Preliminary in vivo investigation of the mesenchymal stromal cell secretome as a novel treatment for methicillin-resistant Staphylococcus aureus in equine skin wounds.

OBJECTIVE: We aimed to study the antimicrobial and pro-healing potential of equine mesenchymal stromal cell secreted products (i.e. secretome), collected as conditioned media (mesenchymal stromal cell-conditioned media, MSC CM), in a novel in vivo model of methicillin-resistant Staphylococcus aureus (MRSA)-inoculated equine thorax wounds. STUDY DESIGN: Prospective in vivo study. ANIMALS: Two Thoroughbred geldings. METHODS: Six full-thickness cutaneous wounds were created bilaterally on the dorsal thorax of two horses (n = 12 wounds/horse). Wounds on the left thoraces were inoculated with MRSA on day 0. All wounds were then treated with either mupirocin ointment, MSC CM, or vehicle control (n = 4 wounds per group) once daily for 3 days. Photographs were taken to quantify wound scores and sizes, as well as samples to determine bacterial colony forming units (CFUs), at days 0, 1, 2, 3, 7, 14, 21, and 28. The wound edge was biopsied on days 0, 7, and 28, and scored histologically. RESULTS: Inoculated wounds had more bacterial CFUs at day 1 (p < .0001) and were larger in size at day 28 (p = .0009) than noninoculated wounds. Mupirocin-treated wounds were smaller than MSC CM and vehicle control-treated wounds at day 28 (p = .003). Mesenchymal stromal cell-conditioned media did not affect CFU numbers in inoculated and noninoculated wounds. Moreover, MSC CM did not affect the parameters of wound size or gross or microscopic wound scores over time. CONCLUSION: Mesenchymal stromal cell-conditioned media did not exhibit antimicrobial or pro-healing properties in the current study; however, the in vivo model of inoculated equine thorax wounds requires further optimization. CLINICAL SIGNIFICANCE: This pilot study contributes to a growing understanding of the equine MSC secretome as an antimicrobial and pro-healing therapeutic for equine wounds.

Emergency Standing Laparoscopic Treatment of Uncontrolled Post-Castration Hemorrhage in Two Geldings.

BACKGROUND: Persistent hemorrhage of testicular vessels is a potentially life-threatening complication of equine castration. Frequently, general anesthesia is required to retrieve and ligate the bleeding vasculature when standing wound packing and retrieval of the spermatic cord are unsuccessful. We propose standing laparoscopic ligation of the testicular arteries via the paralumbar fossa as a rapid, effective means of halting hemorrhage while avoiding castration site trauma as well as the cardiovascular and recovery risks of general anesthesia. METHODS: Two geldings, 6 and 9 months old, presented for emergency treatment of severe post-castration hemorrhage of 10 and 24 h durations, respectively. Both geldings underwent standing laparoscopy under light sedation and the testicular vessels were ligated using a bipolar vessel-sealing device. RESULTS: Testicular vessel sealing was successfully performed in both geldings by standing laparoscopy and resulted in immediate cessation of hemorrhage. In one case, a left paralumbar fossa approach allowed coagulation of both the left and right spermatic vessels. The procedure time was 25 and 35 min. No complications occurred, and both geldings recovered uneventfully. CONCLUSIONS: Standing, laparoscopic ligation of the testicular arteries is a feasible emergency treatment in young geldings and can be applied in cases of uncontrolled post-castration hemorrhage.

Restoration of function following traumatic superior eyelid avulsion in a horse treated with advancement flap blepharoplasty (H-plasty) and subdermal hyaluronic acid filler.

OBJECTIVE: To describe the successful restoration of superior eyelid function in a horse following traumatic avulsion using an advancement flap blepharoplasty and subdermal hyaluronic acid filler. ANIMAL STUDIED: A 21-year-old American Paint Horse stallion who was attacked by a fellow stallion resulting in numerous traumatic injuries including avulsion of approximately 75% of the left superior eyelid. PROCEDURES: With standing sedation and locoregional anesthesia, the superior eyelid wound was debrided and an advancement flap blepharoplasty (H-plasty) and temporary tarsorrhaphy were performed. Routine healing of the surgical site occurred over the subsequent weeks, though lagophthalmos persisted. At 2 and 4 weeks post-operatively, 2.4% cross-linked hyaluronic acid was injected subdermally into the superior eyelid to attempt to improve corneal coverage. At 8 weeks post-operatively, a complete blink was restored and the cosmetic outcome was good. CONCLUSIONS: Injection of subdermal hyaluronic acid filler following eyelid injuries or blepharoplastic procedures that result in lagophthalmos can improve corneal coverage by the eyelids and allow for maintenance of a comfortable and visual eye.

Peri-anesthetic hypothermia in rodents: A factor to consider for accurate and reproducible outcomes in orthopedic device-related infection studies.

Orthopedic device-related infection (ODRI) preclinical models are widely used in translational research. Most ODRI models require induction of general anesthesia, which frequently results in hypothermia in rodents. This study aimed to evaluate the impact of peri-anesthetic hypothermia in rodents on outcomes in preclinical ODRI studies. A retrospective analysis of all rodents that underwent surgery under general anesthesia to induce an ODRI model with inoculation of Staphylococcus epidermidis between 2016 and 2020 was conducted. A one-way multivariate analysis of covariance (one-way MANCOVA) was used to determine the fixed effect of peri-anesthetic hypothermia (hypothermic defined as rectal temperature <35°C) on the combined harvested tissue and implant colony-forming unit (CFU) counts, and having controlled for the study groups including treatments received, duration of surgery and anesthesia, and study period. The results showed a significant effect of peri-anesthetic hypothermia on the post-mortem combined CFU counts from the harvested tissue and implant(s) (p = 0.01) when comparing normo- versus hypothermic rodents. Using Wilks' Λ as a criterion to determine the contribution of independent variables to the model, peri-anesthetic hypothermia was the most significant, though still a weak predictor, of increased harvested CFU counts. Altogether, the data corroborate the concept that bacterial colonization is affected by abnormal body temperature during general anesthesia at the time of bacterial inoculation in rodents, which needs to be taken into consideration to decrease infection data variability and improve experimental reproducibility.

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.

Influence of screw head diameter on ex vivo fixation of equine lateral condylar fractures with 5.5 mm cortical screws.

OBJECTIVE: To determine the influence of screw head diameter on equine condylar fracture fixation with 5.5 mm cortical screws. STUDY DESIGN: Ex vivo, biomechanical study, blinded, matched-pair design. SAMPLE POPULATION: Fifteen pairs of equine third metacarpal (MC3) bones. METHODS: Lateral condylar fractures were simulated by parasagittal osteotomies and repaired pairwise by 2 × 5.5 mm cortical screws of 8 mm (standard) or 10 mm (modified) head diameter. Interfragmentary compression at maximum screw insertion torque was measured. The instrumented specimens were pairwise stratified for biomechanical testing under the following modalities (n = 5): (1) screw insertion torque to failure, (2) quasi-static axial load to failure, and (3) cyclic axial load to 2 mm displacement followed by failure. Tests (1) and (2) were analyzed for yield, maximum, and failure torque/angle and load/displacement, respectively. Number of cycles to 2 mm displacement and failure was assessed from test (3). RESULTS: Maximum insertion torque was greater, and failure angle smaller, when constructs repaired with modified screws were tested (8.1 ± 0.5 vs. 7.4 ± 0.5 Nm; P = .0047 and 550 ± 104 vs. 1130 ± 230; P = .008). Axial yield (7118 ± 707 vs. 5740 ± 2267 N; P = .043) and failure load (12 347 ± 3359 vs. 8695 ± 2277 N; P = .043) were greater for specimens repaired with modified screws. No difference was detected between constructs in the number of cycles to 2 mm displacement. CONCLUSION: Condylar MC3 osteotomies repaired with modified 5.5 mm cortical screws sustained greater maximal hand torque insertion, smaller insertion failure angle and 1.4 fold greater quasi-static failure forces than constructs repaired with standard 5.5 mm screws. CLINICAL SIGNIFICANCE: Use of modified screws with larger heads may improve the fixation of condylar fractures in horses. These results provide evidence to justify clinical evaluation in horses undergoing fracture repair.

Butyrate Inhibits Osteoclast Activity In Vitro and Regulates Systemic Inflammation and Bone Healing in a Murine Osteotomy Model Compared to Antibiotic-Treated Mice.

Short-chain fatty acids (SCFAs) produced by the gut microbiota have previously been demonstrated to play a role in numerous chronic inflammatory diseases and to be key mediators in the gut-bone signaling axis. However, the role of SCFAs in bone fracture healing and its impact on systemic inflammation during the regeneration process has not been extensively investigated yet. The aim of this study was to first determine the effects of the SCFA butyrate on key cells involved in fracture healing in vitro, namely, osteoclasts and mesenchymal stromal cells (MSCs), and second, to assess if butyrate supplementation or antibiotic therapy impacts bone healing, systemic immune status, and inflammation levels in a murine osteotomy model. Butyrate significantly reduced osteoclast formation and resorption activity in a dose-dependent manner and displayed a trend for increased calcium deposits in MSC cultures. Numerous genes associated with osteoclast differentiation were differentially expressed in osteoclast precursor cells upon butyrate exposure. In vivo, antibiotic-treated mice showed reduced SCFA levels in the cecum, as well as a distinct gut microbiome composition. Furthermore, circulating proinflammatory TNFα, IL-17a, and IL-17f levels, and bone preserving osteoprotegerin (OPG), were increased in antibiotic-treated mice compared to controls. Antibiotic-treated mice also displayed a trend towards delayed bone healing as revealed by reduced mineral apposition at the defect site and higher circulating levels of the bone turnover marker PINP. Butyrate supplementation resulted in a lower abundance of monocyte/macrophages in the bone marrow, as well as reduced circulating proinflammatory IL-6 levels compared to antibiotic- and control-treated mice. In conclusion, this study supports our hypothesis that SCFAs, in particular butyrate, are important contributors to successful bone healing by modulating key cells involved in fracture healing as well as systemic inflammation and immune responses.

Biomechanical and wearability testing of novel legwear for variably limiting extension of the metacarpophalangeal joint of horses.

OBJECTIVE: To evaluate the ability of novel legwear designed to limit extension of the metacarpophalangeal joint (MCPJ) to redirect loading forces from the flexor apparatus during walk, trot, and canter on a treadmill and during unrestrained and restrained activity in a stall. ANIMALS: 6 adult horses without musculoskeletal disease. PROCEDURES: Legwear-derived force data were recorded under 4 conditions: inactive state (unlimited legwear extension) and 3 active (restrictive) states (mild, 30° extension; moderate, 20° extension; or maximum, 10° extension). Associations between peak legwear loads and torques among legwear states and treadmill gaits and stall activities were assessed. The hair coat and skin of the forelimbs were examined for any legwear-induced adverse effects after testing. RESULTS: During the treadmill exercises, moderate restriction of legwear extension resulted in significantly higher peak load and torque than mild restriction, and faster speeds (canter vs walk or trot and trot vs walk) yielded significantly higher peak load and torque. During in-stall activity, maximum restriction of legwear extension yielded significantly higher peak load and torque than moderate restriction. Unrestrained in-stall activity resulted in significantly higher peak load and torque than restrained activity. The legwear caused minimal adverse effects on the hair coat and skin of the forelimbs. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that the legwear variably reduced peak loads on the flexor apparatus. Extension of the MCPJ may be incrementally adjusted through the legwear such that return to activity may be controlled, and controlled return to activity is crucial for rehabilitating flexor apparatus injuries.

Kinematic and kinetic analyses of the gait of horses wearing novel legwear for variably limiting extension of the metacarpophalangeal joint.

OBJECTIVE: To investigate the effects of novel legwear designed to limit metacarpophalangeal joint (MCPJ) extension and redirect loading forces from the flexor apparatus through analyses of 2-D kinematic and kinetic data. ANIMALS: 6 adult horses without musculoskeletal disease. PROCEDURES: Horses were subjected to 4 treatments: control (no legwear), inactive legwear (unlimited legwear extension), and active legwear with mild (30°) and moderate (20°) legwear extension limitation. Two-dimensional kinematic data were collected for the right forelimb (FL) during walk and trot and from leading and trailing FLs during canter on a treadmill. Ground reaction force (GRF) data were collected from FLs during overground walk and trot. Peak MCPJ angle and angular velocity were calculated from kinematic data, and peak force and average loading rate were calculated from vertical GRF data during the stance phase of the gait. Interactions between gait and treatment were determined via ANOVA. RESULTS: Interactions between gait and treatment for peak MCPJ angle were significant. Significant reductions in MCPJ angle were noted between the control treatment and legwear with moderate extension limitation for trot and canter (leading and trailing FL) and between inactive legwear and legwear with moderate extension limitation for trot and leading FL during canter. Interactions among peak MCPJ angular velocity, peak vertical GRF, and average loading rate of the vertical GRF showed nonsignificance. CONCLUSIONS AND CLINICAL RELEVANCE: Significant reductions in MCPJ extension without significant alterations to peak vertical GRF suggested the legwear's ability to redistribute internal forces. Findings suggested that the legwear may be beneficial for horses rehabilitating from flexor apparatus injuries.

Effect of Fatigue on Equine Metacarpophalangeal Joint Kinematics-A Single Horse Pilot Study.

The objective was to validate a scientific method for characterizing equine metacarpophalangeal joint (MCPJ) motion in the nonfatigued and fatigued states using a single horse at trot, slow canter, and fast canter. One healthy Thoroughbred gelding exercised on a treadmill to exhaustion (fatigued state) (heart rate >190 BPM and blood lactate >10 mmol/L) while bilateral MCPJ angular data were acquired using electrogoniometry. Blood lactate and heart rate reflected transition from nonfatigued to fatigued states with increasing exercise duration and treadmill speed. Electrogoniometry consistently demonstrated: increase in mean MCPJ maximum extension angle with onset of fatigue; altered extension and flexion angular velocities with onset of fatigue; and increasing stride duration and decreasing stride frequency with onset of fatigue. The method allowed a preliminary but comprehensive characterization of the dynamic relationship between MCPJ kinematics and fatigue, prompting the need for multisubject studies that may enhance our ability to moderate exercise-related distal limb injury in equine athletes.

Three-dimensional kinematics of the equine metacarpophalangeal joint using x-ray reconstruction of moving morphology - a pilot study.

X-ray reconstruction of moving morphology (XROMM) uses biplanar videoradiography and computed tomography (CT) scanning to capture three-dimensional (3D) bone motion. In XROMM, morphologically accurate 3D bone models derived from CT are animated with motion from videoradiography, yielding a highly accurate and precise reconstruction of skeletal kinematics. We employ this motion analysis technique to characterize metacarpophalangeal joint (MCPJ) motion in the absence and presence of protective legwear in a healthy pony. Our in vivo marker tracking precision was 0.09 mm for walk and trot, and 0.10 mm during jump down exercises. We report MCPJ maximum extension (walk: -27.70 ± 2.78° [standard deviation]; trot: -33.84 ± 4.94°), abduction/adduction (walk: 0.04 ± 0.24°; trot: -0.23 ± 0.35°) and external/internal rotations (walk: 0.30 ± 0.32°; trot: -0.49 ± 1.05°) indicating that the MCPJ in this pony is a stable hinge joint with negligible extra-sagittal rotations. No substantial change in MCPJ maximum extension angles or vertical ground reaction forces (GRFv) were observed upon application of legwear during jump down exercise. Neoprene boot application yielded -65.20 ± 2.06° extension (GRFv = 11.97 ± 0.67 N/kg) and fleece polo wrap application yielded -64.23 ± 1.68° extension (GRFv = 11.36 ± 1.66 N/kg), when compared to naked control (-66.11 ± 0.96°; GRFv = 12.02 ± 0.53 N/kg). Collectively, this proof of concept study illustrates the benefits and practical limitations of using XROMM to document equine MCPJ kinematics in the presence and absence of legwear.

Association between inflammatory airway disease of horses and exposure to respiratory viruses: a case control study.

BACKGROUND: Inflammatory airway disease (IAD) in horses, similar to asthma in humans, is a common cause of chronic poor respiratory health and exercise intolerance due to airway inflammation and exaggerated airway constrictive responses. Human rhinovirus is an important trigger for the development of asthma; a similar role for viral respiratory disease in equine IAD has not been established yet. METHODS: In a case-control study, horses with IAD (n = 24) were compared to control animals from comparable stabling environments (n = 14). Horses were classified using pulmonary function testing and bronchoalveolar lavage. PCR for equine rhinitis virus A and B (ERAV, ERBV), influenza virus (EIV), and herpesviruses 2, 4, and 5 (EHV-2, EHV-4, EHV-5) was performed on nasal swab, buffy coat from whole blood, and cells from BAL fluid (BALF), and serology were performed. Categorical variables were compared between IAD and control using Fisher's exact test; continuous variables were compared with an independent t-test. For all analyses, a value of P <0.05 was considered significant. RESULTS: There was a significant association between diagnosis of IAD and history of cough (P = 0.001) and exercise intolerance (P = 0.003) but not between nasal discharge and IAD. Horses with IAD were significantly more likely to have a positive titer to ERAV (68 %) vs. control horses (32 %). Horses with IAD had higher log-transformed titers to ERAV than did controls (2.28 ± 0.18 v.1.50 ± 0.25, P = 0.038). There was a significant association between nasal shedding (positive PCR) of EHV-2 and diagnosis of IAD (P = 0.002). CONCLUSIONS: IAD remains a persistent problem in the equine population and has strong similarities to the human disease, asthma, for which viral infection is an important trigger. The association between viral respiratory infection and development or exacerbation of IAD in this study suggests that viral infection may contribute to IAD susceptibility; there is, therefore, merit in further investigation into the relationship between respiratory virus exposure and development of IAD.