VTechWorks Repository :: Browsing by Author "Byron, Christopher R."

Browsing by Author "Byron, Christopher R."

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Bone Marrow Mononuclear Cell for Equine Joint Disease
Everett, James Blake (Virginia Tech, 2020-09-04)
Osteoarthritis (OA) can be debilitating and career-ending for horses. Current treatments offer temporary and symptomatic relief, but potentially deleterious side effects. Bone marrow mononuclear cells (BMNC) are a rich source of macrophage progenitors that are anti-inflammatory and promote inflammation resolution. The objective of this study was to evaluate the ability of intra-articular BMNC therapy to improve clinical signs of naturally occurring equine OA. Horses presenting with clinical and radiographic evidence of moderate OA in a single joint were randomly assigned to 1 of 3 treatments: saline (negative control), triamcinolone (positive control), or BMNC (treatment group). Horses were subjectively and objectively evaluated for lameness and synovial fluid collected (cytology and cytokine/growth factor quantification) at 0, 7, and 21 days post-injection. Data were analyzed using General Estimating Equations with significance set at P<0.05. There were no adverse effects noted in any treatment group. No significant differences in synovial fluid cytology parameters, objective/subjective lameness scores, nor joint circumference were found between treatment groups at any time point. Within treatment groups, joint circumference did not change over time for saline- and triamcinolone-treated horses. However, joint circumference and objective lameness decreased significantly within BMNC-treated horses between Days 0 and 21 and Days 7 and 21. Lameness improved in saline-treated horses from 0 to 21 days, but did not improve in triamcinolone-treated horses. The decreased lameness and lack of adverse effects in the BMNC-treated horses in our study support a larger clinical trial using BMNC.
The Chondroid Conundrum: Transpharyngeal Removal of Guttural Pouch Chondroids in Horses
Cardona, Guillermo Andres (Virginia Tech, 2023-08-18)
The Clinical Pharmacology of Acetaminophen in Adult Horses
Mercer, Melissa Ann (Virginia Tech, 2022-08-18)
Non-steroidal anti-inflammatory drugs (NSAIDs) are a mainstay of the management of pain and inflammation associated with musculoskeletal disorders and systemic inflammation in horses. The most utilized NSAIDs in equine practice are non-selective cyclooxygenase (COX) inhibitors, such as flunixin meglumine and phenylbutazone, which act through global inhibition of prostaglandin synthesis and release. While non-selective COX inhibitors are effective as anti-inflammatory agents, they are mired with complications with prolonged or high-dose use, particularly in critically ill patients. Therefore, non-selective COX-inhibitors have been displaced by selective COX-2 inhibitors for many practitioners due to the perceived reduced risk of gastrointestinal complications. It should be noted, however, that the use of COX-2 selective inhibitors in horses is not without risk. Due to the potential for significant adverse events in horses with critical illness treated with traditional NSAIDs, there is clinical need for safe, and effective anti-inflammatories and anti-pyretics for administration in these patients. The studies presented in this dissertation explore the pharmacokinetics, efficacy, and safety of acetaminophen in adult horses for use in musculoskeletal pain and pyrexia. In the first study, the pharmacokinetics and efficacy of oral acetaminophen at two different doses (20 mg/kg and 30 mg/kg) were examined in an experimentally induced lameness model and the analgesic efficacy of acetaminophen was compared to placebo and the non-selective COX inhibitor phenylbutazone. Acetaminophen when administered at 30 mg/kg produced a more rapid onset of greater improvement in subjective lameness scores and heart rate compared to other treatments in this model, and therefore would be more suitable as a monotherapy than acetaminophen dosed at 20 mg/kg. Acetaminophen dosed at 30 mg/kg resulted in a more rapid improvement in lameness scores than phenylbutazone at 2.2 mg/kg and was equivalent to phenylbutazone in lameness score reduction. However, results of this study necessitated further evaluation of the pharmacokinetics and safety of repeated oral dosing of acetaminophen at 30 mg/kg orally every 12 hours to determine clinical utility. In the second study, the pharmacokinetics, efficacy, and safety of oral acetaminophen (30 mg/kg) were examined in adult horses with naturally occurring chronic lameness. In that study, following 21 days of twice daily oral dosing at 30 mg/kg, acetaminophen was found to be safe with no evidence of gastric ulceration or hepatopathy in horses. Acetaminophen at 30 mg/kg twice daily for 21 days provided transient improvement in subjective and objective lameness evaluation when compared to baseline evaluation; however, the study concluded that acetaminophen may not be suitable as a monotherapy for management of moderate to severe orthopedic pain in horses In the third study, the pharmacokinetics and efficacy of oral acetaminophen (30 mg/kg) was examined in adult horses with experimentally induced endotoxemia when compared to placebo and the nonselective COX inhibitor flunixin meglumine. That study found that acetaminophen was superior to placebo and not statistically different from flunixin meglumine in reducing rectal temperature in adult horses with experimentally induced endotoxemia and may be an option for antipyresis in clinical cases, particularly when administration of traditional NSAIDs is contraindicated. Furthermore, acetaminophen administered at 30 mg/kg orally to adult horses with experimentally induced endotoxemia is an effective antipyretic but is unlikely to provide any alteration in systemic inflammatory response.
Comparison of equine tendon-, muscle-, and bone marrow–derived cells cultured on tendon matrix
Stewart, Allison A.; Barrett, Jennifer G.; Byron, Christopher R.; Yates, Angela C.; Durgam, Sushmitha S.; Evans, Richard B.; Stewart, Matthew C. (AVMA, 2009-06)
Objective—To compare viability and biosynthetic capacities of cells isolated from equine tendon, muscle, and bone marrow grown on autogenous tendon matrix.
Comparison of the Effects of Interleukin-1 on Equine Articular Cartilage Explants and Cocultures of Osteochondral and Synovial Explants
Byron, Christopher R.; Trahan, Richard A. (Frontiers, 2017-09-20)
Osteoarthritis (OA) is a ubiquitous disease affecting many horses. The disease causes chronic pain and decreased performance for patients and great cost to owners for diagnosis and treatment. The most common treatments include systemic non-steroidal anti-inflammatory drugs and intra-articular injection of corticosteroids. There is excellent support for the palliative pain relief these treatments provide; however, they do not arrest progression and may in some instances hasten advancement of disease. Orthobiologic treatments have been investigated as potential OA treatments that may not only ameliorate pain but also prevent or reverse pathologic articular tissue changes. Clinical protocols for intra-articular use of such treatments have not been optimized; the high cost of in vivo research and concerns over humane use of research animals may be preventing discovery. The objective of this study was to evaluate a novel in vitro articular coculture system for future use in OA treatment research. Concentrations and fold increases in various markers of inflammation (prostaglandin E2 and tumor necrosis factor-alpha), degradative enzyme activity [matrix metalloproteinase-13 (MMP-13)], cartilage and bone metabolism (bone alkaline phosphatase and dimethyl-methylene blue), and cell death (lactate dehydrogenase) were compared between IL-1-stimulated equine articular cartilage explant cultures and cocultures comprised of osteochondral and synovial explants (OCS). Results suggested that there are differences in responses of culture systems to inflammatory stimulation. In particular, the IL-1-induced fold changes in MMP-13 concentration were significantly different between OCS and cartilage explant culture systems after 96 h. These differences may be relevant to responses of joints to inflammation in vivo and could be important to the biological relevance of in vitro research findings.
The effects of bit chewing on gastric emptying and orocecal transit times in clinically normal horses
Patton, Molly Elizabeth (Virginia Tech, 2023-02-14)
Small intestinal ileus affects up to half of all horses undergoing small intestinal surgery, leading to prolonged gastrointestinal (GI) transit time which can be life-threatening. Various prokinetic medications have been associated with varying side effects, questionable efficacy, and increased cost. Gum chewing as a form of sham feeding is used as a safe, effective, well-tolerated, and inexpensive way to ameliorate ileus following GI surgery in humans. Bit chewing for horses, an analogous activity, has been shown to significantly decrease GI total transit time (TTT); however, a direct effect of bit chewing on gastric emptying time (GET), small intestinal transit time (SITT), and total orocecal transit time (OCTT) has not been investigated. Our objective was to determine whether bit chewing increased small intestinal motility and decreased GE, SITT, and OCTT in clinically normal horses. Gastrointestinal motility was compared in horses that were bit chewing compared to control conditions (no bit chewing) in a prospective crossover design study using acetaminophen as a marker for GET and video endoscopy (ALICAM) capsules to determine GET, SITT, and OCTT. Bit chewing was well tolerated by all horses with no side effects noted. Bit chewing led to a shorter GET, SITT, and significantly shorter OCTT when compared to the control group (P = 0.015). Median times for bit chewing conditions were as follows: GE 2.86 hr, SITT 3.65 hr, and OCTT 6.15 hr whereas the median times for control conditions were as follows: GE: 5 hr, SITT 4.4 hr, and OCTT 9.92 hr. In summary, bit chewing proves to be a potential tool to hasten the motility of the oral GIT. It is safe, inexpensive, and potentially effective prokinetic treatment to horses suffering from postoperative ileus and further investigation is warranted.
Effects of Bit Chewing on Gastric Emptying, Small Intestinal Transit, and Orocecal Transit Times in Clinically Normal Horses
Patton, Molly E.; Andrews, Frank M.; Bogers, Sophie Helen; Wong, David; McKenzie, Harold C.; Werre, Stephen R.; Byron, Christopher R. (MDPI, 2023-08-04)
Ileus is a common life-threatening problem in horses, and currently available treatments may be ineffective. The purpose of this study was to determine whether bit chewing, a form of sham feeding, decreases the gastric emptying time (GET), small intestinal transit time (SITT), and total orocecal transit time (OCTT) in clinically normal horses in a prospective crossover study. Nine healthy horses were acclimated and fed a standardized diet. Following 24 h of fasting, self-contained video endoscopy capsules and acetaminophen were administered into the stomach via a nasogastric tube. Each horse underwent experimental (bit chewing for 20 min every 6 h) or control (no bit chewing) conditions, with a 3-week minimum washout period between conditions. The horses were enrolled in either part of the study until all video capsules were retrieved and/or 30 days lapsed. The video capsules were recovered from manure, and GET, SITT, and OCTT were determined from a video analysis. Bit chewing significantly decreased OCTT (p = 0.015) compared to the control conditions. Bit chewing decreased GET and SITT, but the differences were not significant. The mean (median) times determined via the video capsule analysis for the bit-chewing conditions were as follows: GET, 2.34 h (2.86 h); SITT, 3.22 h (3.65 h); and OCTT, 5.13 h (6.15 h), and for the control conditions, they were as follows: GET, 3.93 h (5 h); SITT, 3.79 h (4.4 h); and OCTT, 8.02 h (9.92 h). Bit chewing decreased OCTT in healthy horses. Because this segment of the gastrointestinal tract is frequently affected by ileus, bit chewing may be a safe and inexpensive intervention for that condition in horses. Further investigation in clinical patients with ileus is warranted.
Effects of Three Corticosteroids on Equine Articular Cocultures In Vitro
Trahan, Richard Angellas (Virginia Tech, 2018-06-08)
The objective was to compare the effects of three corticosteroids at various equimolar concentrations on equine articular explant co-cultures in an inflammatory environment. Synovial and osteochondral explant co-cultures from 6 equine cadavers were exposed to IL-1β (10 ng/mL) and various concentrations (10-4, 10-7, or 10-10 M) of MPA, TA, IPA. Concentrations of PGE2, MMP-13, LDH, and GAG in media were determined at 48 and 96 hours. Results indicated wells with low concentrations of MPA (10-7 and 10-10 M at 48 and 96 hours), TA (10-7 M at 48 hours and 10-7 and 10-10 M at 48 and 96 hours), and IPA (10-10 M at 48 hours) had significantly less PGE2 than positive control samples. Groups with low concentrations (10-7 and 10-10 M) of MPA and TA had significantly less PGE2 than the highest concentration (10-4 M) at 48 hours. Significantly less MMP-13 was detected for all concentrations of MPA, TA, and IPA at 96 hours. The LDH assay results indicated cytotoxicity only for samples treated with IPA at 10-4 M at 48 and 96 hours. GAG was significantly lower for samples treated with TA 10-7 M at 48 hours and MPA 10-10 M at 96 hours versus positive controls. These findings suggest corticosteroids at low concentrations mitigated the inflammatory and catabolic effects of IL-1β to a greater extent than high concentrations. Effects of IPA and MPA were similar to TA at clinically relevant low equimolar concentrations.
Equine Septic Arthritis and Serum Amyloid A
Ludwig, Elsa Karen (Virginia Tech, 2016-07-07)
Bacterial infection within a joint, septic arthritis, is a serious condition in horses that can lead to long-term joint disease if the infection is not resolved quickly. Equine septic arthritis is diagnosed primarily based on clinical signs and synovial fluid cytology. Septic synovial fluid is characterized by significant elevations in total protein (TP) and total nucleated cell count (TNCC). However, in some cases it can be difficult to distinguish between septic arthritis and non-septic joint inflammation (synovitis) based on clinical signs and synovial fluid cytology alone. A rapid assay to help confirm septic arthritis would be advantageous. A new assay to quantify the major equine acute phase protein, serum amyloid A (SAA) may fulfill this need. Serum amyloid A increases in the body in response to injury, infection, and inflammation and shows promise as a useful tool in confirming a diagnosis of sepsis, as inflammation causes mild increases in SAA and infection causes marked elevations. In our study, serial serum and synovial fluid samples were collected from horses with experimental models of synovitis and septic arthritis, synovial fluid cytology was performed, and serum and synovial fluid SAA were quantified. Synovial fluid TNCC and TP concentrations increased significantly following induction of both models. Serum and synovial fluid SAA concentrations remained normal in synovitis horses and increased significantly in septic arthritis horses. Any elevation in serum or synovial fluid SAA above normal values may be supportive of synovial sepsis since synovial inflammation alone did not result in SAA elevations in our model.
Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review
Hendricks-Wenger, Alissa; Arnold, Lauren; Gannon, Jessica; Simon, Alex; Singh, Neha; Sheppard, Hannah; Nagai-Singer, Margaret A.; Imran, Khan Mohammed; Lee, Kiho; Clark-Deener, Sherrie; Byron, Christopher R.; Edwards, Michael R.; Larson, Martha M.; Rossmeisl, John H. Jr.; Coutermarsh-Ott, Sheryl; Eden, Kristin; Dervisis, Nikolaos G.; Klahn, Shawna L.; Tuohy, Joanne L.; Allen, Irving C.; Vlaisavljevich, Eli (IEEE, 2021-09-03)
New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.
Macrophage-mediated regulation of joint homeostasis
Menarim, Bruno C. (Virginia Tech, 2019-11-06)
Osteoarthritis (OA) is the leading cause of musculoskeletal disability in people and horses, and is characterized by progressive joint degeneration. There is a critical need for a better understanding of disease processes leading to OA in order to develop more efficient therapies. A shared feature among different arthritic conditions is chronic synovitis. Macrophages are the main drivers of synovitis and can display pro-inflammatory (M1) or pro-resolving responses (M2). Macrophages promote joint health through phagocytic and secretory activities; however, when these functions are overwhelmed, macrophages upregulate inflammation, recruiting more cells to counteract damage. Once cell recruitment is efficiently accomplished, macrophages coordinate tissue repair and further resolution of inflammation. Bone marrow mononuclear cells (BMNC) are a source of macrophages used to treat inflammation and produce essential molecules for cartilage metabolism; however, little information exists regarding their use in joints. The studies presented in this dissertation focus on understanding the dual role of macrophages in driving and resolving synovitis and how to harness their therapeutic potential. In the first study, patterns of macrophage phenotypes (M1:M2) in healthy and osteoarthritic equine synovium were compared and correlated with gross pathology, histology, and synovial fluid cytokines. M1 and M2 markers were co-expressed in normal and osteoarthritic joints, varying in intensity of expression according to degree of inflammation. Concentrations of synovial fluid IL-10, a macrophage-produced cytokine that is vital for chondrocyte recovery from injury, was lower in OA joints. The combined findings of this study suggest homeostatic mechanisms from synovial macrophages in OA may be overwhelmed, preventing inflammation resolution. In the second study we investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). BMNC cultured in autologous SF or ISF developed into macrophage cultures that were more confluent in ISF (~100%) than SF (~25%), and exhibited phenotypes that were ultimately similar to cells native to normal joints. BMNC cultured in SF or ISF were neither M1 nor M2, but exhibited aspects of both phenotypes and a regulatory response, characterized by increasing counts of IL-10+ macrophages, decreasing concentrations of IL-1β, and progressively increasing concentrations of IL-10 and IGF-1, all more marked in ISF. These findings suggest that homeostatic mechanisms were preserved over time, and potentially favored by macrophage proliferation. Our data suggest that BMNC therapy could potentiate the macrophage- and IL-10-associated mechanisms of joint homeostasis lost in OA. Finally, using an equine model of synovitis, the last study investigated the response of normal and inflamed joints to autologous BMNC injection. Inflamed joints treated with BMNC showed gross and analytical improvements in synovial fluid and synovial membrane, with increasing numbers of regulatory macrophages and synovial fluid concentrations of IL-10, not observed in saline-treated controls. Autologous BMNC are readily available, downregulate synovitis through macrophage-associated effects, and can benefit thousands of patients with OA. Combined, the results of these studies support the role of macrophage-driven synovial homeostasis and identified a therapeutic way to recover homeostatic mechanisms of synovial macrophages lost during chronic inflammation. Our findings also uncover new research directions and methods for future studies targeting modulation of joint inflammation.
Pharmacokinetics and pulmonary distribution of Draxxin® (tulathromycin) in healthy adult horses
Leventhal, Hannah Rani (Virginia Tech, 2021-10-13)
The objective of this study was to determine the pharmacokinetics and tolerance of tulathromycin (Draxxin®; 2.5 mg/kg once) after intramuscular (IM), subcutaneous (SC), and slow intravenous (IV) administration to six adult horses. A three-phase design and 4-week washout period were used. Drug concentrations in blood and bronchoalveolar lavage (BAL) samples were determined by ultra-performance liquid chromatography tandem mass spectrometry and pharmacokinetic parameters calculated using noncompartmental analysis. Following SC and IM administration, all horses exhibited sweating, discomfort, and periods of recumbency. As signs were more severe after SC administration this route was only used in 3/6 horses. Intravenous administration of tulathromycin was well tolerated in all horses. Mean bioavailability was 99.4% IM and 115% SC. Mean maximum plasma concentration was 645 ng/ml IM and 373 ng/ ml SC. Mean half-life was 59.8 h, 54.8 h, and 57.9 h for IV, IM, and SC administration, respectively. Mean clearance was 3.25 ml/kg/min, and mean volume of distribution was 16.8 L/kg following IV administration. Drug was detectable in plasma and BAL samples for 120 h following all routes; however, adverse effects may prevent IM use and SC use is not recommended. Tulathromycin may be a practical and affordable antibacterial for use in adult equine patients.
Porcine urinary bladder matrix in an in vitro equine model of tenogenesis
Khatibzadeh, Sarah M. (Virginia Tech, 2019-08-22)
Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone.
Strategies for Overcoming Shortcomings of Thermal Ablations: A Comprehensive Study of Nanoparticle Transport During Photothermal Chemotherapy Treatments, and High Frequency Irreversible Electroporation
Dewitt, Matthew Ryan (Virginia Tech, 2017-11-09)
Cancer continues to be a leading cause of death worldwide despite the increasing research advances into novel treatments. Thermal ablation of tumors is a relatively established method for the destruction of many tumor types, despite inherent shortcomings including incomplete tumor treatment and non-specific treatment. Novel therapies are currently studied including nanoparticle-based therapies to overcome these limitations. One field of research is focused on utilizing non-lethal hyperthermia to enhance carried chemotherapeutic drugs. Additionally a novel field of non-thermal ablations termed Irreversible Electroporation has recently been developed to treat tumors by irreversibly destroying cell membrane function through short electrical pulses. The goal of the present study is to research two novel potential treatments for cancer that do not require thermal destruction of tissue. Firstly, we developed and tested novel ways to load the antineoplastic agent Cisplatin into SWNHs to test the ability to thermally enhance carried drugs with non-lethal, mild hyperthermia. We attached the imaging agent Quantum Dots (QDs) to the particles to understand how hyperthermia affects cellular uptake, minimizing thermal enhancement. Results of this study highlight the need for better biomimetic in vitro models of the tumors to study how hyperthermia affects tissue level transport of nanoparticles. In the second aim we utilized a perfusable 3D collagen in vitro model of the tumor microenvironment, previously developed by our group to study tumor angiogenesis, to study nanoparticle transport. We demonstrated the ability of this model to study key mass transport obstacles nanoparticles face in the tumor including extravasation from a leaky, pro-angiogenic vasculature, diffusion in the extracellular matrix, and cellular uptake in a 3D environment. This model was then utilized in the third aim to study how mild hyperthermia affects transport of SWNHs. Results from this aim were valuable in showing the utility of the 3D in vitro model to controllably test the effects of external stimuli on transport of particles and shows how mild hyperthermia can selectively allow increased permeability of SWNHs in the tumor, increasing selectivity of nanoparticle transport to the targeted tissue. Lastly, we tested the non-thermal ablation, high-frequency irreversible electroporation (H-FIRE) in a 3D tumor platform and in an in vivo swine model to better understand the ability of H-FIRE to produce repeatable destruction of hepatocellular carcinoma, a disease state growing in incidence rate. We then used H-FIRE in an outpatient treatment for infiltrative skin tumors in equines, showcasing the ability to deliver high voltage, short duration pulses in a clinical setting without muscle contractions. Ultimately, the results of this study the engineering studies that must occur to optimize novel treatments utilizing non-lethal hyperthermia, or non-thermal death mechanism to treat cancer. The studies show the usefulness of more complex 3D in vitro models of tumors for early development of novel therapies and the utility of in vivo models to validate studies.
Tendon Regeneration: Roles of Growth Factors and Phenotypic Diversity in Tendon Stem Cells
Rajpar, Ibtesam Mohamed Husein (Virginia Tech, 2019-03-04)
Tendon injuries significantly impact quality of life and are often career ending. Mesenchymal stem cell (MSC) therapy is known to augment intrinsic tendon healing, however, little is known of the stem cells endogenous to tendon, the microenvironmental cues that induce tendon differentiation, and whether individual cells in an inflammatory milieu respond differently to these cues. To address these questions, a three-dimensional tenogenesis assay was developed as an efficient and reproducible metric of cellular capacity to differentiate toward tendon. In contrast to more complex assays of tenogenesis, this design incorporates a simple apparatus using commercially available plasticware for the application of uniaxial static strain in in a type I collagen cell-seeded hydrogel construct. Tendon-related gene expression, glycosaminoglycan levels, elongated cell morphologies and parallel cell alignments are enhanced with BMP-12 induction over ten days of culture. This dissertation provides novel insight to the roles of growth factors in MSC tenogenesis. Tendon healing in vivo is dependent on endogenous tendon stem cells (TSC) that mediate the inflammatory response to injury and promote synthesis of collagen and matrix remodeling, among other extracellular processes. Recent evidence suggests that these cells exist on a spectrum of differentiation potencies, and may be differently committed to the tendon fate. Individual stem cells were isolated from the tendon, and their capacities for proliferation, tri-lineage differentiation and tenogenesis were evaluated. Three distinct TSC phenotypes were revealed, and significant, positive correlations were found in quadra-differentiation potency (toward four lineages) and the expression of a strong, composite tendon phenotype. These studies have important implications in the current standard-of-care in regenerative therapies for tendon. Our benchtop tenogenesis assay can be used to determine the therapeutic potential of allogeneic MSC lines and MSCs from novel sources for 'off-the-shelf' treatments. Our study of TSCs lends valuable insight to the diversity of cell phenotypes found in tendon, and the potential contributions of each phenotype to tendon healing and homeostasis. These results further strengthen the status of tendon as a superior source of stem cells for tendon repair.
Treatment of Infiltrative Superficial Tumors in Awake Standing Horses Using Novel High-Frequency Pulsed Electrical Fields
Byron, Christopher R.; DeWitt, Matthew R.; Latouche, Eduardo L.; Davalos, Rafael V.; Robertson, John L. (Frontiers, 2019-08-14)
Irreversible electroporation is a proven ablation modality for local ablation of soft tissue tumors in animals and humans. However, the strongmuscle contractions associated with the electrical impulses (duration, 50–100 μs) requires the use of general anesthesia and, in most situations, application of neuromuscular blockade. As such, this technology is not used in an outpatient setting for ablating common cutaneous tumors (e.g., squamous cell carcinoma or melanoma) in humans or animals. Recently, high-frequency irreversible electroporation (H-FIRE) technology has been developed to enable electroporation of tumors without stimulation of nearby skeletal muscle. H-FIRE administers bursts of electrical pulses (duration, 0.5–2 μs) through bipolar electrodes placed in tumor parenchyma. We hypothesized that H-FIRE could be used to safely ablate superficial tumors in standing, awake horses without the need for general anesthesia. Here, we describe the treatment of superficial tumors in five horses using this novel ablation therapy without the need for general anesthesia. In each case, H-FIRE therapy predictably ablated tumor volume. All patients tolerated the procedure, no complications developed, and veterinary personnel safety was maintained. The H-FIRE treatment may be useful for treatment in veterinary and human patients in an outpatient setting without the need for hospitalization, general anesthesia, and advanced monitoring techniques.
Turning Round: Optimizing the Anti-Inflammatory Properties of Equine Bone Marrow Derived Mesenchymal Stem Cells for Osteoarthritis Through Three-Dimensional Culture
Bogers, Sophie Helen (Virginia Tech, 2017-04-19)
Osteoarthritis (OA) is a degenerative disease of diarthrodial joints causing pain and loss of joint function. Etiology is heterogeneous, but commonly involves inflammation arising from impairment of normal tissue homeostasis and/or function. A cycle of low-grade inflammation and global tissue degradation causes alteration of tissue morphology and function via primary mechanisms or inability to withstand physiological forces. Current therapies variably ameliorate symptoms but do not modify progression. Mesenchymal stem cells (MSCs) have multi-modal properties but are ineffective in ameliorating equine OA. However, anti-inflammatory activities of bone marrow derived MSCs (BMSCs) are enhanced by three-dimensional spheroid culture so equine BMSC (eBMSC) spheroids could inhibit intra-articular inflammation. The overarching hypothesis is that eBMSCs can be enhanced to produce an allogeneic eBMSC therapy that inhibits intra-articular inflammation. In vitro experiments compared differences in anti-inflammatory phenotype between spheroid and traditionally cultured monolayer eBMSCs, the viability and health of eBMSC spheroids administered through needles, and the effects of allogeneic donor on the anti-inflammatory potential of eBMSC spheroids. A model of equine LPS induced synovitis was used to investigate anti-inflammatory efficacy of spheroid eBMSCs compared to placebo or monolayer eBMSCs in vivo. eBMSCs aggregate into spheroids that have stable stem cell marker expression with increased secretion and gene expression of IL-6 and PGE2, and gene expression of SDF-1 and TSG-6. IFN𝛾 and TNFα were not produced by eBMSC spheroids and IL-10 production varied between individuals. Spheroids maintain higher viability and lower senescence than monolayer eBMSCs after injection through a needle and form in high-throughput culture without detrimental effects on expression of TSG-6, IL-6 and PGE synthases that denote an anti-inflammatory phenotype. Additionally, there is significant variation in this phenotype depending on the eBMSC donor. eBMSC spheroids reduced total nucleated cell counts and objective lameness measurements at peak levels of intra-articular inflammation compared to monolayer cultured eBMSCs in vivo. In summary, spheroids increase anti-inflammatory potential of eBMSCs and are practical for clinical use. Increased anti-inflammatory efficacy was demonstrated in a model of in vivo inflammation. This dissertation provides an understanding of the anti-inflammatory activities of eBMSC spheroids that can be used to develop an OA therapy.
The Use of Amnion in Equine Wound Healing
Moyer, Christine T. (Virginia Tech, 2018-06-25)
Objective: To assess the safety and efficacy of lyophilized milled human amnion as a wound dressing of experimentally created equine distal limb wounds. Animals: Four clinically normal adult horses (3 Thoroughbred and 1 Paint, median age 11 years) obtained via donation. Procedures: One forelimb of each horse was randomly assigned to the treatment group, and the contralateral limb was assigned as the control. Full-thickness skin wounds were created on each metacarpus. Treatment limb wounds were dressed with lyophilized, milled, human-derived amnion material delivered under triple antibiotic ointment. Control wounds were dressed with triple antibiotic ointment. All wounds were covered in non-adherent dressings and distal limb bandages were applied. Digital photographs were taken of the wounds at each bandage change, performed every 2-4 days throughout a 98-day study period. Biopsies were collected at days 7, 21, 35, and 84. Results: One horse developed unilateral cellulitis that resolved with additional treatment. All treatment limbs exhibited an inflammatory response characterized by focal edema and discharge from the wounds. Wounds were completely epithelialized in control limbs sooner than treatment limbs in all horses, although there was no statistical difference between control (mean 46.8 days) and treatment (mean 51.8 days) wounds. Histologic scores were better in control wounds than in amnion-treated wounds at all time points. Conclusions and Clinical Relevance: Because wounds treated with amnion material in this study exhibited an inappropriate inflammatory response that resulted in delayed time to wound closure, human lyophilized milled amnion is not recommended for use in equine wound management.

Browsing by Author "Byron, Christopher R."

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