Investigation of integrated time nanosecond pulse irreversible electroporation against spontaneous equine melanoma.

Introduction: Integrated time nanosecond pulse irreversible electroporation (INSPIRE) is a novel tumor ablation modality that employs high voltage, alternating polarity waveforms to induce cell death in a well-defined volume while sparing the underlying tissue. This study aimed to demonstrate the in vivo efficacy of INSPIRE against spontaneous melanoma in standing, awake horses. Methods: A custom applicator and a pulse generation system were utilized in a pilot study to treat horses presenting with spontaneous melanoma. INSPIRE treatments were administered to 32 tumors across 6 horses and an additional 13 tumors were followed to act as untreated controls. Tumors were tracked over a 43-85 day period following a single INSPIRE treatment. Pulse widths of 500ns and 2000ns with voltages between 1000 V and 2000 V were investigated to determine the effect of these variables on treatment outcomes. Results: Treatments administered at the lowest voltage (1000 V) reduced tumor volumes by 11 to 15%. Higher voltage (2000 V) treatments reduced tumor volumes by 84 to 88% and eliminated 33% and 80% of tumors when 500 ns and 2000 ns pulses were administered, respectively. Discussion: Promising results were achieved without the use of chemotherapeutics, the use of general anesthesia, or the need for surgical resection in regions which are challenging to keep sterile. This novel therapeutic approach has the potential to expand the role of pulsed electric fields in veterinary patients, especially when general anesthesia is contraindicated, and warrants future studies to demonstrate the efficacy of INSPIRE as a solid tumor treatment.

Determination of correlation of proximal sesamoid bone osteoarthritis with high-speed furlong exercise and catastrophic sesamoid bone fracture in Thoroughbred racehorses.

OBJECTIVE: To examine whether proximal sesamoid bone (PSB) articular cartilage and bone osteoarthritic changes or palmar osteochondral disease (POD) scores were associated with exercise history and catastrophic PSB fracture in Thoroughbred racehorses. SAMPLES: PSBs from 16 Thoroughbred racehorses (8 with and 8 without PSB fracture). PROCEDURES: Exercise history was collected, and total career high-speed furlongs was used as the measure of total exercise per horse. At necropsy, medial and lateral condyles of the third metacarpus from each forelimb were assigned a POD score, followed by imaging with micro-CT for evaluation of osteophyte size. Three investigators that were blinded to the type of PSB (fracture or no fracture) used the Osteoarthritis Research Society International (OARSI) scoring system to evaluate acellularity, chondrocyte necrosis, cartilage fibrillation, chondrone formation, safranin O stain uptake, and tidemark advancement of 1 central sagittal tissue section/PSB (4 PSBs/horse). Cartilage thickness and bone necrosis were scored on the basis of histologic examination. RESULTS: POD score, osteophyte size score, percentage of bone necrosis, tidemark advancement, chondrone formation, and total OARSI score were greater in horses with more accrued total career high-speed furlongs. Scores for POD, osteophyte size, fibrillation, acellularity, chondrone formation, and total OARSI were greater for horses with PSB fracture. CONCLUSIONS AND CLINICAL REVELANCE: OARSI scoring revealed that more advanced osteoarthritic changes strongly correlated with total career high-speed furlongs and PSB fracture. However, the effect of exercise was dominant, suggesting that exercise history will be important to include in future models that aim to assess risk factors for catastrophic PSB fracture.

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.

Temporal changes in synovial fluid composition and elastoviscous lubrication in the equine carpal fracture model.

The objective of this study was to examine temporal variations in synovial fluid composition and lubrication following articular fracture. Post-traumatic osteoarthritis (PTOA) was induced by creating an osteochondral fracture in the middle carpal joint of four horses while the contralateral limb served as a sham-operated control. Horses were exercised on a high-speed treadmill, and synovial fluid was collected pre-operatively and at serial timepoints until 75 days post-operatively. Lubricin and hyaluronic acid (HA) concentrations were measured using sandwich ELISAs, and the molecular weight distribution of HA was analyzed via gel electrophoresis. Synovial fluid viscosity and cartilage friction coefficients across all modes of lubrication were measured on days 0, 19, 33, and 61 using a commercial rheometer and a custom tribometer, respectively. HA concentrations were significantly decreased post-operatively, and high molecular weight HA (>6.1MDa) did not recover to pre-operative values by the study termination at day 75. Lubricin concentrations increased after surgery to a greater extent in the OA as compared to sham-operated limbs. Viscosity was significantly reduced after surgery. While boundary and elastoviscous mode friction coefficients did not vary, the transition number, representing the shift between these modes, was lower. Although more pronounced in the OA limbs, similar derangements in HA, HA molecular weight distribution, viscosity, and transition number were observed in the sham-operated limbs, which may be explained by synovial fluid washout during arthroscopy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor.

Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art.