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.

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.