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Objective: To compare 3 perioperative feeding regimens and their effect on anesthetic complications, manure output, and colic proportion in healthy horses., Methods: 45 horses presenting for elective orthopedic procedures were randomly assigned to 1 of 3 groups: not fasted (NF; continuous access to hay perioperatively), fasted muzzled (FM; 10-hour preoperative fast with slow refeeding postoperatively and muzzle placement), or fasted not muzzled (FNM; same as FM without muzzle placement). Anesthetic protocol was standardized. Outcomes compared between groups included anesthesia time, arterial oxygenation, duration of hypotension, perioperative manure output, time to first passage of manure postoperatively, pain scores, and colic proportion. Comparisons were made with a mixed model and Fisher exact test with statistical significance considered at P ≤ .05., Results: No differences were seen in pain scores, oxygenation, hypotension, or colic between groups. Groups FM and FNM had a significantly greater mean reduction in postoperative manure weight (-81% and -70%; P = .003) and number of manure piles (-63% and -55%; P = .005) compared to group NF (-39% and -22%; P < .001; weight and piles, respectively). Mean ± SD minutes to passage of manure postoperatively was significantly shorter in group NF (238 ± 13 minutes) than groups FM (502 ± 174 minutes; P < .001) and FNM (444 ± 171 minutes; P = .003)., Clinical Relevance: Horses with continuous access to hay prior to and following recovery from anesthesia passed more manure and passed manure sooner after surgery than their fasted counterparts without detrimental effect on anesthetic parameters and postoperative complications. Continuous access to hay perioperatively supports manure production in healthy horses without increase in anesthetic complications.

Objective: To evaluate the effects of a gene transfer approach to IL-1β inhibition in an equine osteochondral chip fragment model of joint injury using a self-complementary adeno-associated virus with interleukin receptor antagonist transgene cassette (scAAVIL-1ra), as posttraumatic osteoarthritis in horses, similar to people, is a significant clinical problem., Animals: 16 horses were utilized for the study., Methods: All horses had an osteochondral chip fragment induced arthroscopically in one middle carpal joint while the contralateral joint was sham operated. Eight horses received either scAAVIL-1ra or saline in the osteoarthritis joint. Horses were evaluated over 70 days clinically (lameness, imaging, and biomarker analysis) and euthanized at 70 days and evaluated grossly, with imaging and histopathology., Results: The following findings were statistically significant. Injection of scAAVIL-1ra resulted in high synovial fluid levels of IL-1ra (0.5 to 9 μg/mL) throughout the duration of the experiment (70 days). Over the duration, we observed scAAVIL-1ra to improve lameness (lameness score relative improvement of 1.2 on a scale of 0 to 5), cause suppression of prostaglandin E2 (a relative decline of 30 pg/mL), and result in histological improvement in articular cartilage (decreased chondrocyte loss and chondrone formation) and subchondral bone (less osteochondral splitting and osteochondral lesions). Within the synovial membrane of scAAVIL-1ra-treated joints, we also observed perivascular infiltration with CD3-positive WBCs, suggesting lymphocytic T-cell perivascular infiltration commonly observed with viral transduction., Clinical Relevance: These data provide support for further evaluation and optimization of scAAVIL-1ra gene therapy to treat equine osteoarthritis.

Introduction: Alpha 2 macroglobulin (A2M), a multi-functional protein in the plasma protease inhibitor class, regulates proinflammatory cytokines and the clearance of chondrodestructive enzymes in cases of joint injury and osteoarthritis (OA). The purpose of this study was to compare A2M concentrations in equine plasma samples processed by three commercial devices developed for stall-side regenerative joint therapy., Methods: Plasma samples were obtained from healthy adult horses ( N  = 13). Mass spectrometry analysis was used to determine the concentration of protein analytes in each sample. Selected reaction monitoring measured a specific A2M peptide as a surrogate of the whole A2M protein. A2M concentrations produced by each test device were compared for two sample types: a pre-concentrate or platelet-poor (PP) component and a final component for use in the horse., Results: There was no significant difference ( p  > 0.05) in the geometric mean (GM) concentration of A2M in the final concentration samples produced by the Alpha2EQ ® device (N horses = 13) and the single-centrifugation PP samples produced by the Pro-Stride ® APS (autologous protein solution) device ( N  = 13) and the Restigen ® PRP (platelet-rich plasma) device ( N  = 11). When A2M content in final concentration samples produced by each device was compared, the Pro-Stride APS and Restigen PRP samples had significantly greater GM A2M content ( p  < 0.0001) compared to the Alpha2EQ samples, and the Pro-Stride APS final concentration samples had significantly greater GM A2M concentration ( p  < 0.0001) versus that for the Restigen PRP final samples., Discussion: This comparison demonstrated that the volume and A2M concentration of an Alpha2EQ final concentrate are no different than the volume and concentration of A2M in the PP from Pro-Stride or Restigen devices., Competing Interests: LA, BC, JRF, and DA were employed by Zoetis, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ortved, Alward, Cowles, Linardi, Barot, Usimaki, Fedie, Amodie and Goodrich.)

Objective: To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology., Animals: 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4)., Methods: Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints., Results: Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20)., Clinical Relevance: Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.

Objective: To raise awareness of the potential for intra-articular subchondral bone sequestrum formation secondary to a traumatic or septic process to enable more rapid identification of this uncommon but possible outcome in future cases., Animal: A client-owned 12-year-old Appaloosa mare., Clinical Presentation, Progression, and Procedures: The mare had a wound to the lateral aspect of the fourth metatarsal bone (MT4) that communicated with the distal tarsal joints. Radiographs revealed a displaced, comminuted fracture of MT4., Treatment and Outcome: The horse underwent aggressive debridement of the wound and MT4 as well as, on 2 occasions, needle joint lavage. Systemic, regional, and IA antibiotic therapy was also performed together with a bone graft from the tuber coxae. The horse's comfort improved, and the wound appeared to be healing. Five weeks following discharge, the horse re-presented with a non-weight-bearing lameness and radiographs revealed marked osteomyelitis of the tarsometatarsal and distal intertarsal joints. Postmortem examination of the limb identified a sequestrum within the proximal articular surface of the third metatarsal bone., Clinical Relevance: The present report highlights the importance of arthroscopic lavage to visualize the cartilage surface and the benefits of advanced imaging to detect associated changes within the bone earlier than conventional radiographs. To our knowledge, no reports exist of intra-articular subchondral bone sequestra in the tarsometatarsal joint in horses.

The use of radiofrequency energy (RFE) has become increasingly popular in equine orthopedic surgery in recent years, particularly for the debridement of cartilage lesions and soft tissue resection. However, despite considerable advancements in the technology, the safety and efficacy of RFE have continued to be questioned. While studies investigating the use of RFE for chondroplasty in the equine population are lacking, there is an abundance of research studies in the human literature assessing its effect on healthy chondrocytes, and researchers are seeking to develop guidelines to minimize collateral damage. This review article provides a concise and thorough summary of the current use of RFE in equine orthopedics, in addition to discussing the recent evidence surrounding its use for chondroplasty in both the human and equine populations.

Objective: To perform a pilot study with the intent of assessing the feasibility of a modified subchondroplasty (mSCP) technique in a validated preclinical equine model of full-thickness articular cartilage loss and evaluate the short-term patient response to the injected materials., Animals: 3 adult horses., Procedures: Two 15-mm-diameter full-thickness cartilage defects were created on the medial trochlear ridge of each femur. Defects were treated with microfracture and then filled by 1 of 4 techniques: (1) autologous fibrin graft (FG) via subchondral injection of fibrin glue (FG), (2) autologous fibrin graft via direct injection of FG, (3) subchondral injection of a calcium phosphate bone substitute material (BSM) with direct injection of FG, and (4) untreated control. Horses were euthanized after 2 weeks. Patient response was evaluated via serial lameness examination, radiography, magnetic resonance imaging, computed tomography, gross evaluation, microcomputed tomography, and histopathology., Results: All treatments were successfully administered. The injected material perfused through the underlying bone into the respective defects without adversely affecting the surrounding bone and articular cartilage. Increased new bone formation was seen at the margins of the trabecular spaces containing BSM. There was no treatment effect on the amount or composition of tissue within defects., Clinical Relevance: The mSCP technique was a simple, well-tolerated technique in this equine articular cartilage defect model without significant adverse effects to host tissues after 2 weeks. Larger studies with long-term follow-ups are warranted.

Introduction: Multiple biological therapies for orthopedic injuries are marketed to veterinarians, despite a lack of rigorous comparative biological activity data to guide informed decisions in selecting a most effective compound. Therefore, the goal of this study was to use relevant bioassay systems to directly compare the anti-inflammatory and immunomodulatory activity of three commonly used orthobiological therapies (OTs): mesenchymal stromal cells (MSC), autologous conditioned serum (ACS), and platelet rich plasma (PRP)., Methods: Equine monocyte-derived macrophages were used as the readout system to compare therapies, including cytokine production and transcriptomic responses. Macrophages were stimulated with IL-1ß and treated 24 h with OTs, washed and cultured an additional 24 h to generate supernatants. Secreted cytokines were measured by multiplex immunoassay and ELISA. To assess global transcriptomic responses to treatments, RNA was extracted from macrophages and subjected to full RNA sequencing, using an Illumina-based platform. Data analysis included comparison of differentially expressed genes and pathway analysis in treated vs. untreated macrophages., Results: All treatments reduced production of IL-1ß by macrophages. Secretion of IL-10 was highest in MSC-CM treated macrophages, while PRP lysate and ACS resulted in greater downregulation of IL-6 and IP-10. Transcriptomic analysis revealed that ACS triggered multiple inflammatory response pathways in macrophages based on GSEA, while MSC generated significant downregulation of inflammatory pathways, and PRP lysate induced a mixed immune response profile. Key downregulated genes in MSC-treated cultures included type 1 and type 2 interferon response, TNF-α and IL-6. PRP lysate cultures demonstrated downregulation of inflammation-related genes IL-1RA, SLAMF9, ENSECAG00000022247 but concurrent upregulation of TNF-α, IL-2 signaling, and Myc targets. ACS induced upregulation of inflammatory IL-2 signaling, TNFα and KRAS signaling and hypoxia, but downregulation of MTOR signaling and type 1 interferon signaling., Discussion: These findings, representing the first comprehensive look at immune response pathways for popular equine OTs, reveal distinct differences between therapies. These studies address a critical gap in our understanding of the relative immunomodulatory properties of regenerative therapies commonly used in equine practice to treat musculoskeletal disease and will serve as a platform from which further in vivo comparisons may build., Competing Interests: LP, LG, LC, and SD acknowledge that they hold stock options in eQCell Inc. (LP, LG, and SD), Validus (SD), and ART Advanced Regenerative Therapies (LG), and have filed provisional patents covering immune activated MSC technology for treatment of musculoskeletal disease (SD, LP, LC, and LG). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pezzanite, Chow, Griffenhagen, Bass, Goodrich, Impastato and Dow.)

Background: Rapid development of antibiotic resistance necessitates advancement of novel therapeutic strategies to treat infection. Mesenchymal stromal cells (MSC) possess antimicrobial and immunomodulatory properties, mediated through antimicrobial peptide secretion and recruitment of innate immune cells including neutrophils and monocytes. TLR-3 activation of human, canine and equine MSC has been shown to enhance bacterial killing and clearance in vitro , in rodent Staphylococcal biofilm infection models and dogs with spontaneous multi-drug-resistant infections. The objective of this study was to determine if intra-articular (IA) TLR-3-activated MSC with antibiotics improved clinical parameters and reduced bacterial counts and inflammatory cytokine concentrations in synovial fluid (SF) of horses with induced septic arthritis., Methods: Eight horses were inoculated in one tarsocrural joint with multidrug-resistant Staphylococcus aureus ( S. aureus ). Bone marrow-derived MSC from three unrelated donors were activated with TLR-3 agonist polyinosinic, polycytidylic acid (pIC). Recipient horses received MSC plus vancomycin (TLR-MSC-VAN), or vancomycin (VAN) alone, on days 1, 4, 7 post-inoculation and systemic gentamicin. Pain scores, quantitative bacterial counts (SF, synovium), SF analyses, complete blood counts, cytokine concentrations (SF, plasma), imaging changes (MRI, ultrasound, radiographs), macroscopic joint scores and histologic changes were assessed. Results were reported as mean ± SEM., Results: Pain scores (d7, P=0.01, 15.2±0.2 vs. 17.9±0.5), ultrasound (d7, P=0.03, 9.0±0.6 vs. 11.8±0.5), quantitative bacterial counts (SF d7, P=0.02, 0±0 vs. 3.4±0.4; synovium P=0.003, 0.4±0.4 vs. 162.7±18.4), systemic neutrophil (d4, P=0.03, 4.6±0.6 vs. 7.8±0.6) and serum amyloid A (SAA) (d4, P=0.01, 1,106.0±659.0 vs. 2,858.8±141.3; d7, P=0.02, 761.8±746.2 vs. 2,357.3±304.3), and SF lactate (d7, P<0.0001, 5.4±0.2 vs. 15.0±0.3), SAA (endterm, P=0.01, 0.0 vs. 2,094.0±601.6), IL-6 (P=0.03, 313.0±119.2 vs. 1,328.2±208.9), and IL-18 (P=0.02, 11.1±0.5 vs. 13.3±3.8) were improved in TLR-MSC-VAN vs. VAN horses. Study limitations include the small horse sample size, short study duration, and lack of additional control groups., Conclusions: Combined TLR-activated MSC with antibiotic therapy may be a promising approach to manage joint infections with drug resistant bacteria., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-1746/coif). LMP, LC, SD, and LRG declare that a patent application has been filed covering the antimicrobial cellular therapy technology described here. LMP reports that support for this work was provided by the Grayson Jockey Club Research Foundation, ACVS Zoetis Dual Training Grant, NIH/NCATS CTSA 5TL1TR002533-02, NIH 5T32ODO010437-19, Verdad Foundation, Charles Shipley Family Foundation and Carolyn Quan and Porter Bennett. The StableLab serum amyloid A testing material was kindly provided by Zoetis. LMP reports that she holds stock options in eQCell Inc. SD reports that he holds stock options in eQCell Inc. LRG reports that she holds stock options in eQCell and Advanced Regenerative Therapies. The other authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

With an intrinsically low ability for self-repair, articular cartilage injuries often progress to cartilage loss and joint degeneration resulting in osteoarthritis (OA). Osteoarthritis and the associated articular cartilage changes can be debilitating, resulting in lameness and functional disability both in human and equine patients. While articular cartilage damage plays a central role in the pathogenesis of OA, the contribution of other joint tissues to the pathogenesis of OA has increasingly been recognized thus prompting a whole organ approach for therapeutic strategies. Gene therapy methods have generated significant interest in OA therapy in recent years. These utilize viral or non-viral vectors to deliver therapeutic molecules directly into the joint space with the goal of reprogramming the cells' machinery to secrete high levels of the target protein at the site of injection. Several viral vector-based approaches have demonstrated successful gene transfer with persistent therapeutic levels of transgene expression in the equine joint. As an experimental model, horses represent the pathology of human OA more accurately compared to other animal models. The anatomical and biomechanical similarities between equine and human joints also allow for the use of similar imaging and diagnostic methods as used in humans. In addition, horses experience naturally occurring OA and undergo similar therapies as human patients and, therefore, are a clinically relevant patient population. Thus, further studies utilizing this equine model would not only help advance the field of human OA therapy but also benefit the clinical equine patients with naturally occurring joint disease. In this review, we discuss the advancements in gene therapeutic approaches for the treatment of OA with the horse as a relevant patient population as well as an effective and commonly utilized species as a translational model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thampi, Samulski, Grieger, Phillips, McIlwraith and Goodrich.)

Background: Allogenic mesenchymal stem cell (MSC) secretome is a novel intra-articular therapeutic that has shown promise in in vitro and small animal models and warrants further investigation., Objectives: To investigate if intra-articular allogenic MSC-secretome has anti-inflammatory effects using an equine model of joint inflammation., Study Design: Randomized positively and negatively controlled experimental study., Method: In phase 1, joint inflammation was induced bilaterally in radiocarpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After 2 h, the secretome of INFy and TNFα stimulated allogeneic equine MSCs was injected in one randomly assigned joint, while the contralateral joint was injected with medium (negative control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded, and synovial fluid samples were analyzed for biomarkers (total protein, WBCC; eicosanoid mediators, CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed post-injection hours (PIH 0, 8, 24, 72, and 168 h). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were evaluated. For phase 2, allogeneic MSC-secretome vs. allogeneic equine MSCs (positive control) was tested using a similar methodology., Results: In phase 1, the joint circumference was significantly ( p < 0.05) lower in the MSC-secretome treated group compared to the medium control group at PIH 24, and significantly higher peak synovial GAG values were noted at PIH 24 ( p < 0.001). In phase 2, no significant differences were noted between the treatment effects of MSC-secretome and MSCs., Main Limitations: This study is a controlled experimental study and therefore cannot fully reflect natural joint disease. In phase 2, two therapeutics are directly compared and there is no negative control., Conclusions: In this model of joint inflammation, intra-articular MSC-secretome injection had some clinical anti-inflammatory effects. An effect on cartilage metabolism, evident as a rise in GAG levels was also noted, although it is unclear whether this could be considered a beneficial or detrimental effect. When directly comparing MSC-secretome to MSCs in this model results were comparable, indicating that MSC-secretome could be a viable off-the-shelf alternative to MSC treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kearney, Khatab, van Buul, Plomp, Korthagen, Labberté, Goodrich, Kisiday, Van Weeren, van Osch and Brama.)

Septic arthritis causes significant morbidity and mortality in veterinary and human clinical practice and is increasingly complicated by multidrug-resistant infections. Intra-articular (IA) antibiotic administration achieves high local drug concentrations but is considered off-label usage, and appropriate doses have not been defined. Using an equine joint model, we investigated the effects of amikacin injected at three different doses (500, 125, and 31.25 mg) on the immune and cartilage responses in tibiotarsal joints. Synovial fluid (SF) was sampled at multiple time points over 24 h, the cell counts determined, and amikacin concentrations measured by liquid chromatography-mass spectrometry. Cytokine concentrations and collagen degradation products in SF were measured by ELISA and multiplex immunoassays. The mean amikacin concentrations in SF were greater than or equal to the minimum inhibitory concentration (MIC) (0.004 mg/ml) for most common equine joint pathogens at all time points tested to 24 h for all three amikacin doses evaluated. The inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) increased significantly in SF in the highest amikacin dose group, despite the fact that increases in SF cell counts were not observed. Similarly, the biomarkers of cartilage type II collagen cleavage (C2C and C12C) were increased in SF following amikacin injection. Mechanistically, we further demonstrated using in vitro studies that chondrocytes and synoviocytes killed by exposure to amikacin underwent apoptotic cell death and were phagocytosed by macrophages in a non-inflammatory process resembling efferocytosis. Neutrophils and T cells were susceptible to amikacin cytotoxicity at clinically relevant doses, which may result in blunting of cellular inflammatory responses in SF and account for the lack of increase in total nucleated cell counts following amikacin injection. In summary, decisions on whether to inject cytotoxic antibiotics such as aminoglycosides intra-articularly and what doses to use should take into account the potential harm that antibiotics may cause and consider lower doses than those previously reported in equine practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pezzanite, Chow, Hendrickson, Gustafson, Russell Moore, Stoneback, Griffenhagen, Piquini, Phillips, Lunghofer, Dow and Goodrich.)

Objective: To investigate the diffusion trajectory of a cationic contrast medium (CA4+) into equine articular cartilage, and to assess normal and degenerative equine articular cartilage using cationic contrast-enhanced computed tomography (CECT)., Design: In the first experiment (Exp1), equine osteochondral specimens were serially imaged with cationic CECT to establish the diffusion time constant and time to reach equilibrium in healthy articular cartilage. In a separate experiment (Exp2), articular cartilage defects were created on the femoral trochlea (defect joint) in a juvenile horse, while the opposite joint was a sham-operated control. After 7 weeks, osteochondral biopsies were collected throughout the articular surfaces of both joints. Biopsies were analyzed for cationic CECT attenuation, glycosaminoglycan (GAG) content, mechanical stiffness (E eq ), and histology. Imaging, biochemical and mechanical data were compared between defect and control joints., Results: Exp1: The mean diffusion time constant was longer for medial condyle cartilage (3.05 ± 0.1 hours) than lateral condyle cartilage (1.54 ± 0.3 hours, P = 0.04). Exp2: Cationic CECT attenuation was lower in the defect joint than the control joint ( P = 0.005) and also varied by anatomic location ( P = 0.045). Mean cationic CECT attenuation from the lateral trochlear ridge was lower in the defect joint than in the control joint (2223 ± 329 HU and 2667 ± 540 HU, respectively; P = 0.02). Cationic CECT attenuation was strongly correlated with both GAG (ρ = 0.79, P < 0.0001) and E eq (ρ = 0.61, P < 0.0001)., Conclusions: The equilibration time of CA4+ into equine articular cartilage is affected by tissue volume. Quantitative cationic CECT imaging reflects the biochemical, biomechanical and histological state of normal and degenerative equine articular cartilage.

Objective: Rats are an early preclinical model for cartilage tissue engineering, and a practical species for investigating the effects of aging. However, rats may be a poor aging model for mesenchymal stem cells (MSCs) based on laboratory reports of a severe decline in chondrogenesis beyond young adulthood. Such testing has not been conducted with MSCs seeded in a scaffold, which can improve the propensity of MSCs to undergo chondrogenesis. Therefore, the objective of this study was to evaluate chondrogenesis of middle-aged rat MSCs encapsulated in agarose., Design: MSCs from 14- to 15-month-old rats were expanded, seeded into agarose, and cultured in chondrogenic medium with or without 5% serum for 15 days. Samples were evaluated for cell viability and cartilaginous extracellular matrix (ECM) accumulation. Experiments were repeated using MSCs from 6-week-old rats., Results: During expansion, middle-aged rat MSCs demonstrated a diminishing proliferation rate that was improved ~2-fold in part by transient exposure to chondrogenic medium. In agarose culture in defined medium, middle-aged rat MSCs accumulated ECM to a much greater extent than negative controls. Serum supplementation improved cell survival ~2-fold, and increased ECM accumulation ~3-fold. Histological analysis indicated that defined medium supported chondrogenesis in a subset of cells, while serum-supplementation increased the frequency of chondrogenic cells. In contrast, young rat MSCs experienced robust chondrogenesis in defined medium that was not improved with serum-supplementation., Conclusions: These data demonstrate a previously-unreported propensity of middle-aged rat MSCs to undergo chondrogenesis, and the potential of serum to enhance chondrogenesis of aging MSCs.

Regenerative medicine is commonly used in human and equine athletes. Potential therapies include culture expanded stem cells, stromal vascular fraction of adipose tissue, platelet-rich plasma, bone marrow concentrate, or autologous conditioned serum. The purpose of this manuscript is to disseminate findings from a workshop on the development of translational regenerative medicine in the equine field. Five themes emerged: stem cell characterization and tenogenic differentiation; interactions between mesenchymal stem cells, other cells and the environment; scaffolds and cell packaging; blood- and bone marrow-based regenerative medicines; clinical use of regenerative therapies. Evidence gained through the use of regenerative medicine applications in the horse should continue to translate to the human patient, bringing novel regenerative therapies to both humans and horses.

Objective: Cationic agent contrast-enhanced computed tomography (cationic CECT) characterizes articular cartilage ex vivo, however, its capacity to detect post-traumatic injury is unknown. The study objectives were to correlate cationic CECT attenuation with biochemical, mechanical and histological properties of cartilage and morphologic computed tomography (CT) measures of bone, and to determine the ability of cationic CECT to distinguish subtly damaged from normal cartilage in an in vivo equine model., Design: Mechanical impact injury was initiated in equine femoropatellar joints in vivo to establish subtle cartilage degeneration with site-matched controls. Cationic CECT was performed in vivo (clinical) and postmortem (microCT). Articular cartilage was characterized by glycosaminoglycan (GAG) content, biochemical moduli and histological scores. Bone was characterized by volume density (BV/TV) and trabecular number (Tb.N.), thickness (Tb.Th.) and spacing (Tb.Sp.)., Results: Cationic CECT attenuation (microCT) of cartilage correlated with GAG (r = 0.74, P < 0.0001), compressive modulus (E eq ) (r = 0.79, P < 0.0001) and safranin-O histological score (r = -0.66, P < 0.0001) of cartilage, and correlated with BV/TV (r = 0.37, P = 0.0005), Tb.N. (r = 0.39, P = 0.0003), Tb.Th. (r = 0.28, P = 0.0095) and Tb.Sp. (r = -0.44, P < 0.0001) of bone. Mean [95% CI] cationic CECT attenuation at the impact site (2215 [1987, 2443] Hounsfield Units [HUs]) was lower than site-matched controls (2836 [2490, 3182] HUs, P = 0.036). Clinical cationic CECT attenuation correlated with GAG (r = 0.23, P = 0.049), E eq (r = 0.26, P = 0.025) and safranin-O histology score (r = -0.32, P = 0.0046)., Conclusions: Cationic CECT (microCT) reflects articular cartilage properties enabling segregation of subtly degenerated from healthy tissue and also reflects bone morphometric properties on CT. Cationic CECT is capable of characterizing articular cartilage in clinical scanners., (Copyright © 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

CASE DESCRIPTION A 12-year-old mixed-breed mare (horse 1) and 6-year-old Friesian gelding (horse 2) were examined for chronic lameness associated with the stifle joint. CLINICAL FINDINGS Lameness examination revealed effusion of the right (horse 1) or left (horse 2) femoropatellar and medial femorotibial joints and grade 3/5 (horse 1) or 4/5 (horse 2) lameness. A diagnosis of cranial cruciate ligament (CCL) injury with associated mineralization and avulsion (horse 1) or mineralization alone (horse 2) was facilitated in both horses with a caudomedial-craniolateral oblique radiographic view obtained 45° medial to the caudocranial line, which highlighted the origin of the ligament on the caudoaxial aspect of the lateral femoral condyle within the intercondylar fossa. These lesions were subsequently confirmed via CT. TREATMENT AND OUTCOME Arthroscopy of the medial and lateral femorotibial joints was performed for horse 1 and revealed the osseous fragment associated with the CCL, but the fragment could not be removed. Horse 2 was euthanized while anesthetized following CT owing to the poor prognosis. CONCLUSIONS AND CLINICAL RELEVANCE Radiography is typically the first imaging modality attempted for horses with CCL injury, particularly outside the hospital setting. A 45° caudomedial-craniolateral oblique radiographic view may aid in diagnosis of CCL injury when avulsion or mineralization is present. Although this view is not commonly included in the typical radiographic series for imaging of the stifle joint in horses, it should be considered when CCL injury is suspected.

OBJECTIVE To describe clinical use of a locking compression plate (LCP) for proximal interphalangeal joint (PIPJ) arthrodesis in horses and compare outcomes for horses that underwent the procedure as treatment for fracture of the middle phalanx (P2) versus other causes. DESIGN Retrospective case series. ANIMALS 29 client-owned horses. PROCEDURES Medical records of 2 veterinary teaching hospitals from 2008 through 2014 were reviewed to identify horses that underwent PIPJ arthrodesis of 1 limb. Signalment, surgical, and outcome-related variables were recorded. Owners were contacted from 1 to 6 years after surgery to determine rehabilitation time, current use of the horse, and overall owner satisfaction with the procedure. Success was determined on the basis of owner satisfaction and outcome for intended use. Variables of interest were compared statistically between horses that underwent surgery for P2 fracture versus other reasons. RESULTS 14 horses underwent surgery for treatment of P2 fracture, and 15 had surgery because of osteoarthritis, subluxation, or osteochondrosis. Median convalescent time after surgery (with no riding or unrestricted exercise) was 7 months. Four horses were euthanized; of 23 known alive at follow-up, 22 were not lame, and 18 had returned to their intended use (8 and 10 at higher and lower owner-reported levels of work, respectively). Horses undergoing arthrodesis for reasons other than fracture were significantly more likely to return to their previous level of work. Twenty-two of 24 owners contacted indicated satisfaction with the procedure. CONCLUSIONS AND CLINICAL RELEVANCE Surgical arthrodesis of the PIPJ was successful in most horses of the study population. Various nuances of the system for fracture repair need to be understood prior to its use.

Background: The effects of recombinant interleukin-1β (rIL-1β) have been described for the middle carpal joint (MCJ). However, we are unaware of any studies that have described the cytological response of the tibiotarsal joint (TTJ) to rIL-1β or compared the clinical and cytological responses of the MCJ to the TTJ following the administration of intra-articular rIL-1β. Such information is critical for researchers planning to use rIL-1β to create acute synovitis models in horses. Objectives: To compare the clinical and cytological responses of the MCJ to the TTJ following administration of intra-articular rIL-1β. Methods: Twelve horses were used for the study. Eight horses received 75 ng of rIL-1β into the MCJ and four horses received 75 ng of rIL-1β into the TTJ. Clinical and cytological outcome parameters including lameness, joint circumference, joint effusion score, total nucleated cell count, cellular differentials, C-reactive protein, and prostaglandin-E2 concentrations were determined at baseline and multiple post-treatment time points over a 336 h period (2 weeks). Results: Recombinant IL-1β administered into the TTJ resulted in a significantly greater respiratory rate at 24 h and heart rate at 12 h when compared to rIL-1β administered into the MCJ. In addition, the TTJ had a significantly greater increase in joint circumference at 24 post-injection hour (PIH) and subjective effusion grade at 24 PIH and 336 PIH. The MCJ had significantly higher total protein concentration at 6 PIH, and a significantly higher NCC at 24 and 72 PIH when compared to the TTJ. Conversely, the TTJ had significantly higher neutrophilic infiltration than the MCJ at 6 PIH and 168 PIH. Conclusions: This study establishes that the same intra-articular dose of rIL-1 β elicits significantly different clinical and cytological responses in the MCJ compared to the TTJ in the equine model of intra-articular synovitis. In addition, clinical and cytological evidence of synovitis may persist up to or >1 week following intra-articular administration of rIL-1 β.

Background: Meniscal root tears cause menisci and their insertions to inadequately distribute loads and potentially leave underlying articular cartilage unprotected. Untreated meniscal root tears are becoming increasingly recognized to induce joint degradation; however, little information is known about anterior meniscal root tears and how they affect joint tissue., Purpose: To observe the early degenerative changes within the synovial fluid, menisci, tibial articular cartilage, and subchondral bone after arthroscopic creation of untreated anterior meniscal root tears., Study Design: Controlled laboratory study., Methods: Anterolateral meniscal root tears were created in 1 knee joint of 5 adult Flemish Giant rabbits, and anteromedial meniscal root tears were created in 4 additional rabbits. The contralateral limbs were used as nonoperated controls. The animals were euthanized at 8 weeks postoperatively; synovial fluid was aspirated, and tissue samples of menisci and tibial articular cartilage were collected and processed for multiple analyses to detect signs of early degeneration., Results: Significant changes were found within the synovial fluid, meniscal tissue, and tibial subchondral bone of the knees with anterior meniscal root tears when compared with controls. There were no significant changes identified in the tibial articular cartilage when comparing the tear groups with controls., Conclusion: This study demonstrated early degenerative changes within the synovial fluid, menisci, and tibial subchondral bone when leaving anterior meniscal root tears untreated for 8 weeks. The results suggest that meniscal tissue presents measurable, degenerative changes prior to changes within the articular cartilage after anterior meniscal root tears. Anterior destabilization of the meniscus arthroscopically may lead to measurable degenerative changes and be useful for future in vivo natural history and animal repair studies., Clinical Relevance: The present study is the first to investigate various tissue changes after anterior meniscal root tears of both the medial and lateral menisci. The results from this study suggest that degenerative changes occur within the synovial fluid, meniscus, and tibial subchondral bone prior to any measurable changes to the tibial articular cartilage. Further studies should expand on this study to evaluate how these components continue to progress when left untreated for long periods., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: R.F.L. receives royalties from Arthrex, Ossur, and Smith & Nephew; is a paid consultant for Arthrex, Ossur, and Smith & Nephew; and receives research support from Arthrex, Smith & Nephew, Ossur, and Linvatec. Funding for this project was provided by the Steadman Philippon Research Institute in Vail, Colorado.

Objective: IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I., Design: Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes., Results: Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis., Conclusions: This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response., (Copyright © 2015. Published by Elsevier Ltd.)

Diseases of the musculoskeletal system are a major cause of loss of use and retirement in sport horses. The use of bone marrow-derived mesenchymal stem cells (BMDMSCs) for healing of traumatized tissue has gained substantial favor in clinical settings and can assist healing and tissue regeneration in orthopedic injuries. There are two common sites of harvest of BMDMSCs, the sternum and the ilium. Our objective was to determine if any differences exist in BMDMSCs acquired from the sternum and the ilium. We compared the two harvest sites in their propensity to undergo multilineage differentiation, differences in cell surface markers, or gene transduction efficiencies. BMDMSCs were isolated and culture-expanded from 5 ml aspirates of bone marrow from sternum and ilium. The cells were then plated and cultured with appropriate differentiation medium to result in multi-lineage differentiation and cell characteristics were compared between sternal and ilial samples. Cell surface antibody expression of CD11a/18, CD34, CD44, and CD90 were evaluated using flow cytometry, and gene transduction efficiencies were evaluated using GFP scAAV. There were no statistically significant differences in cell characteristics between MSCs cultured from the sternum and the ilium under any circumstances.

A gene therapeutic approach to treat osteoarthritis (OA) appears to be on the horizon for millions of people who suffer from this disease. Previously we described optimization of a scAAVIL-1ra gene therapeutic vector and initially tested this in an equine model verifying long-term intrasynovial IL-1ra protein at therapeutic levels. Using this vector, we carried out a dosing trial in six horses to verify protein levels and establish a dose that would express relevant levels of therapeutic protein for extended periods of time (8 months). A novel arthroscopic procedure used to detect green fluorescence protein (GFP) fluorescence intrasynovially confirmed successful transduction of the scAAVGFP vector in both the synovial and cartilage tissues. No evidence of intra-articular toxicity was detected. Immune responses to vector revealed development of neutralizing antibodies (Nabs) within 2 weeks of administration, which persisted for the duration of the study but did not lower protein expression intra-articularly. Re-dosing with a different serotype to attain therapeutic levels of protein confirmed establishment of successful transduction. This is the first study in an equine model to establish a dosing/redosing protocol, as well as examine the Nab response to capsid and supports further clinical investigation to determine the clinical efficacy of scAAVIL-1ra to treat OA.

Adeno-associated virus (AAV) receptors range from heparan sulfate proteoglycan to sialic acid moieties present on cell surfaces. Abundance of the glycan profiles is greatly influenced by animal species, cell type, and culture conditions. The objective of this study was to determine whether AAV serotypes' transduction efficiencies specifically in the equine monolayer culture model are an accurate representation of transduction efficiencies in tissue explants, a model more closely related to in vivo transduction. It was found that AAV 2 and 2.5 transduced cells more efficiently in explants than in monolayers. Through experiments involving assessing enzyme degradation of cell surface proteoglycans, this change could not be attributed to differences in the extra cellular matrix (ECM), but a similar change in AAV 5 transduction efficiency could be readily explained by differences in cell surface sialylated glycan. Unexpectedly it was found that in a small but diverse sample of horses evidence for serum neutralizing antibodies was only found to AAV 5. This suggests a unique relationship between this capsid and the equine host or an unresolved relationship between similar bovine AAV and the AAV 5 capsid immune response.

Background: Following various types of naturally-occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan and proteoglycan-4. However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown., Hypothesis: Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin+mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and proteoglycan-4 composition., Study Design: Controlled Laboratory Study., Methods: Equine synovial fluid was aspirated from normal joints at a pre-injury state (0 days) and at 10 days and 3 months following fibrin or fibrin+mesenchymal stem cell repair of full thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and proteoglycan-4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high-molecular weight hyaluronan to restore lubrication function., Results: Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to pre-injury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (-64%) and shifted towards lower molecular weight forms, while proteoglycan-4 concentration was increased (+210%) in 10-day samples relative to pre-injury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high molecular weight hyaluronan. Addition of high molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0mg/ml restored kinetic friction to pre-injury levels., Conclusion: Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high molecular weight hyaluronan., Clinical Relevance: Synovial fluid lubrication is deficient shortly following arthroscopic cartilage repair surgery, and supplementation with high molecular weight hyaluronan may be beneficial.

Objective: To compare the mesenchymal stem cell (MSC) yield and chondrogenic and osteogenic differentiation from 5- and 50-mL bone marrow aspirates from horses., Animals: Six 2- to 5-year-old mixed-breed horses. Procedures-2 sequential 5-mL aspirates were drawn from 1 ilium or sternebra. A single 50-mL aspirate was drawn from the contralateral ilium, and 2 sequential 50-mL aspirates were drawn from a second sternebra. The MSC yield was determined through the culture expansion process. Chondrogenesis and osteogenesis were evaluated by means of conventional laboratory methods., Results: The second of the 2 sequential 50-mL sternal aspirates yielded few to no MSCs. Independent of location, the highest density of MSCs was in the first of the 2 sequential 5-mL fractions, although with subsequent culture expansion, the overall yield was not significantly different between the first 5-mL and first 50-mL fractions. Independent of location, chondrogenesis and osteogenesis were not significantly different among fractions. Independent of fraction, the overall cell yield and chondrogenesis from the ilium were significantly higher than that from the sternum., Conclusions and Clinical Relevance: This study failed to detect an additional benefit of 50-mL aspirates over 5-mL aspirates for culture-expanding MSCs for equine clinical applications. Chondrogenesis was highest for MSCs from ilial aspirates, although it is not known whether chondrogenesis is indicative of activation of other proposed pathways by which MSCs heal tissues.

Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200-800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA.Molecular Therapy - Nucleic Acids (2013) 2, e70; doi:10.1038/mtna.2012.61; published online 5 February 2013.

Objective: To evaluate the effect of fibrin concentrations on mesenchymal stem cell (MSC) migration out of autologous and commercial fibrin hydrogels., Sample: Blood and bone marrow from six 2- to 4-year-old horses., Procedures: Autologous fibrinogen was precipitated from plasma and solubilized into a concentrated solution. Mesenchymal stem cells were resuspended in fibrinogen solutions containing 100%, 75%, 50%, and 25% of the fibrinogen precipitate solution. Fibrin hydrogels were created by mixing the fibrinogen solutions with MSCs and thrombin on tissue culture plates. After incubation for 24 hours in cell culture medium, the MSCs that had migrated onto the tissue culture surface and beyond the boundary of the hydrogels were counted. This procedure was repeated with a commercial fibrin sealant., Results: Hydrogel-to-surface MSC migration was detected for all fibrin hydrogels. Migration from the 25% autologous hydrogels was 7.3-, 5.2-, and 4.6-fold higher than migration from 100%, 75%, and 50% autologous hydrogels, respectively. The number of migrating cells from 100%, 75%, and 50% autologous hydrogels did not differ significantly. With commercial fibrin sealant, the highest magnitude of migration was from the 25% hydrogels, and it was 26-fold higher than migration from 100% hydrogels. The 75% and 50% hydrogels resulted in migration that was 9.5- and 4.2-fold higher than migration from the 100% hydrogels, respectively., Conclusions and Clinical Relevance: MSC migration from fibrin hydrogels increased with dilution of the fibrinogen component for both autologous and commercial sources. These data supported the feasibility of using diluted fibrin hydrogels for rapid delivery of MSCs to the surface of damaged tissues.

Cell transplantation for the treatment of joint disease is an important clinical tool. Genetic modification of cells before transplantation has shown enhanced healing. Ex vivo genetic modification of joint tissue cells with various adeno-associated virus (AAV) serotypes has not been investigated. The transduction efficiencies of self-complementary AAV serotypes (1-6 and 8) were determined in joint tissue containing chondrocytes and synoviocytes isolated from equine models. When comparing scAAV serotypes for efficient transduction ex vivo, in chondrocytes versus synoviocytes, serotypes 6 and 2, and serotypes 3 and 2, respectively, appeared superior for gene expression. Unlike adenoviral vectors, no upregulation of inflammatory markers, such as matrix metalloproteinases and aggrecanase, was seen on treatment of joint tissue with AAV vectors ex vivo. Our findings also corroborate that ex vivo transduction of joint tissue can result in high transgene protein levels over time, and transplantation modalities might be feasible using AAV vectors in the treatment of joint-related diseases.

Insulin-like growth factor-I (IGF-I) is one of the most influential growth factors in cartilage repair. Maintenance of adequate IGF-I levels after articular repair procedures is complicated by the short biological half-life of IGF-I in vivo. This study investigated the potential for more prolonged IGF-I delivery through direct adenoviral mediated transduction of synovial tissues in the metacarpophalangeal (MCP) joints of horses. The use of a large animal model provided a structurally similar and metabolically relevant corollary to the human knee. The complete IGF-I coding sequence was packaged into an E1-E3 deleted adenovirus-5 vector under cytomegalovirus promoter control (AdIGF-I), and injected at varying total joint doses to the MCP joints of 14 horses. Direct injection of 20 and 50 x 10(10) AdIGF-I resulted in significant elevations of IGF-I in synovial fluid for approximately 21 days. Synovial tissue taken from injected joints at day 35 following injection and compared to tissue taken preinjection from the same joints revealed elevated synoviocyte IGF-I mRNA levels for the highest viral dose by in situ hybridization and real-time PCR techniques. AdIGF-I injections did not result in significant lameness, joint effusion or elevated total protein concentrations in the synovial fluid. Mild mononuclear infiltration of white blood cells was evident in histologic sections of the synovium in the second highest adenoviral IGF-I dose of 20 x 10(10) particles. Cartilage biopsies taken from all injected joints did not reveal any significant changes in proteoglycan levels nor in histological morphology, which included chondrocyte cloning, architecture, cell type or toluidine blue staining, when compared to control joints. Based on these findings, gene transfer of IGF-I to the synovium of joints can result in significant and persistent elevations of IGF-I ligand in synovial fluid with minimal detrimental effects. Direct IGF-I gene therapy may offer a simple approach in treating patients with acute cartilage injury.

Background: Chondral defects may lead to degradative changes in the surrounding cartilage, predisposing patients to developing osteoarthritis., Purpose: To quantify changes in the biomechanical and biochemical properties of the articular cartilage adjacent to chondral defects after experimental defect repair., Study Design: Controlled laboratory study., Methods: Specimens were harvested from tissue within (lesion), immediately adjacent to, and at a distance from (remote area) a full-thickness cartilage defect 8 months after cartilage repair with genetically modified chondrocytes expressing insulin-like growth factor-I or unmodified, control chondrocytes. Biomechanical properties, including instantaneous Young's and equilibrium aggregate moduli, were determined by confined compression testing. Biochemical properties, such as water and proteoglycan content, were also measured., Results: The instantaneous Young's modulus, equilibrium modulus, and proteoglycan content increased, whereas water content decreased with increasing distance from the repaired lesion. The instantaneous Young's and equilibrium moduli of the adjacent articular cartilage were 80% and 50% that of remote area samples, respectively, whereas water content increased 0.9% and proteoglycan content was decreased by 35%. No significant changes in biomechanical and biochemical properties were found either in the lesion tissue or in adjacent cartilage with genetic modification of the chondrocytes., Conclusion: Articular cartilage adjacent to repaired chondral defects showed significant remodeling 8 months after chondral defect repair, regardless of whether genetically modified or unmodified cells were implanted., Clinical Relevance: Changes in the biochemical and biomechanical properties of articular cartilage adjacent to repaired chondral defects may represent remodeling as part of an adaptive process or degeneration secondary to an altered distribution of joint forces. Quantification of these changes could provide important parameters for assessing progress after operative chondral defect repair.

Objective: To determine clinical and ultrasonographic abnormalities in horses with primary desmitis of the palmar or plantar annular ligament (PAL) and the response to treatment., Design: Retrospective study., Animals: 25 horses., Procedure: Data collected from medical records included signalment, horse use, affected limb, severity and duration of lameness, results of diagnostic tests performed, ultrasonographic findings, treatment, and outcome., Results: All horses had a prominent swelling in the region of the affected PAL, and signs of pain were evident during palpation of the swelling. In all horses, the affected PAL was thicker than normal as determined ultrasonographically. Twenty horses had hypoechoic regions in the PAL. Four horses, including 1 horse with 3 affected limbs, were treated by means of PAL desmoplasty, 9 were treated by means of PAL desmotomy, and 12 were treated with rest. Follow-up information was available for 21 horses. All 4 horses that underwent PAL desmoplasty, 4 of 7 horses that underwent PAL desmotomy, and 7 of 10 horses treated with rest alone became sound., Conclusions and Clinical Relevance: Results suggest that primary desmitis of the PAL may be a cause of lameness in horses. Although significant differences in outcome between horses treated with rest alone versus PAL desmoplasty versus PAL desmotomy were not identified, because it is less invasive, PAL desmoplasty should be considered for treatment of horses with PAL desmitis that do not respond to conservative treatment and do not have any evidence of constriction of the digital flexor tendons.

Cell-based cartilage-resurfacing procedures may be enhanced by the addition of insulin-like growth factor I (IGF-I) to the transplant biomatrix. Given the relatively short half-life of IGF-I in biological systems, however, maintenance of effective concentrations of this peptide necessitates either high initial doses, or repeated treatment. This study investigated IGF-I delivery via adenoviral gene therapy, targeting graftable articular chondrocytes. Cultured articular chondrocytes were infected with an E1-deleted adenoviral vector containing IGF-I-coding sequence under CMV promoter control. Increased adenovirus-IGF-I concentrations resulted in coordinate increase in IGF-I mRNA and ligand expression; however, chondrocyte matrix synthesis was maximized by the lower adenovirus-IGF-I concentration (100 MOI) without additional increase at 200 or 500 MOI. Using 100 MOI, infected monolayers produced medium IGF-I content of at least 10 ng/ml in each 48-hr period for 28 days, reaching a day 4 peak concentration of 66 +/- 4.0 ng/ml. These concentrations were sufficient to produce significant stimulation of normal cartilage matrix gene expression. The concentration of secreted matrix products in medium from infected monolayers was increased up to 8-fold over uninfected control cultures. Moreover, compared with uninfected cultures, cells in infected cultures were more resistant to de-differentiation over time under serum-starved conditions, maintaining a normal chondrocyte molecular phenotype for at least 28 days. These data indicate that cultured chondrocytes are readily transduced by recombinant adenoviral vectors. The adenoviral-IGF transgene is abundantly expressed and its product secreted at therapeutic concentrations for at least 28 days, resulting in increased matrix biosynthesis and maintenance of the chondrocytic phenotype. Combined, this information suggests that there may be significant value in preimplantation adenoviral-IGF gene therapy for chondrocytes destined for cartilage resurfacing.

Objective: To evaluate healing of pinch-grafted wounds on the distal aspect of the limbs of ponies bandaged with equine amnion or a standard nonadherent wound dressing material., Animals: 6 ponies., Procedure: A 2.5x2.5-cm full-thickness section of skin was removed from the dorsal aspect of each limb at the midpoint of the metacarpus or metatarsus. Six days later, wounds were grafted with partial-thickness pinch grafts. Half the wounds were bandaged with amnion, and the other half were bandaged with a nonadherent dressing. Bandages were changed every 3 days until wound healing was complete. At each bandage change, numbers of grafts lost were recorded, and wounds were measured., Results: Percentage of grafts lost from wounds bandaged with amnion was not significantly different from percentage lost from wounds bandaged with the nonadherent dressing. Median healing time for wounds bandaged with amnion (30 days) was significantly less than median healing time for wounds bandaged with the nonadherent dressing (39 days). All wounds were healed by day 45., Conclusions and Clinical Relevance: Results suggest that amnion can be used for bandaging pinch-grafted wounds on the distal aspect of the limbs of ponies.

Objective: To determine clinical and radiographic findings in and treatment and outcome of large animals with shoulder joint luxations., Design: Retrospective study., Animals: 5 horses, 3 goats, 1 calf, 1 sheep, 1 Himalayan tahr, 1 pot-bellied pig, 1 reindeer, and 1 white-tailed deer., Procedure: Medical records and radiographs were reviewed to determine signalment, history, physical examination findings, type of luxation, treatment, and outcome. Owners and referring veterinarians were contracted for follow-up information., Results: Goats, sexually intact males, and animals < 1 year old were overrepresented, compared with the general hospital population during the study period. Closed reduction was attempted in 3 animals and was successful in 1. Open reduction and internal stabilization was attempted in 4 animals, including 1 in which closed reduction was unsuccessful. Long-term stabilization of the joint was achieved in 3 animals, but overall results were poor because of osteoarthritis and chronic lameness. Three animals were not treated, and 5 were euthanatized because of a poor prognosis., Clinical Implications: Large animals with shoulder joint luxation and concurrent fractures had a poorer prognosis than did those with shoulder joint luxation alone.

With the exception of lipoma, neoplasia of the gastrointestinal tract is rare in horses. Lymphosarcoma is the most common neoplasm of the hematopoietic system in horses. In horses with lymphosarcoma of the large colon, clinical signs may include intermittent signs of mild abdominal pain, weight loss, pyrexia, and pelvic flexure impaction caused by impingement of the colonic lumen by the mass. Peritoneal fluid analysis may be normal or have a high total protein concentration. If signs of metastasis are not evident, resection of the large colon affected by the mass may prolong survival.