Modulation of diabetic wound healing using carbon monoxide gas-entrapping materials.

Diabetic wound healing is uniquely challenging to manage due to chronic inflammation and heightened microbial growth from elevated interstitial glucose. Carbon monoxide (CO), widely acknowledged as a toxic gas, is also known to provide unique therapeutic immune modulating effects. To facilitate delivery of CO, we have designed hyaluronic acid-based CO-gas-entrapping materials (CO-GEMs) for topical and prolonged gas delivery to the wound bed. We demonstrate that CO-GEMs promote the healing response in murine diabetic wound models (full-thickness wounds and pressure ulcers) compared to N2-GEMs and untreated controls.

Pancreatic duct organoid swelling is chloride-dependent.

Pancreatic secretions become viscous and acidic in Cystic fibrosis (CF), highlighting the role of CFTR in pancreatic fluid and bicarbonate secretion. Forskolin-induced swelling (FIS) assay developed in intestinal organoids measures residual CFTR function. It is not known whether FIS reflects bicarbonate secretion in pancreas, an organ that secretes near-isotonic NaHCO3 levels. To investigate this, we generated pancreatic duct organoids from CF and non-CF pigs. Epithelial and ductal origin was confirmed with epithelial markers, ion transporters and lack of acinar, islet cell markers. CF organoids were small with no identifiable lumen; CFTR was expressed only in non-CF organoids. Utilizing FIS, organoid size increased only in response to chloride, not bicarbonate. This report highlights pancreatic duct organoids isolated for the first time from CF pigs and evidence for chloride and not bicarbonate driving pancreatic organoid swelling. These organoids would be useful to test chloride permeability of CFTR mutations that cause CF pancreatic disease.

Tubular mitochondrial pyruvate carrier disruption elicits redox adaptations that protect from acute kidney injury.

OBJECTIVE: Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Although tubular metabolism changes markedly following acute kidney injury (AKI), it remains unclear which metabolic alterations are beneficial or detrimental. By analyzing large-scale, publicly available datasets, we observed that AKI consistently leads to downregulation of the mitochondrial pyruvate carrier (MPC). This investigation aimed to understand the contribution of the tubular MPC to kidney function, metabolism, and acute injury severity. METHODS: We generated tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice and employed renal function tests, in vivo renal 13C-glucose tracing, mechanistic enzyme activity assays, and tests of injury and survival in an established rhabdomyolysis model of AKI. RESULTS: MPC TubKO mice retained normal kidney function, displayed unchanged markers of kidney injury, but exhibited coordinately increased enzyme activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, compared to WT control mice, MPC TubKO mice showed increased glycolysis, decreased kidney injury and oxidative stress markers, and strikingly increased survival. CONCLUSIONS: Our findings suggest that decreased renal tubular mitochondrial pyruvate uptake hormetically upregulates oxidant defense systems before AKI and is a beneficial adaptive response after rhabdomyolysis-induced AKI. This raises the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Plant physiological indicators for optimizing conservation outcomes.

Plant species of concern often occupy narrow habitat ranges, making climate change an outsized potential threat to their conservation and restoration. Understanding the physiological status of a species during stress has the potential to elucidate current risk and provide an outlook on population maintenance. However, the physiological status of a plant can be difficult to interpret without a reference point, such as the capacity to tolerate stress before loss of function, or mortality. We address the application of plant physiology to conservation biology by distinguishing between two physiological approaches that together determine plant status in relation to environmental conditions and evaluate the capacity to avoid stress-induced loss of function. Plant physiological status indices, such as instantaneous rates of photosynthetic gas exchange, describe the level of physiological activity in the plant and are indicative of physiological health. When such measurements are combined with a reference point that reflects the maximum value or environmental limits of a parameter, such as the temperature at which photosynthesis begins to decline due to high temperature stress, we can better diagnose the proximity to potentially damaging thresholds. Here, we review a collection of useful plant status and reference point measurements related to photosynthesis, water relations and mineral nutrition, which can contribute to plant conservation physiology. We propose that these measurements can serve as important additional information to more commonly used phenological and morphological parameters, as the proposed parameters will reveal early warning signals before they are visible. We discuss their implications in the context of changing temperature, water and nutrient supply.

Effects of Radiotherapy Upon Bone Structure-Strength Relationships Vary With Sex and Fractionation of Dosing.

Background: Radiotherapy for tumor treatment in or near bones often causes osteopenia and/or osteoporosis, and the resulting increased bone fragility can lead to pathologic fractures. Bone mineral density (BMD) is often used to screen for fracture risk, but no conclusive relationship has been established between BMD and the microstructural/ biomechanical changes in irradiated bone. Understanding the effects of radiation dosing regimen on the bone structure-strength relationship would improve the ability to reduce fracture-related complications resulting from cancer treatment. Methods: Thirty-two C57B6J mice aged 10 - 12 weeks old were randomized to single dose (1 x 25 Gy) and fractionated dose (5 x 5 Gy) irradiation groups. Right hindlimbs were irradiated while the contralateral hindlimbs served as the non-irradiated control. Twelve weeks after irradiation, BMD and bone microstructure were assessed with micro-computed tomography, and mechanical strength/stiffness was assessed with a torsion test. The effects of radiation dosing regimen on bone microstructure and strength were assessed using ANOVA, and bone strength-structure relationships were investigated through correlation analysis of microstructural and mechanical parameters. Results: Fractionated irradiation induced significantly greater losses in BMD in the femur (23% - male mice, p=0.016; 19% - female mice) and the tibia (18% - male mice; 6% - female mice) than the single-dose radiation. The associated reductions in trabecular bone volume (-38%) and trabecular number (-34% to -42%), and the increase in trabecular separation (23% to 29%) were only significant in the male mice with fractionated dosing. There was a significant reduction in fracture torque in the femurs of male (p=0.021) and female (p=0.0017) mice within the fractionated radiation group, but not in the single dose radiation groups. There was moderate correlation between bone microstructure and mechanical strength in the single-dose radiation group (r = 0.54 to 0.73), but no correlation in the fractionated dosing group (r=0.02 to 0.03). Conclusion: Our data indicate more detrimental changes in bone microstructure and mechanical parameters in the fractionated irradiation group compared to the single dose group. This may suggest the potential for protecting bone if a needed therapeutic radiation dose can be delivered in a single session rather than administered in fractions.

Tubular Mitochondrial Pyruvate Carrier Disruption Elicits Redox Adaptations that Protect from Acute Kidney Injury.

Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Manipulation of Redox Metabolism Using Pharmacologic Ascorbate Opens a Therapeutic Window for Radio-Sensitization by ATM Inhibitors in Colorectal Cancer.

PURPOSE: Ataxia telangiectasia mutated kinase (ATM) inhibitors are potent radiosensitizers that regulate DNA damage responses and redox metabolism, but they have not been translated clinically because of the potential for excess normal tissue toxicity. Pharmacologic ascorbate (P-AscH-; intravenous administration achieving mM plasma concentrations) selectively enhances H2O2-induced oxidative stress and radiosensitization in tumors while acting as an antioxidant and mitigating radiation damage in normal tissues including the bowel. We hypothesized that P-AscH- could enhance the therapeutic index of ATM inhibitor-based chemoradiation by simultaneously enhancing the intended effects of ATM inhibitors in tumors and mitigating off-target effects in adjacent normal tissues. METHODS AND MATERIALS: Clonogenic survival was assessed in human (human colon tumor [HCT]116, SW480, HT29) and murine (CT26, MC38) colorectal tumor lines and normal cells (human umbilical vein endothelial cell, FHs74) after radiation ± DNA repair inhibitors ± P-AscH-. Tumor growth delay was assessed in mice with HCT116 or MC38 tumors after fractionated radiation (5 Gy × 3) ± the ATM inhibitor KU60019 ± P-AscH-. Intestinal injury, oxidative damage, and transforming growth factor ß immunoreactivity were quantified using immunohistochemistry after whole abdominal radiation (10 Gy) ± KU60019 ± P-AscH-. Cell cycle distribution and ATM subcellular localization were assessed using flow cytometry and immunohistochemistry. The role of intracellular H2O2 fluxes was assessed using a stably expressed doxycycline-inducible catalase transgene. RESULTS: KU60019 with P-AscH- enhanced radiosensitization in colorectal cancer models in vitro and in vivo by H2O2-dependent oxidative damage to proteins and enhanced DNA damage, abrogation of the postradiation G2 cell cycle checkpoint, and inhibition of ATM nuclear localization. In contrast, concurrent P-AscH- markedly reduced intestinal toxicity and oxidative damage with KU60019. CONCLUSIONS: We provide evidence that redox modulating drugs, such as P-AscH-, may facilitate the clinical translation of ATM inhibitors by enhancing tumor radiosensitization while simultaneously protecting normal tissues.

Tensile strain and altered synovial tissue metabolism in human knee osteoarthritis.

Synovium is critical for maintaining joint homeostasis and may contribute to mechanobiological responses during joint movement. We investigated mechanobiological responses of whole synovium from patients with late-stage knee osteoarthritis (OA). Synovium samples were collected during total knee arthroplasty and assigned to histopathology or cyclic 10% tensile strain loading, including (1) static (control); (2) low-frequency (0.3 Hz); and iii) high-frequency (1.0 Hz) for 30-min. After 6-h incubation, tissues were bisected for RNA isolation and immunostaining (3-nitrotyrosine; 3-NT). RNA sequencing was analyzed for differentially expressed genes and pathway enrichment. Cytokines and lactate were measured in conditioned media. Compared to controls, low-frequency strain induced enrichment of pathways related to interferon response, Fc-receptor signaling, and cell metabolism. High-frequency strain induced enrichment of pathways related to NOD-like receptor signaling, high metabolic demand, and redox signaling/stress. Metabolic and redox cell stress was confirmed by increased release of lactate into conditioned media and increased 3-NT formation in the synovial lining. Late-stage OA synovial tissue responses to tensile strain include frequency-dependent increases in inflammatory signaling, metabolism, and redox biology. Based on these findings, we speculate that some synovial mechanobiological responses to strain may be beneficial, but OA likely disturbs synovial homeostasis leading to aberrant responses to mechanical stimuli, which requires further validation.

Oxidative stress and impaired insulin secretion in cystic fibrosis pig pancreas.

Cystic fibrosis-related diabetes (CFRD) is one the most common comorbidities in cystic fibrosis (CF). Pancreatic oxidative stress has been postulated in the pathogenesis of CFRD, but no studies have been done to show an association. The main obstacle is the lack of suitable animal models and no immediate availability of pancreas tissue in humans. In the CF porcine model, we found increased pancreatic total glutathione (GSH), glutathione disulfide (GSSG), 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins, and decreased copper zinc superoxide dismutase (CuZnSOD) activity, all indicative of oxidative stress. CF pig pancreas demonstrated increased DHE oxidation (as a surrogate marker of superoxide) in situ compared to non-CF and this was inhibited by a SOD-mimetic (GC4401). Catalase and glutathione peroxidase activities were not different between CF and non-CF pancreas. Isolated CF pig islets had significantly increased DHE oxidation, peroxide production, reduced insulin secretion in response to high glucose and diminished secretory index compared to non-CF islets. Acute treatment with apocynin or an SOD mimetic failed to restore insulin secretion. These results are consistent with the hypothesis that CF pig pancreas is under significant oxidative stress as a result of increased O2 ●- and peroxides combined with reduced antioxidant defenses against reactive oxygen species (ROS). We speculate that insulin secretory defects in CF may be due to oxidative stress.

Extracellular biomolecular free radical formation during injury.

OBJECTIVE: Determine if oxidative damage increases in articular cartilage as a result of injury and matrix failure and whether modulation of the local redox environment influences this damage. Osteoarthritis is an age associated disease with no current disease modifying approaches available. Mechanisms of cartilage damage in vitro suggest tissue free radical production could be critical to early degeneration, but these mechanisms have not been described in intact tissue. To assess free radical production as a result of traumatic injury, we measured biomolecular free radical generation via immuno-spin trapping (IST) of protein/proteoglycan/lipid free radicals after a 2 J/cm2 impact to swine articular cartilage explants. This technique allows visualization of free radical formation upon a wide variety of molecules using formalin-fixed, paraffin-embedded approaches. Scoring of extracellular staining by trained, blinded scorers demonstrated significant increases with impact injury, particularly at sites of cartilage cracking. Increases remain in the absence of live chondrocytes but are diminished; thus, they appear to be a cell-dependent and -independent feature of injury. We then modulated the extracellular environment with a pulse of heparin to demonstrate the responsiveness of the IST signal to changes in cartilage biology. Addition of heparin caused a distinct change in the distribution of protein/lipid free radicals at sites of failure alongside a variety of pertinent redox changes related to osteoarthritis. This study directly confirms the production of biomolecular free radicals from articular trauma, providing a rigorous characterization of their formation by injury.

Delivery of therapeutic carbon monoxide by gas-entrapping materials.

Carbon monoxide (CO) has long been considered a toxic gas but is now a recognized bioactive gasotransmitter with potent immunomodulatory effects. Although inhaled CO is currently under investigation for use in patients with lung disease, this mode of administration can present clinical challenges. The capacity to deliver CO directly and safely to the gastrointestinal (GI) tract could transform the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, inspired by molecular gastronomy techniques, we have developed a family of gas-entrapping materials (GEMs) for delivery of CO to the GI tract. We show highly tunable and potent delivery of CO, achieving clinically relevant CO concentrations in vivo in rodent and swine models. To support the potential range of applications of foam GEMs, we evaluated the system in three distinct disease models. We show that a GEM containing CO dose-dependently reduced acetaminophen-induced hepatocellular injury, dampened colitis-associated inflammation and oxidative tissue injury, and mitigated radiation-induced gut epithelial damage in rodents. Collectively, foam GEMs have potential paradigm-shifting implications for the safe therapeutic use of CO across a range of indications.

Sex-based differences in the severity of radiation-induced arthrofibrosis.

As cancer survivorship increases, so does the number of patients that suffer from the late effects of radiation therapy. This includes arthrofibrosis, the development of stiff joints near the field of radiation. Previous reports have concentrated on skin fibrosis around the joint but largely ignored the deeper tissues of the joint. We hypothesized that fat, muscle, and the joint tissues themselves would play a more significant role in joint contracture after radiation than the skin surrounding the joint. To address this hypothesis, we irradiated the right hind flanks of mice with fractionated and unfractionated dose schedules, then monitored the mice for 3 months postradiotherapy. Mice were euthanized and physiological indications of arthrofibrosis including limb contracture and joint resting position were assessed. Stifle (knee) joints demonstrated significant arthrofibrosis, but none was observed in the hock (ankle) joints. During these studies, we were surprised to find that male and female mice showed a significantly different response to radiation injury. Female mice developed more injuries, had significantly worse contracture, and showed a greater difference in the expression of all markers studied. These results suggest that women undergoing radiation therapy might be at significantly greater risk for developing arthrofibrosis and may require specific adjustments to their care.

Automated quantification of live articular chondrocyte fluorescent staining using a custom image analysis framework.

The goal of this study was to develop, validate, and implement an image analysis framework to automatically analyze chondrocytes in 3D image stacks of cartilage acquired using a fluorescent confocal microscope. Source specimens consist of viable osteochondral tissue co-stained with multiple live-cell dyes. Our framework utilizes a seeded watershed-based algorithm to automatically segment individual chondrocytes in each 2D slice of the confocal image stack. The resulting cell segmentations are colocalized in 3D to eliminate duplicate segmentation of the same cell resulting from the visibility of fluorescence signal in multiple imaging planes, and the 3D cell distribution is used to automatically define the cartilage tissue volume. The algorithm then provides chondrocyte density data, and the associated segmentation can be used as a mask to extract and quantify per cell intensity of a secondary, functional dye co-staining the chondrocytes. The accuracy of the automated chondrocyte segmentation was validated against manual segmentations (average IOU = 0.79). When applied to a cartilage surrogate, this analysis framework estimated chondrocyte density within 10% of the true density and demonstrated a good agreement between framework's counts and manual counts (R2 = 0.99). In a real application, the framework was able to detect the increased dye signal of monochlorobimane (MCB) in chondrocytes treated with N-acetylcysteine (NAC) after mechanical injury, quantifying intracellular biochemical changes in living cells. This new framework allows for fast and accurate quantification of intracellular activities of chondrocytes, and it can be adapted for broader application in many imaging and treatment modalities, including therapeutic OA research.

Mid-infrared absorption by soft tissue sarcoma and cell ablation utilizing a mid-infrared interband cascade laser.

SIGNIFICANCE: Mid-infrared (MIR) light refers to wavelengths ranging from 3 to 30 µm and is the most attractive spectral region for ablation of soft and hard tissues. This is because building blocks of biological tissue, such as water, proteins, and lipids, exhibit molecular vibrational modes in the MIR wavelengths that result in strong MIR light absorption. To date, researchers investigating MIR lasers for surgical applications have used bulky light sources, such as free electron lasers, nonlinear light generators, and carbon dioxide lasers. We demonstrate the use of a tiny (a few microns wide, a few millimeters long) MIR interband cascade laser (ICL) for surgical thermal ablation applications. AIM: Our goal is to demonstrate the use of an ICL for surgical thermal ablation and demonstrate its efficacy in ablating normal fibroblasts and primary undifferentiated pleomorphic sarcoma tumor cells (C1619). APPROACH: We conducted Fourier transform infrared spectroscopy analysis of healthy and cancerous tissue samples, which indicated that the absorption of tumor tissue is higher than healthy tissue around 3.3-µm wavelength. These results enabled us to select an ICL emission wavelength, λ, of 3.3 µm to probe normal fibroblast and primary undifferentiated pleomorphic sarcoma cell survival after ICL exposure. RESULTS: We show that the absorption of tumorous tissue is higher than that of healthy tissues around the 3-µm MIR wavelength. We demonstrate that the ICL is able to ablate cancer cells at very low-power levels that can be clinically implemented but that this effect does not appear to be specific to C1619 when compared to normal fibroblasts. CONCLUSIONS: Our study demonstrates that ICLs may represent an exciting new avenue toward precise laser-based thermal ablation.

Intersections Between Mitochondrial Metabolism and Redox Biology Mediate Posttraumatic Osteoarthritis.

PURPOSE OF REVIEW: This review will cover foundational studies and recent findings that established key concepts for understanding the importance of redox biology to chondrocyte mitochondrial function and osteoarthritis pathophysiology after injury. RECENT FINDINGS: Articular chondrocyte mitochondria can be protected with a wide variety of antioxidants that will be discussed within a framework suggested by classic studies. These agents not only underscore the importance of thiol metabolism and associated redox function for chondrocyte mitochondria but also suggest complex interactions with signal transduction pathways and other molecular features of osteoarthritis that require more thorough investigation. Emerging evidence also indicates that reductive stress could occur alongside oxidative stress. Recent studies have shed new light on historic paradoxes in chondrocyte redox and mitochondrial physiology, leading to the development of promising disease-modifying therapies for posttraumatic osteoarthritis.

Targeting oxidative stress with amobarbital to prevent intervertebral disc degeneration: Part I. in vitro and ex vivo studies.

BACKGROUND: Mounting evidence that oxidative stress contributes to the pathogenesis of intervertebral disc (IVD) degeneration (IDD) suggests that therapies targeting oxidative stress may slow or prevent disease progression. PURPOSE: The objective of this study was to investigate the inhibitory effects of amobarbital (Amo) on the mitochondria of nucleus pulposus (NP) cells under tert-butyl hydrogen peroxide (tBHP)-induced oxidative stress or in NP tissues under oxidative stress from tissue injury as a means of identifying therapeutic targets for IDD. STUDY DESIGN/SETTING: We tested the effects inhibiting mitochondria, a major source of oxidants, with Amo in NP cells subjected to two different forms of insult: exposure to tBHP, and physical injury induced by disc transection. N-acetylcysteine (NAC), an antioxidant known to protect NP cells, was compared to the complex I inhibitor, Amo. METHODS: NP cells were pre-treated for 2 hours with Amo, NAC, or both, and then exposed to tBHP for 1 hour. Apoptosis, necrosis, and reactive oxygen species (ROS) production were assessed using confocal microscopy and fluorescent probes (Annexin V, propidium iodide, and MitoSox Red, respectively). The activation of mitogen-activated protein kinases (MAPKs) involved in oxidative stress responses were interrogated by confocal imaging of immunofluorescence stains using phospho-specific antibodies to extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38. Mitochondrial function was assessed by imaging JC-1 staining, a probe for membrane potential. RESULTS: Amo was modestly more protective than NAC by some measures, while both agents improved mitochondrial function and lowered tBHP-induced apoptosis, necrosis, and ROS production. Activation of MAPK by tBHP was significantly suppressed by both drugs. Physically injured IVDs were treated immediately after transection with Amo or NAC for 24 hours, and then stained with dihydroethidium (DHE), a fluorescent probe for ROS production. Immunofluorescence was used to track the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor that induces the expression of antioxidant genes. Amo and NAC significantly reduced ROS production and increased Nrf2 expression. CONCLUSION: These findings suggest that the progression of IDD may be forestalled by Amo via protection of NP cells from oxidative stress following IVD injury. CLINICAL SIGNIFICANCE: This study will define the extent to which a novel, minimally invasive procedure targeting oxidative stress in NP cells can augment surgical interventions intended to retard IVD degeneration.

Neoadjuvant Radiotherapy-Related Wound Morbidity in Soft Tissue Sarcoma: Perspectives for Radioprotective Agents.

Historically, patients with localized soft tissue sarcomas (STS) of the extremities would undergo limb amputation. It was subsequently determined that the addition of radiation therapy (RT) delivered prior to (neoadjuvant) or after (adjuvant) a limb-sparing surgical resection yielded equivalent survival outcomes to amputation in appropriate patients. Generally, neoadjuvant radiation offers decreased volume and dose of high-intensity radiation to normal tissue and increased chance of achieving negative surgical margins-but also increases wound healing complications when compared to adjuvant radiotherapy. This review elaborates on the current neoadjuvant/adjuvant RT approaches, wound healing complications in STS, and the potential application of novel radioprotective agents to minimize radiation-induced normal tissue toxicity.

Iatrogenic Articular Cartilage Injury in Arthroscopic Hip and Knee Videos and the Potential for Cartilage Cell Death When Simulated in a Bovine Model.

PURPOSE: To determine the incidence and characterize the severity of iatrogenic cartilage injuries. METHODS: Technique videos of arthroscopic femoral acetabular impingement procedures and meniscus repairs on VuMedi (n = 85) and Arthroscopy Techniques (n = 45) were reviewed and iatrogenic cartilage injuries were identified and graded (minor, intermediate, and major injury) by 2 independent reviewers. To demonstrate that even minor injuries on a cellular scale result in damage, a bovine osteochondral explant was used to create comparable minor iatrogenic injuries at varied forces that do not disrupt the articular surface (1.5 N, 2.5 N, and 9.8 N). Dead chondrocytes at the site of injury were stained with ethidium homodimer-2 and imaged with an Olympus FV1000 confocal microscope. χ2 tests were used for analysis; all results with P < .05 were considered significant. RESULTS: In total, 130 videos of arthroscopic meniscus and femoral acetabular impingement procedures were analyzed and the incidence of iatrogenic cartilage injury was 73.8%. There were 110 (70.0%) minor, 35 (22.3%) intermediate, and 11 (7.0%) major iatrogenic injuries. All forces tested in the minor injury bovine model resulted in chondrocyte death at the site of contact. CONCLUSIONS: Iatrogenic articular cartilage injuries are common in arthroscopy, occurring in more than 70% of the surgeon-published instructional videos analyzed. At least some chondrocyte death occurs with minor simulated iatrogenic injuries (1.5 N). CLINICAL RELEVANCE: The high rate of cartilage damage during arthroscopic technique videos likely under-represents the true incidence in clinical practice. Cell death occurs in the bovine minor injury model with minimal contact forces. This suggests iatrogenic cartilage damage during arthroscopy could contribute to clinical outcomes.

Deep Learning for Chondrocyte Identification in Automated Histological Analysis of Articular Cartilage.

Background: Histology-based methods are commonly used in osteoarthritis (OA) research because they provide detailed information about cartilage health at the cellular and tissue level. Computer-based cartilage scoring systems have previously been developed using standard image analysis techniques to give more objective and reliable evaluations of OA severity. The goal of this work was to develop a deep learning-based method to segment chondrocytes from histological images of cartilage and validate the resulting method via comparison with human segmentation. Methods: The U-Net approach was adapted for the task of chondrocyte segmentation. A training dataset consisting of 235 images and a validation set consisting of 25 images in which individual chondrocytes had been manually segmented, were used for training the U-Net. Chondrocyte count, detection accuracy, and boundary segmentation of the trained U-Net was evaluated by comparing its results with those of human observers. Results: The U-Net chondrocyte counts were not significantly different (p = 0.361 in a paired t-test) than the algorithm trainer counts (Pearson correlation coefficient = 0.92). The five expert observers had good agreement on chondrocyte counts (intraclass correlation coefficient = 0.868), however the resulting U-Net counted a significantly fewer chondrocytes than the average of those expert observers (p < 0.001 in a paired t-test). Chondrocytes were accurately detected by the U-Net (F1 scores = 0.86, 0.90, with respect to the selected expert observer and algorithm trainer). Segmentation accuracy was also high (IOU = 0.828) relative to the algorithm trainer. Conclusions: This work developed a method for chondrocyte segmentation from histological images of arthritic cartilage using a deep learning approach. The resulting method detected chondrocytes and delineated them with high accuracy. The method will continue to be improved through expansion to detect more complex cellular features representative of OA such as cell cloning. Clinical Relevance: The imaging tool developed in this work can be integrated into an automated cartilage health scoring system and helps provide a robust, objective and reliable assessment of OA severity in cartilage.

Persistent increase in mitochondrial superoxide mediates cisplatin-induced chronic kidney disease.

Severe and recurrent cisplatin-induced acute kidney injury (AKI) as part of standard cancer therapy is a known risk factor for development of chronic kidney disease (CKD). The specific role of superoxide (O2•-)-mediated disruption of mitochondrial oxidative metabolism in CKD after cisplatin treatment is unexplored. Cisplatin is typically administered in weekly or tri-weekly cycles as part of standard cancer therapy. To investigate the role of O2•- in predisposing patients to future renal injury and in CKD, mice were treated with cisplatin and a mitochondrial-specific, superoxide dismutase (SOD) mimetic, GC4419. Renal function, biomarkers of oxidative stress, mitochondrial oxidative metabolism, and kidney injury markers, as well as renal histology, were assessed to evaluate the cellular changes that occur one week and one month (CKD phase) after the cisplatin insult. Cisplatin treatment resulted in persistent upregulation of kidney injury markers, increased steady-state levels of O2•-, increased O2•--mediated renal tubules damage, and upregulation of mitochondrial electron transport chain (ETC) complex I activity both one week and one month following cisplatin treatment. Treatment with a novel, clinically relevant, small-molecule superoxide dismutase (SOD) mimetic, GC4419, restored mitochondrial ETC complex I activity to control levels without affecting complexes II-IV activity, as well as ameliorated cisplatin-induced kidney injury. These data support the hypothesis that increased mitochondrial O2•- following cisplatin administration, as a result of disruptions of mitochondrial metabolism, may be an important contributor to both AKI and CKD progression.