. Limitations of MSCs include variability in cell phenotype, low engraftment and retention rates, and inconsistent clinical outcomes. Therefore, acellular biologic therapies such as extracellular vesicles (EVs) are currently being investigated. MSC-derived EVs have been found to replicate many of the therapeutic effects of their cells of origin, but the mechanisms driving this remain unclear. Recent evidence in non-orthopedic tissues suggests MSCs can rescue injured cells by donating mitochondria, restoring mitochondrial function in recipient cells, preserving cell viability, and promoting tissue repair. Our group hypothesized that MSCs package mitochondria for export into EVs, and that these so-called "mitoEVs" could provide a delivery strategy for cell-free mitochondria-targeted therapy. Therefore, the goals of this study were to: 1) characterize the vesicle fractions of the MSCs secretome with respect to mitochondrial cargoes, 2) determine if MSC-EVs contain functional mitochondria, and 3) determine if chondrocytes can take up MSC-derived mitoEVs. We isolated exosome, microvesicle, and vesicle-free fractions from MSC-conditioned media. Using a combination of dynamic light scattering and nanoparticle tracking, we determined that MSC-EV populations fall within the three size categories typically used to classify EVs (exosomes, microvesicles, apoptotic bodies). Fluorescent nanoparticle tracking, immunoblotting, and flow cytometry revealed that mitochondrial cargoes are abundant across all EV size populations, and mitoEVs are nearly ubiquitous among the largest EVs. Polarization staining indicated a subset of mitoEVs contain functional mitochondria. Finally, flow cytometry and fluorescent imaging confirmed uptake of mitoEVs by chondrocytes undergoing rotenone/antimycin-induced mitochondrial dysfunction. These data indicate that MSCs package intact, functional mitochondria into EVs, which can be transferred to chondrocytes in the absence of direct cell-cell interactions. This work suggests intercellular transfer of healthy MT to chondrocytes could represent a new, acellular approach to augment mitochondrial content and function in poorly-healing avascular skeletal soft tissues.

Biological Mechanisms for Cartilage Repair Using a BioCartilage Scaffold: Cellular Adhesion/Migration and Bioactive Proteins.

Objective. BioCartilage is a desiccated, particulated cartilage allograft used for repair of focal cartilage defects. It is mixed with a biologic such as bone marrow concentrate (BMC), pressed into a contained defect, and sealed with fibrin glue. The objective of this study was to assess if BioCartilage could serve as a bioactive scaffold by affecting cellular adhesion, cellular migration, or the release interleukin-1 receptor antagonist protein (IL-1RA), and to identify its full proteomic makeup. Design. Cartilage explants were used to model confined defects. BioCartilage was mixed with BMC, grafted into defects, and sealed with 1 of 5 fibrin glues. Constructs were cultured for 24 or 48 hours and then processed for live/dead microscopy. Chondrocyte and mesenchymal stem cell (MSC) adhesion on BioCartilage was assessed using scanning electron microscopy. Conditioned medium from cultures and the biologics used in the study were assayed for IL-1RA. The protein footprint of BioCartilage was determined using bottom-up proteomics. Results. BioCartilage supported chondrocyte and MSC attachment within 24 hours, and cell viability was retained in all constructs at 24 and 48 hours. Fibrin glue did not inhibit cell attachment. BMC had the highest concentration of IL-1RA. Proteomics yielded 254 proteins, including collagens, proteoglycans, and several bioactive proteins with known anabolic roles including cartilage oligomeric matrix protein. Conclusions. This study suggests that BioCartilage has the chemical composition and architecture to support cell adherence and migration and to provide bioactive proteins, which together should have biologics advantages in cartilage repair beyond its role as a scaffold.

Cartilage articulation exacerbates chondrocyte damage and death after impact injury.

Posttraumatic osteoarthritis (PTOA) is typically initiated by momentary supraphysiologic shear and compressive forces delivered to articular cartilage during acute joint injury and develops through subsequent degradation of cartilage matrix components and tissue remodeling. PTOA affects 12% of the population who experience osteoarthritis and is attributed to over $3 billion dollars annually in healthcare costs. It is currently unknown whether articulation of the joint post-injury helps tissue healing or exacerbates cellular dysfunction and eventual death. We hypothesize that post-injury cartilage articulation will lead to increased cartilage damage. Our objective was to test this hypothesis by mimicking the mechanical environment of the joint during and post-injury and determining if subsequent joint articulation exacerbates damage produced by initial injury. We use a model of PTOA that combines impact injury and repetitive sliding with confocal microscopy to quantify and track chondrocyte viability, apoptosis, and mitochondrial depolarization in a depth-dependent manner. Cartilage explants were harvested from neonatal bovine knee joints and subjected to either rapid impact injury (17.34 ± 0.99 MPa, 21.6 ± 2.45 GPa/s), sliding (60 min at 1 mm/s, under 15% axial compression), or rapid impact injury followed by sliding. Explants were then bisected and fluorescently stained for cell viability, caspase activity (apoptosis), and mitochondria polarization. Results show that compared to either impact or sliding alone, explants that were both impacted and slid experienced higher magnitudes of damage spanning greater tissue depths.

Proteomic Analysis and Cell Viability of Nine Amnion, Chorion, Umbilical Cord, and Amniotic Fluid-Derived Products.

OBJECTIVE: Amnion products are used in various musculoskeletal surgeries and as injections for joint pain with conflicting reports of cell viability and protein contents. The objective of this study was to determine the full proteome and examine cell viability in 9 commercial amnion products using an unbiased bottom-up shotgun proteomics approach and confocal microscopy. DESIGN: Products were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and searched against a UniProt Homo sapiens database. Relative protein abundance was determined for each sample. Based on proteomics results, lumican was measured by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis was performed for interleukin-1 receptor antagonist (IL-1Ra) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). Cell viability was determined by calcein AM (live) and ethidium homodimer (dead) staining and confocal microscopy. RESULTS: Proteomic analysis revealed 919 proteins in the nine products. Proteins were primarily collagens, keratin, and albumin. Lumican, a small leucine-rich proteoglycan (SLRP) was found in all samples. Western blot analysis for IL-1Ra and TIMP-2 indicated presence of both proteins, with nonspecific antibody binding also present in all samples. No live cells were identified in any product. CONCLUSIONS: Several novel proteins were identified through proteomics that might impart the beneficial effects of amnion products, including SLRPs, collagens, and regulators of fibroblast activity. IL-1Ra and TIMP-2 were identified, but concentrations measured by ELISA may be falsely increased due to nonspecific antibody binding. The concept that the amnion tissues provide live cells to aid in tissue regeneration cannot be supported by the findings of this study.

Amnion and Umbilical Cord-Derived Products in Sports Medicine: From Basic Science to Clinical Application.

BACKGROUND: Birth tissue products from amnion, chorion, umbilical cord, amniotic fluid, or cord blood are frequently marketed as viable sources of stem cells and growth factors. It can be difficult for health care professionals to differentiate implied from explicit conclusions in reported product analyses. PURPOSE: To provide an educational platform for health care professionals to interpret data presented in the promotion of birth tissue products. STUDY DESIGN: Descriptive laboratory study and expert opinion; Level of evidence, 5. METHODS: A cord blood product was analyzed by 3 methods for cell viability, 2 methods for assessment of cell morphology and cell type, multicolor flow cytometry to identify stem cells, and enzyme-linked immunosorbent assay (ELISA) plus Western blot for analysis of interleukin 1 receptor antagonist protein (IL-1ra). These data were compared with analyses reported by the manufacturer. RESULTS: Cell viability in the cord blood product was less than reported by the manufacturer, the cells were primarily leukocytes, no stem cells were present, and the concentration of IL-1ra was falsely increased due to nonspecific antibody binding in the sample. CONCLUSION: To assess birth tissue products, health care professionals should consider the following: (1) Understanding fluorescent dyes is important for assessing cell viability data-green does not always mean alive. (2) The report of "cells" in the product does not necessarily mean "stem cells"; microscopic images of at least ×20 or a hemogram should be evaluated to determine cell type (leukocyte, red blood cells, etc). (3) There is no single cluster of differentiation (CD) marker on flow cytometry to identify stem cells. (4) Biological tissues are complex substances, and inaccurately increased measurements of growth factors could be present in ELISA results because most ELISAs are not designed or validated for use in biologics. Furthermore, the reported measurement of growth factors should be considered relative to concentrations in native biological tissues and plasma. CLINICAL RELEVANCE: Health care professionals should be able to interpret cell viability, cell morphology, stem cell analysis using CD markers, and growth factor analysis when considering use of a birth tissue product in patients.

Synovial fluid lubricin and hyaluronan are altered in equine osteochondral fragmentation, cartilage impact injury, and full-thickness cartilage defect models.

The objectives of this study were to evaluate temporal changes in lubricin, hyaluronan (HA), and HA molecular weight (MW) distributions in three distinct models of equine joint injury affecting the carpal (wrist), tarsal (ankle), and femoropatellar (knee) joints. To establish ranges for lubricin, HA, and HA MW distributions across multiple joints, we first evaluated clinically healthy, high-motion equine joints. Synovial fluid was collected from high-motion joints in horses without clinical signs of joint disease (n = 11 horses, 102 joints) and from research horses undergoing carpal osteochondral fragmentation (n = 8), talar cartilage impact injury (n = 7), and femoral trochlear ridge full-thickness cartilage injury (n = 22) prior to and following arthroscopically induced joint injury. Lubricin and HA concentrations were measured via enzyme-linked immunosorbent assays, and gel electrophoresis was performed to evaluate HA MW distributions. Synovial fluid parameters were analyzed via linear regression models, revealing that lubricin and HA concentrations were conserved across healthy, high-motion joints. Lubricin concentrations increased post-injury in all osteoarthritis models (carpal fragmentation P = .001; talar impact P < .001; femoral trochlear ridge cartilage defect P = .03). Sustained loss of HA was noted post-arthroscopy following carpal osteochondral fragmentation (P < .0001) and talar impact injury (P < .001). Lubricin may be elevated to compensate for the loss of HA and to protect cartilage post-injury. Further investigation into the mechanisms regulating lubricin and HA following joint injury and their effects on joint homeostasis is warranted, including whether lubricin has value as a biomarker for post-traumatic osteoarthritis.

Mitoprotective therapy prevents rapid, strain-dependent mitochondrial dysfunction after articular cartilage injury.

Posttraumatic osteoarthritis (PTOA) involves the mechanical and biological deterioration of articular cartilage that occurs following joint injury. PTOA is a growing problem in health care due to the lack of effective therapies combined with an aging population with high activity levels. Recently, acute mitochondrial dysfunction and altered cellular respiration have been associated with cartilage degeneration after injury. This finding is particularly important because recently developed mitoprotective drugs, including SS peptides, can preserve mitochondrial structure and function after acute injury in other tissues. It is not known, however, if cartilage injury induces rapid structural changes in mitochondria, to what degree mitochondrial dysfunction in cartilage depends on the mechanics of injury or the time frame over which such dysfunction develops. Similarly, it is unknown if SS-peptide treatment can preserve mitochondrial structure and function after cartilage injury. Here, we combined fast camera elastography, longitudinal fluorescence assays, and computer vision techniques to track the fates of thousands of individual cells. Our results show that impact induces mechanically dependent mitochondrial depolarization within a few minutes after injury. Electron microscopy revealed that impact causes rapid structural changes in mitochondria that are related to reduced mitochondrial function, namely, fission and loss of cristae structure. We found that SS-peptide treatment prior to impact protects the mitochondrial structure and preserves mitochondrial function at levels comparable with that of unimpacted control samples. Overall, this study reveals the vital role of mitochondria in mediating cartilage's peracute (within minutes) response to traumatic injury and demonstrates mitoprotection as a promising therapeutic strategy for injury-induced cartilage damage.

Integrin α10ß1-Selected Mesenchymal Stem Cells Mitigate the Progression of Osteoarthritis in an Equine Talar Impact Model.

BACKGROUND: Early intervention with mesenchymal stem cells (MSCs) after articular trauma has the potential to limit progression of focal lesions and prevent ongoing cartilage degeneration by modulating the joint environment and/or contributing to repair. Integrin α10ß1 is the main collagen type II binding receptor on chondrocytes, and MSCs that are selected for high expression of the α10 subunit have improved chondrogenic potential. The ability of α10ß1-selected (integrin α10high) MSCs to protect cartilage after injury has not been investigated. PURPOSE: To investigate integrin α10high MSCs to prevent posttraumatic osteoarthritis in an equine model of impact-induced talar injury. STUDY DESIGN: Controlled laboratory study. METHODS: Focal cartilage injuries were created on the tali of horses (2-5 years, n = 8) by using an impacting device equipped to measure impact stress. Joints were treated with 20 × 106 allogenic adipose-derived α10high MSCs or saline vehicle (control) 4 days after injury. Synovial fluid was collected serially and analyzed for protein content, cell counts, markers of inflammation (prostaglandin E2, tumor necrosis factor α) and collagen homeostasis (procollagen II C-propeptide, collagen type II cleavage product), and glycosaminoglycan content. Second-look arthroscopy was performed at 6 weeks, and horses were euthanized at 6 months. Joints were imaged with radiographs and quantitative 3-T magnetic resonance imaging. Postmortem examinations were performed, and India ink was applied to the talar articular surface to identify areas of cartilage fibrillation. Synovial membrane and osteochondral histology was performed, and immunohistochemistry was used to assess type I and II collagen and lubricin. A mixed effect model with Tukey post hoc and linear contrasts or paired t tests were used, as appropriate. RESULTS: Integrin α10high MSC-treated joints had less subchondral bone sclerosis on radiographs (P = .04) and histology (P = .006) and less cartilage fibrillation (P = .04) as compared with control joints. On gross pathology, less India ink adhered to impact sites in treated joints than in controls, which may be explained by the finding of more prominent lubricin immunostaining in treated joints. Prostaglandin E2 concentration in synovial fluid and mononuclear cell synovial infiltrate were increased in treated joints, suggesting possible immunomodulation by integrin α10high MSCs. CONCLUSION: Intra-articular administration of integrin α10high MSCs is safe, and evidence suggests that the cells mitigate the effects of joint trauma. CLINICAL RELEVANCE: This preclinical study indicates that intra-articular therapy with integrin α10high MSCs after joint trauma may be protective against posttraumatic osteoarthritis.

Mitochondrial dysfunction is an acute response of articular chondrocytes to mechanical injury.

Mitochondrial (MT) dysfunction is known to occur in chondrocytes isolated from end-stage osteoarthritis (OA) patients, but the role of MT dysfunction in the initiation and early pathogenesis of post-traumatic OA (PTOA) remains unclear. The objective of this study was to investigate chondrocyte MT function immediately following mechanical injury in cartilage, and to determine if the response to injury differed between a weight bearing region (medial femoral condyle; MFC) and a non-weight bearing region (distal patellofemoral groove; PFG) of the same joint. Cartilage was harvested from the MFC and PFG of 10 neonatal bovids, and subjected to injurious compression at varying magnitudes (5-17 MPa, 5-34 GPa/s) using a rapid single-impact model. Chondrocyte MT respiratory function, MT membrane polarity, chondrocyte viability, and cell membrane damage were assessed in situ. Cartilage impact resulted in MT depolarization and impaired MT respiratory function within 2 h of injury. Cartilage from a non-weight bearing region of the joint (PFG) was more sensitive to impact-induced MT dysfunction and chondrocyte death than cartilage from a weight-bearing surface (MFC). Our findings suggest that MT dysfunction is an acute response of chondrocytes to cartilage injury, and that MT may play a key mechanobiological role in the initiation and early pathogenesis of PTOA. CLINICAL SIGNIFICANCE: Direct therapeutic targeting of MT function in the early post-injury time frame may provide a strategy to block perpetuation of tissue damage and prevent the development of PTOA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:739-750, 2018.

Mitoprotective therapy preserves chondrocyte viability and prevents cartilage degeneration in an ex vivo model of posttraumatic osteoarthritis.

No disease-modifying osteoarthritis (OA) drugs are available to prevent posttraumatic osteoarthritis (PTOA). Mitochondria (MT) mediate the pathogenesis of many degenerative diseases, and recent evidence indicates that MT dysfunction is a peracute (within minutes to hours) response of cartilage to mechanical injury. The goal of this study was to investigate cardiolipin-targeted mitoprotection as a new strategy to prevent chondrocyte death and cartilage degeneration after injury. Cartilage was harvested from bovine knee joints and subjected to a single, rapid impact injury (24.0 ±1.4 MPa, 53.8 ± 5.3 GPa/s). Explants were then treated with a mitoprotective peptide, SS-31 (1µM), immediately post-impact, or at 1, 6, or 12 h after injury, and then cultured for up to 7 days. Chondrocyte viability and apoptosis were quantified in situ using confocal microscopy. Cell membrane damage (lactate dehydrogenase activity) and cartilage matrix degradation (glycosaminoglycan loss) were quantified in cartilage-conditioned media. SS-31 treatment at all time points after impact resulted in chondrocyte viability similar to that of un-injured controls. This effect was sustained for up to a week in culture. Further, SS-31 prevented impact-induced chondrocyte apoptosis, cell membrane damage, and cartilage matrix degeneration. CLINICAL SIGNIFICANCE: This study is the first investigation of cardiolipin-targeted mitoprotective therapy in cartilage. These results suggest that even when treatment is delayed by up to 12 h after injury, mitoprotection may be a useful strategy in the prevention of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-10, 2018.

Microscale frictional strains determine chondrocyte fate in loaded cartilage.

Mounting evidence suggests that altered lubricant levels within synovial fluid have acute biological consequences on chondrocyte homeostasis. While these responses have been connected to increased friction, the mechanisms behind this response remain unknown. Here, we combine a frictional bioreactor with confocal elastography and image-based cellular assays to establish the link between cartilage friction, microscale shear strain, and acute, adverse cellular responses. Our incorporation of cell-scale strain measurements reveals that elevated friction generates high shear strains localized near the tissue surface, and that these elevated strains are closely associated with mitochondrial dysfunction, apoptosis, and cell death. Collectively, our data establish two pathways by which chondrocytes negatively respond to friction: an immediate necrotic response and a longer term pathway involving mitochondrial dysfunction and apoptosis. Specifically, in the surface region, where shear strains can exceed 0.07, cells are predisposed to acute death; however, below this surface region, cells exhibit a pathway consistent with apoptosis in a manner predicted by local shear strains. These data reveal a mechanism through which cellular damage in cartilage arises from compromised lubrication and show that in addition to boundary lubricants, there are opportunities upstream of apoptosis to preserve chondrocyte health in arthritis therapy.

Pharmacokinetics and in vitro effects of tegaserod, a serotonin 5-hydroxytryptamine 4 (5-HT4) receptor agonist with prokinetic activity in horses.

Tegaserod, a serotonin agonist, has been shown to have prokinetic effects in horses, but pharmacokinetic information is not currently available. The pharmacokinetics and in vitro effects of tegaserod were evaluated. Tegaserod increased the contractile activity of smooth muscle preparations of the equine pelvic flexure. Pertinent pharmacokinetic parameters for a single IV and oral dose were determined. Therapeutic plasma concentrations of tegaserod were achieved by a single oral dose at 0.27 mg/kg. These findings indicate that further clinical studies are warranted to investigate potential benefits in cases of functional gastrointestinal motility disorders in horses.

Post-traumatic osteoarthritis of the ankle: A distinct clinical entity requiring new research approaches.

The diagnosis of ankle osteoarthritis (OA) is increasing as a result of advancements in non-invasive imaging modalities such as magnetic resonance imaging, improved arthroscopic surgical technology and heightened awareness among clinicians. Unlike OA of the knee, primary or age-related ankle OA is rare, with the majority of ankle OA classified as post-traumatic (PTOA). Ankle trauma, more specifically ankle sprain, is the single most common athletic injury, and no effective therapies are available to prevent or slow progression of PTOA. Despite the high incidence of ankle trauma and OA, ankle-related OA research is sparse, with the majority of clinical and basic studies pertaining to the knee joint. Fundamental differences exist between joints including their structure and molecular composition, response to trauma, susceptibility to OA, clinical manifestations of disease, and response to treatment. Considerable evidence suggests that research findings from knee should not be extrapolated to the ankle, however few ankle-specific preclinical models of PTOA are currently available. The objective of this article is to review the current state of ankle OA investigation, highlighting important differences between the ankle and knee that may limit the extent to which research findings from knee models are applicable to the ankle joint. Considerations for the development of new ankle-specific, clinically relevant animal models are discussed. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:440-453, 2017.

Sub-critical impact inhibits the lubricating mechanisms of articular cartilage.

Although post-traumatic osteoarthritis accounts for a significant proportion of all osteoarthritis, the understanding of both biological and mechanical phenomena that lead to cartilage degeneration in the years to decades after trauma is still lacking. In this study, we evaluate how cartilage lubrication is altered after a sub-critical impact (i.e., an impact to the cartilage surface that produces surface cracking but not full thickness fissuring). Through utilizing a Stribeck-like framework, the elastoviscous transition, we evaluated changes to both the innate boundary lubricating ability of cartilage after impact and also the effectiveness of high viscosity lubricants to lower friction after impact. Increases in boundary friction coincided with changes in lubricin localization after impact. However, larger increases in friction coefficient were observed in mixed-mode lubrication which can be predicted by increases in surface roughness due to cartilage fissuring. The data here reveal distinct mechanisms of cartilage lubrication that can fail after traumatic impact and may explain a key mechanical phenomenon that predisposes cartilage to development of osteoarthritis after injury.