Injury to the palmar supporting structures of the fetlock alters limb stiffness and fetlock angle.

BACKGROUND: In vivo measurement of limb stiffness and conformation provides a non-invasive proxy assessment of superficial digital flexor tendon (SDFT) and suspensory ligament (SL) function. Here, we compared it in fore and hindlimbs and after injury. OBJECTIVES: To compare the limb stiffness and conformation in forelimbs and hindlimbs, changes with age, and following injury to the SDFT and SL. STUDY DESIGN: Retrospective cohort study. METHODS: Limb stiffness was calculated using floor scales and an electrogoniometer taped to the dorsal fetlock. The fetlock angle and weight were simultaneously recorded five times with the limb weight-bearing and when the opposite limb was picked up (increased load). Limb stiffness of both limbs was calculated from the gradient of the regression line of angle versus load. Fetlock angle when the weight was zero was extrapolated from the graph and used as a measure of conformation. Limb stiffness was measured in uninjured forelimbs (n = 42 limbs), hindlimbs (n = 19 limbs), forelimbs with SDFT injury (n = 18) and hindlimbs with SL injury (n = 5). RESULTS: Limb stiffness correlated with weight in forelimbs as shown previously (p < 0.001) but also in hindlimbs (p = 0.006). When normalised to the horse's weight (503 kg, IQR 471.5-560), forelimb stiffness was significantly higher (22.3 [±4.5] × 10-3 degree-1) than for the hindlimb (16.4 [±4.0] × 10-3 degree-1; p < 0.001). While there were no significant differences between forelimb and hindlimb conformation in unaffected or SDFT injury, both limb stiffness and conformation was significantly greater in limbs with SL injury (p = 0.009 and p = 0.002, respectively). MAIN LIMITATIONS: Small sample size, lack of clinical data including lameness and quantification of injuries. CONCLUSIONS: Injury to the forelimb SDFT does not alter limb stiffness or conformation in the long-term, while hindlimb SL injury simultaneously increases limb stiffness and fetlock angle, suggesting an increase in SL length following injury.

A SIRT1-independent mechanism mediates protection against steroid-induced senescence by resveralogues in equine tenocytes.

Tendinopathy is a common age-related disease which causes significant morbidity for both human athletes and performance horses. In the latter, the superficial digital flexor tendon is an excellent model for human tendinopathies because it is a functional homologue of the human Achilles tendon and a primary site of injuries with strong similarities to the human disease. Corticosteroids have been previously used clinically to treat tendinopathic inflammation, but they upregulate the p53-p21 axis with concomitant reductions in cell proliferation and collagen synthesis in human tenocytes. This phenotype is consistent with the induction of cellular senescence in vitro and in vivo and probably represents an important clinical barrier to their effective use. Because of the many differences in senescence mechanisms between species, this study aimed to evaluate these mechanisms after corticosteroid treatment in equine tenocytes. Exposure to clinically reflective levels of dexamethasone for 48 hours drove equine tenocytes into steroid induced senescence (SIS). This was characterised by permanent growth arrest and upregulation of p53, the cyclin dependent kinase inhibitors p21waf and p16ink4a as well as the matrix degrading enzymes MMP1, MMP2 and MMP13. SIS also induced a distinctive equine senescence associated secretory phenotype (eSASP) characterised by enhanced secretion of IL-8 and MCP-1. Preincubation with resveratrol or the potent SIRT1 activator SRT1720 prevented SIS in equine tenocytes, while treatment with the non-SIRT1 activating resveratrol analogue V29 was equally protective against SIS, consistent with a novel, as yet uncharacterised SIRT1-indendent mechanism which has relevance for the development of future preventative and therapeutic strategies.

Autologous bone marrow derived mesenchymal stem cells are safe for the treatment of Achilles tendinopathy.

Achilles tendinopathy is a disabling condition that affects more than 50% of runners. Pre-clinical studies in a large animal model of naturally-occurring tendinopathy similar to human Achilles tendinopathy has shown benefits of autologous bone marrow-derived mesenchymal stem cell (MSC) implantation. However, MSCs are advanced therapies medicinal products (ATMPs), with strict regulatory requirements. Guided by the regulator we carried out a first in man study to assess the safety and efficacy of autologous MSC injection in human patients with non-insertional Achilles tendinopathy. Ten patients, mean age 47 with mid-portion Achilles tendon pain and swelling for more than 6 months, underwent autologous cultured cell injections (median 12.2 × 106, range 5-19 × 106 cells) into their Achilles tendon. At 24 weeks follow-up, no serious adverse reactions or important medical events were observed. MOXFQ, EQ-5D-5L, and VISA-A scores improved clinically at 12 and 24 weeks. VAS pain improved increasingly at 6, 12 and 24 weeks. MOXFQ Pain and VISA-A Scores improved > 12 points from baseline to 24 weeks in 8 patients. Maximum anteroposterior tendon thickness as measured by greyscale US decreased by mean 0.8 mm at 24 weeks. This phase IIa study demonstrated the safety of autologous MSC injection for non-insertional Achilles tendinopathy and provides proof-of-concept of the technique in patients, all of whom had previously failed conservative treatments for chronic disease and leads the way for a larger randomised controlled trial.

Effects of resveratrol and its analogues on the cell cycle of equine mesenchymal stem/stromal cells.

Resveratrol (RSV; trans-3,5,4'-trihydroxystilbene) strongly activates sirtuin 1, and it and its analogue V29 enhance the proliferation of mesenchymal stem/stromal cells (MSCs).Although culture medium containing 5-azacytydine and RSV inhibits senescence of adipose tissue-derived MSCs isolated from horses with metabolic syndrome, few studies have reported the effects of RSV on equine bone marrow-derived MSCs (eBMMSCs) isolated from horses without metabolic syndrome. The aim of this study was to investigate the effects of RSV and V29 on the cell cycle of eBMMSCs. Following treatment with 5 µM RSV or 10 µM V29, the cell proliferation capacity of eBMMSCs derived from seven horses was evaluated by EdU (5-ethynyl-2'-deoxyuridine) and Ki-67 antibody assays. Brightfield images of cells and immunofluorescent images of EdU, Ki-67, and DAPI staining were recorded by fluorescence microscopy, and the number of cells positive for each was quantified and compared by Friedman's test at P<0.05. The growth fraction of eBMMSCs was significantly increased by RSV and V29 as measured by the EdU assay (control 28.1% ± 13.8%, V29 31.8% ± 14.6%, RSV 32.0% ± 10.8%; mean ± SD; P<0.05) but not as measured by the Ki-67 antibody assay (control 27.0% ± 11.2%, V29 27.4% ± 10.8%, RSV 27.7% ± 6.8%). RSV and V29 promoted progression of the cell cycle of eBMMSCs into the S phase and may be useful for eBMMSC expansion.

Desmitis of the accessory ligament of the deep digital flexor tendon in the forelimb: A retrospective case study of 91 horses.

BACKGROUND: Desmitis of the accessory ligament of the deep digital flexor tendon (ALDDFT) is a commonly reported injury. Despite the commonality of this injury, the literature is limited to small case series, with the reported success following treatment varying from 18% to 75%. OBJECTIVES: To identify the prognosis and factors associated with a return to work following ALDDFT injury. STUDY DESIGN: Retrospective case series. METHODS: Medical records of horses from four equine hospitals (January 2000 and December 2018) with a diagnosis of desmitis of ALDDFT were reviewed. Data retrieved included case detail, use, history, lameness treatment and follow-up. Success was defined as returning to work. Backward stepwise logistic regression was used to identify variables significantly associated with return to work. RESULTS: Ninety-one horses were included. The mean age was 13.5 years (standard deviation 4.9 years). Thirty-four percent (28/91) of horses were sound at the initial presentation. Sixty-eight percent (62/91) of horses were managed using controlled exercise alone, 28% (29/91) were treated with intra-lesional injection, therapeutic ultrasound, extracorporeal shockwave therapy or desmectomy of the ALDDFT and 3% (3/91) were euthanased without treatment. Sixty-four percent (54/85) of horses returned to work. Horses that were lame at follow-up were less likely to return to work (odds ratio [OR] 107.93, 95% confidence interval [CI] 20.06-580.61, p < 0.001) than those that returned to soundness. Identification of adhesions on ultrasonography was also associated with having reduced odds for return to work when compared to horses without adhesions (OR 0.10, 95% CI 0.01-0.76, p = 0.03). MAIN LIMITATIONS: Retrospective nature of the study, the potential of selection bias with regards to follow-up. CONCLUSION: Sixty-four percent (54/85) of horses returned to work following injury of the ALDDFT. Persistence of lameness and adhesion formation were significantly associated with a poor outcome.

Transdermal drug delivery in horses: An in vitro comparison of skin structure and permeation of two model drugs at various anatomical sites.

BACKGROUND: Oral and parenteral drug delivery in horses can be difficult. Equine-specific transdermal drug formulations offer improved ease of treatment; development of such formulations requires a deeper understanding of the structural and chemical tissue barrier of horse skin. HYPOTHESIS/OBJECTIVES: To compare the structural composition and barrier properties of equine skin. ANIMALS: Six warmblood horses (two males, four females) with no skin diseases. MATERIALS AND METHODS: Routine histological and microscopic analyses were carried out with image analysis for skin from six different anatomical locations. In vitro drug permeation was analysed using a standard Franz diffusion cell protocol coupled with reversed phase-high-performance liquid chromatography detailing flux, lag times and tissue partitioning ratios of two model drug compounds. RESULTS: Epidermal and dermal thicknesses varied between sites. The dermal and epidermal thicknesses of the croup were 1764 ± 115 µm and 36 ± 3.6 µm, respectively, and were significantly different (p < 0.05) from the inner thigh thicknesses which were 824 ± 35 µm and 49 ± 3.6 µm. Follicular density and size also varied. The highest flux for the model hydrophilic molecule (caffeine) was for the flank (3.22 ± 0.36 µg/cm2 /h), while that for the lipophilic molecule (ibuprofen) was for the inner thigh (0.12 ± 0.02 µg/cm2 /h). CONCLUSIONS AND CLINICAL RELEVANCE: Anatomical location differences in equine skin structure and small molecule permeability were demonstrated. These results can aid in the development of transdermal therapies for horses.

Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell-derived factors.

Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1ß, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells' ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1ß using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1ß stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB.

Changes in Head, Withers, and Pelvis Movement Asymmetry in Lame Horses as a Function of Diagnostic Anesthesia Outcome, Surface and Direction.

Evaluation of diagnostic anesthesia during equine lameness examination requires comparison of complex movement patterns and can be influenced by expectation bias. There is limited research about how changes in movement asymmetries after successful analgesia are affected by different exercise conditions. Movement asymmetry of head, withers and pelvis was quantified in N = 31 horses undergoing forelimb or hindlimb diagnostic anesthesia. Evaluation on a straight line and a circle was performed with subjective diagnostic anesthesia outcome and quantitative changes recorded. Mixed linear models (P < .05) analyzed the differences in movement asymmetry before/after diagnostic anesthesia - random factor: horse, fixed factors: surface (soft, hard), direction (straight, inside, outside, inside-outside average), diagnostic anesthesia outcome (negative, partially positive, positive) and two-way interactions. Forelimb diagnostic anesthesia influenced primary movement asymmetry (all head and withers parameters) and compensatory movement asymmetry (two pelvic parameters) either individually (P≤.009) or in interaction with surface (P≤.03). Hindlimb diagnostic anesthesia influenced primary movement asymmetry (all pelvic parameters) and compensatory movement asymmetry (two head and two withers parameters) either individually (P≤.04) or in interaction with surface (P≤.01;) or direction (P≤.006). Direction was also significant individually for two pelvic parameters (P≤.04). Changes in primary movement asymmetries after partially positive or positive outcomes indicated improvement in the blocked limb. Compensatory changes were mostly in agreement with the 'law of sides'. The changes were more pronounced on the hard surface for hindlimb lameness and on the soft surface for forelimb lameness. Withers asymmetry showed distinct patterns for forelimb and hindlimb lameness potentially aiding clinical decision-making.

Linear Discriminant Analysis for Investigating Differences in Upper Body Movement Symmetry in Horses before/after Diagnostic Analgesia in Relation to Expert Judgement.

Diagnostic analgesia and lunging are parts of the equine lameness examination, aiding veterinarians in localizing the anatomical region(s) causing pain-related movement deficits. Expectation bias of visual assessment and complex movement asymmetry changes in lame horses on the lunge highlight the need to investigate data-driven approaches for optimally integrating quantitative gait data into veterinary decision-making to remove bias. A retrospective analysis was conducted with inertial sensor movement symmetry data before/after diagnostic analgesia relative to subjective judgement of efficacy of diagnostic analgesia in 53 horses. Horses were trotted on the straight and on the lunge. Linear discriminant analysis (LDA) applied to ten movement asymmetry features quantified the accuracy of classifying negative, partial and complete responses to diagnostic analgesia and investigated the influence of movement direction and surface type on the quality of the data-driven separation between diagnostic analgesia categories. The contribution of movement asymmetry features to decision-making was also studied. Leave-one-out classification accuracy varied considerably (38.3-57.4% for forelimb and 36.1-56.1% for hindlimb diagnostic analgesia). The highest inter-category distances (best separation) were found with the blocked limb on the inside of the circle, on hard ground for forelimb diagnostic analgesia and on soft ground for hindlimb diagnostic analgesia. These exercises deserve special attention when consulting quantitative gait data in lame horses. Head and pelvic upward movement and withers minimum differences were the features with the highest weighting within the first canonical LDA function across exercises and forelimb and hindlimb diagnostic analgesia. This highlights that movement changes after diagnostic analgesia affect the whole upper body. Classification accuracies based on quantitative movement asymmetry changes indicate considerable overlap between subjective diagnostic analgesia categories.

"One Health" in tendinopathy research: Current concepts.

Tendinopathy remains one of the most common musculoskeletal disorders affecting both human and equine athletes and presents a considerable therapeutic challenge. The following workshop report comes from the third Dorothy Havemeyer Symposium of Tendinopathy which provided a unique overview of our current understanding of both the basic science and the clinical challenges for diagnosing and treating tendinopathy in both species. Pathologically, tendon demonstrates alterations in both cellular, molecular, structural, and biomechanical features, leading to a spectrum of pathological endotypes. To develop novel interventions to manage, treat or prevent tendinopathies it is vital to understand the underlying mechanisms that lead to both tendon failure, and also regeneration and resolution of inflammation. The horse shows analogous pathology with both human Achilles tendinopathy (superficial digital flexor tendon) and intrathecal rotator cuff tears (deep digital flexor tendon tears) enabling scientists and clinicians from both medical and veterinary fields to work jointly on matching naturally occurring disease models. The experience in human medicine on the design, conduct, and impact of clinical trials has much to inform clinical trials in horses. There is a need to design appropriate studies to address clear questions, socialize the study to achieve good enrollment, and consider the significance and impact of the clinical question as well as the cost of addressing it. Because economics is often a limitation in equine medicine the use of observational studies, and specifically registries, should be given careful consideration.

Histopathological and immunohistochemical evaluation of cellular response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch for tendon repair.

We investigated endogenous tissue response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch intended for tendon repair. A sheep tendon injury model characterised by a natural history of consistent failure of healing was chosen to assess the biological potential of woven and aligned electrospun fibres to induce a reparative response. Patches were implanted into 8 female adult English Mule sheep. Significant infiltration of tendon fibroblasts was observed within the electrospun component of the patch but not within the woven component. The cellular infiltrate into the electrospun fibres was accompanied by an extensive network of new blood vessel formation. Tendon fibroblasts were the most abundant scaffold-populating cell type. CD45+, CD4+ and CD14+ cells were also present, with few foreign body giant cells. There were no local or systemic signs of excessive inflammation with normal hematology and serology for inflammatory markers three months after scaffold implantation. In conclusion, we demonstrate that an endogenous healing response can be safely induced in tendon by means of biophysical cues using a woven and electrospun patch.

Bone marrow mesenchymal stem cells do not enhance intra-synovial tendon healing despite engraftment and homing to niches within the synovium.

BACKGROUND: Intra-synovial tendon injuries display poor healing, which often results in reduced functionality and pain. A lack of effective therapeutic options has led to experimental approaches to augment natural tendon repair with autologous mesenchymal stem cells (MSCs) although the effects of the intra-synovial environment on the distribution, engraftment and functionality of implanted MSCs is not known. This study utilised a novel sheep model which, although in an anatomically different location, more accurately mimics the mechanical and synovial environment of the human rotator cuff, to determine the effects of intra-synovial implantation of MSCs. METHODS: A lesion was made in the lateral border of the lateral branch of the ovine deep digital flexor tendon within the digital sheath and 2 weeks later 5 million autologous bone marrow MSCs were injected under ultrasound guidance into the digital sheath. Tendons were recovered post mortem at 1 day, and 1-2, 4, 12 and 24 weeks after MSC injection. For the 1-day and 1-2-week groups, MSCs labelled with fluorescent-conjugated magnetic iron-oxide nanoparticles (MIONs) were tracked with MRI, histology and flow cytometry. The 4, 12 and 24-week groups were implanted with non-labelled cells and compared with saline-injected controls for healing. RESULTS: The MSCs displayed no reduced viability in vitro to an uptake of 20.0 ± 4.6 pg MIONs per cell, which was detectable by MRI at minimal density of ~ 3 × 104 cells. Treated limbs indicated cellular distribution throughout the tendon synovial sheath but restricted to the synovial tissues, with no MSCs detected in the tendon or surgical lesion. The lesion was associated with negligible morbidity with minimal inflammation post surgery. Evaluation of both treated and control lesions showed no evidence of healing of the lesion at 4, 12 and 24 weeks on gross and histological examination. CONCLUSIONS: Unlike other laboratory animal models of tendon injury, this novel model mimics the failed tendon healing seen clinically intra-synovially. Importantly, however, implanted stem cells exhibited homing to synovium niches where they survived for at least 14 days. This phenomenon could be utilised in the development of novel physical or biological approaches to enhance localisation of cells in augmenting intra-synovial tendon repair.

Mineralization can be an incidental ultrasonographic finding in equine tendons and ligaments.

Tendon/ligament mineralization is recognized in horses but information regarding its clinical significance is limited. The aims of this observational study were to report the structures most commonly affected by ultrasonographically detectable mineralization and, for these, determine frequency of diagnosis and key clinical features. Cases presented at our hospital in April 1999-April 2013 and September 2014-November 2015 were included: a total of 27 horses (22 retrospective, five prospective). Mineralizations were most common in deep digital flexor tendons (10) and suspensory ligament branches (eight), representing 10% and 7% (estimated), respectively, of horses diagnosed with injuries to these structures during the study. Two deep digital flexor tendon and three suspensory ligament branch cases showed bilateral mineralization. Deep digital flexor tendon mineralization was restricted to the digital flexor tendon sheath, most commonly in the proximal sheath (±sesamoidean canal), and seven of 10 cases involved hindlimbs. Suspensory ligament branch mineralization was visible in the same ultrasound window as the proximal sesamoid bones in 10/11 limbs and six of eight cases involved forelimbs. Previous corticosteroid medication was a feature of one deep digital flexor tendon and one suspensory ligament branch case. Mineralization was associated with lameness in some but not all limbs. Mineralized foci within the deep digital flexor tendon preceded hypoechoic lesion formation in two limbs. Of the cases with deep digital flexor tendon or suspensory ligament branch injury only, one of three and two of three cases, respectively, became sound. Findings indicated that tendon/ligament mineralization can be associated with lameness in some horses, but can also be an incidental finding.

Exposure of a tendon extracellular matrix to synovial fluid triggers endogenous and engrafted cell death: A mechanism for failed healing of intrathecal tendon injuries.

AIM: The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDCs) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix. METHODS: Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFTs) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSCs and TDCs in SF-media and co-culture with DDFT explants was determined by alamarblue®. Non-viable Native Tendon matrices (NNTs) were re-populated with MSCs or TDCs and cultured in SF-media. Immunohistochemical staining of tendon sections for the apoptotic proteins caspase-3, -8, and -9 was performed. RESULTS: Contact with autologous or allogeneic SF resulted in rapid death of resident tenocytes in equine and human tendon. SF did not affect the viability of equine epitenon cells, or of MSCs and TDCs in the monolayer or indirect explant co-culture. MSCs and TDCs, engrafted into NNTs, died when cultured in SF. Caspase-3, -8, and -9 expression was the greatest in SDFT explants exposed to allogeneic SF. CONCLUSIONS: The efficacy of cells administered intra-synovially for tendon lesion repair is likely to be limited, since once incorporated into the matrix, cells become vlnerable to the adverse effects of SF. These observations could account for the poor success rate of intra-synovial tendon healing following damage to the epitenon and contact with SF, common with most soft tissue intra-synovial pathologies.

Modulation of mesenchymal stem cell genotype and phenotype by extracellular matrix proteins.

AIM: To investigate the effect of extracellular matrix (ECM) proteins on characteristics of mesenchymal stem cells (MSCs) and tendon-derived cells (TDCs). MATERIALS AND METHODS: MSCs and TDCs, cultured in a monolayer (2D) or hydrogels (3D), with or without ECM protein supplementation, and on a non-viable native tendon (NNT) matrix were assayed for adhesion, proliferation, gene expression, and integrin expression. RESULTS: MSCs exhibited a fibroblastic, spindle-shaped morphology on 2D matrices except in the presence of fibronectin. In 3D matrices, MSCs displayed a rounded phenotype except when cultured on NNTs where cells aligned along the collagen fibrils but, unlike TDCs, did not form inter-cellular cytoplasmic processes. MSC proliferation was significantly (p < 0.01) increased by collagen type I in 2D culture and fibronectin in 3D culture. TDC proliferation was unaffected by substrata. MSCs and TDCs differentially expressed α2 integrin. Adhesion to substrata was reduced by RGD-blocking peptide and ß1 integrin antibody. The presence of collagen I or fibronectin upregulated MSC expression of collagen type I and collagen type III, COMP, decorin, osteopontin, and fibronectin. CONCLUSIONS: The morphology, gene expression, and adhesion of both MSCs and TDCs are sensitive to the presence of specific ECM components. Interaction with the ECM is, therefore, likely to affect the mechanism of action of MSCs in vitro and may contribute to phenotypic modulation in vivo.

Mineralization of the Equine Palmar/Plantar Annular Ligament Treated by Surgical Resection.

OBJECTIVE: To document the clinical presentation, diagnosis, and surgical treatment of mineralization of the equine palmar/plantar annular ligament (PAL). STUDY DESIGN: Retrospective study. ANIMALS: Ponies (n=7). METHODS: Case records from 2 referral hospitals were examined to identify cases with lameness associated with PAL mineralization treated surgically. Follow-up information was obtained from the owners by telephone questionnaire. RESULTS: Duration of lameness before referral ranged from 5 weeks to 6 months, and degree of lameness from grade 1 to 5 out of 10. In 3 cases, records noted obvious pain when pressure was applied over the PAL. Pain resulting in lameness was localized to this area and all cases were treated surgically, although the extent of resected tissue varied among cases. Histological examination of resected tissue (4 cases) revealed fibrocartilaginous and/or osseous metaplasia. Following surgery, 6 of the 7 ponies became sound. CONCLUSION: Based on this limited case series, surgical treatment for mineralization of the PAL offers a favorable success rate without severe complications where conservative methods have failed.

Immunophenotypic characterization of ovine mesenchymal stem cells.

The clinical potential of multipotent mesenchymal stem cells (MSCs) has led to the essential development of analytical tools such as antibodies against membrane-bound proteins for the immunophenotypic characterization of human and rodent cells. Such tools are frequently lacking for emerging large animal models like the sheep that have greater relevance for the study of human musculoskeletal diseases. The present study identified a set of commercial nonspecies specific monoclonal antibodies for the immunophenotypic characterization of ovine MSCs. A protocol combining the less destructive proteolytic activity of accutase and EDTA was initially developed for the detachment of cells from plastic with minimum loss of cell surface antigens. A range of commercially available antibodies against human or rodent MSC antigens were then tested in single and multistain-based assays for their cross-reactivity to bone marrow derived ovine MSCs. Antibody clones cross-reactive to ovine CD73 (96.9% ± 5.9), CD90 (99.6% ± 0.3), CD105 (99.1 ± 1.5), CD271 (97.7 ± 2.0), and MHC1 (94.0% ± 7.2) antigens were identified using previously reported CD29, CD44, and CD166 as positive controls. Multistaining analysis indicated the colocalization of these antigens on MSCs. Furthermore, antibody clones identified to cross-react against white blood cell antigens exhibited either negative (CD117 (0.1% ± 0.1)) or low (MHCII (10.5% ± 16.0); CD31 (14.6% ± 4.2), and CD45 (39.4% ± 31.8)) cross-reactivity with ovine MSCs. The validation of these antibody clones to sheep MSC antigens is essential for studies utilizing this large animal model for stem cell-based therapies. © 2016 International Society for Advancement of Cytometry.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (<10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers. This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.