Equine sarcoid of the glans penis with bovine papillomavirus type 1 in a miniature horse (Falabella).

A 23-year-old Falabella gelding kept in Tochigi, Japan, for more than 20 years presented with a recurrent mass of the glans penis that was first noticed about a year earlier. Partial phallectomy was performed with no adjunctive therapy for local regrowth of the mass. The horse was euthanized 3 months after surgery for urinary retention due to suspected regrowth. The resected mass affected the genital and urethral mucosa of the glans penis, and was diagnosed as equine sarcoid by histopathology and identification of bovine papillomavirus (BPV) DNA. Phylogenetic analysis of the BPV genome of the sarcoid showed high sequence homology to BPV type 1 (BPV-1) from Hokkaido, Japan, suggesting a geographical relationship for BPV-1 in Japan.

Risk factors for superficial digital flexor tendinopathy in Thoroughbred racing horses in Japan.

Thoroughbred racehorses are commonly affected with superficial digital flexor (SDF) tendinopathy. This study aimed to identify risk factors for SDF tendinopathy in racing horses. The authors selected racehorses (n=292) with SDF tendinopathy from the medical records of a racetrack. As a risk factor associated with track-related variables, the SDF tendinopathy odds ratio (OR) was significantly high for a sloppy track surface compared with a standard track surface. Regarding risk factors associated with race-related variables, the SDF tendinopathy OR was significantly high in the following cases: when the order of arrival was worse than or equal to the 10th place; when the racehorses started to run a short race and when the racehorses' favourites were worse than or equal to the 8th place. Regarding risk factors associated with racehorse-related variables, the body weight of racehorses with SDF tendinopathy was significantly heavier than that of control horses. When there was a decrease in body weight since previous racing, the SDF tendinopathy OR was significantly high. Regarding risk factors associated with race career-related variables, when the charge in the race distance was short, the SDF tendinopathy OR was significantly high. As a countermeasure to prevent SDF tendinopathy, a sloppy track surface should be avoided during the race by guiding the horse toward to more solid track surface. Selecting long-distance races with slow speed, if possible, could reduce the risk of SDF tendinopathy.

Displacement of the large colon in a horse with enterolithiasis due to changed positions observed by computed tomography.

Computed tomography (CT) was performed for an 18-year-old female pony with enterolithiasis in the prone and supine positions. CT images from the prone position revealed displacement of the large dorsal colon, which contained an enterolith to the ventral side of the abdomen, and those from the supine position revealed displacement to the dorsal side. A high-density material suggestive of a metallic foreign body was also observed in the enterolith core. An enterolith (422 g, 104 mm) was surgically removed from the large dorsal colon. This caused no complications after surgery and increased the horse's weight. Changing positions during CT helps identify the exact location of enterolith and intestinal displacement due to enterolith weight, as well as size and number.

Evaluation of the ability of a gravitational filtration system to enhance recovery of equine bone marrow elements.

OBJECTIVE: To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells. ANIMALS: 12 healthy adult horses. PROCEDURES: Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility complex (MHC) class I and II antigens. RESULTS: Mean cell viability of harvested bone marrow was 95.9%. Total WBCs and platelets were efficiently captured on the filter (> 95%), and mean recovery in harvested bone marrow was 30%. Cytologic cell differential counts indicated that the percentage of neutrophils was significantly less and the progenitor cell population was significantly higher and concentrated 1.56-fold in harvested bone marrow, compared with results for raw bone marrow. Flow cytometry and cell culture were used to characterize harvested bone marrow cells as positive for expression of CD90 and negative for MHCI and MHCII, which indicated stem cells with a multipotent phenotype that differentiated into chondrocytes, osteocytes, adipocytes, and tenocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Gravitational filtration of bone marrow efficiently yielded platelets and cells and produced a progenitor-enriched, leukocyte-reduced product, compared with raw bone marrow.

Autologous implantation of BMP2-expressing dermal fibroblasts to improve bone mineral density and architecture in rabbit long bones.

Cell-mediated gene therapy may treat bone fragility disorders. Dermal fibroblasts (DFb) may be an alternative cell source to stem cells for orthopedic gene therapy because of their rapid cell yield and excellent plasticity with bone morphogenetic protein-2 (BMP2) gene transduction. Autologous DFb or BMP2-expressing autologous DFb were administered in twelve rabbits by two delivery routes; a transcortical intra-medullar infusion into tibiae and delayed intra-osseous injection into femoral drill defects. Both delivery methods of DFb-BMP2 resulted in a successful cell engraftment, increased bone volume, bone mineral density, improved trabecular bone microarchitecture, greater bone defect filling, external callus formation, and trabecular surface area, compared to non-transduced DFb or no cells. Cell engraftment within trabecular bone and bone marrow tissue was most efficiently achieved by intra-osseous injection of DFb-BMP2. Our results suggested that BMP2-expressing autologous DFb have enhanced efficiency of engraftment in target bones resulting in a measurable biologic response by the bone of improved bone mineral density and bone microarchitecture. These results support that autologous implantation of DFb-BMP2 warrants further study on animal models of bone fragility disorders, such as osteogenesis imperfecta and osteoporosis to potentially enhance bone quality, particularly along with other gene modification of these diseases.

Effect of Intra-Medullar and Intra-Venous Infusions of Mesenchymal Stem Cells on Cell Engraftment by In-Vivo Cell Tracking and Osteoinductivity in Rabbit Long Bones: A Pilot Study.

OBJECTIVE: Stem cell therapy can be an efficacious treatment option for bone fragility disorders (eg, osteogenesis imperfecta, disuse osteopenia, and osteoporosis), and successful cell therapy application may be dependent on optimal cell engraftment in target bones. The objective of this study was to compare the efficiency of intra-medullar and intra-venous delivery of mesenchymal stem cells (MSC) to improve cell engraftment rate, bone mineral density, and micro-architecture. METHODS: By using six healthy juvenile New Zealand White rabbits, MSC were isolated from cancellous bone harvests and confirmed to have osteogenic capacity by inducing ectopic bone formation. The MSC were cultured, transduced by foamy viral vectors with marker genes for in vivo cell tracking, and expanded. All rabbits had one randomly selected limb receive intra-medullar infusion of 3×107 to 1×108 autologous MSC in the distal femur or the distal femur and proximal tibia. Two of six rabbits also received an intra-venous MSC infusion. At 28 days, MSC bone engraftment was assessed by PCR and the bone density and microstructure assessed by computed tomography and histomorphometry. RESULTS: The intra-medullar-infused MSC were detected in epiphysis or diaphysis of the distal femurs and/or proximal tibiae. Infused MSC comprised 0.01 to 0.3% of all cells in the bone tissues. The intra-venous-infused MSC were not detected in any location. Neither intra-medullar nor intra-venous MSC infusion altered bone volume, bone mineral density, or cortical bone porosity/thickness. Systemic biodistribution of intra-medullar-infused MSC was not evident. CONCLUSIONS: Our results indicated that intra-medullar infusion can be an effective cell delivery route for stem cell therapy potentially for orthopedic disorders, in preference to systemic administration. Further research is warranted to demonstrate an efficacy of intra-medullar MSC infusion on bone density and micro-architecture using animal models of bone disorders.

Analgesic effects of intraneural injection of ethyl alcohol or formaldehyde in the palmar digital nerves of horses.

OBJECTIVE: To determine analgesic effects of intraneural injection of ethyl alcohol or formaldehyde in the palmar digital nerves of horses. ANIMALS: 6 horses. PROCEDURES: Ethyl alcohol was injected in the medial palmar digital nerve of 1 forelimb, and formaldehyde was injected in the contralateral nerve. The lateral palmar digital nerve in 1 forelimb was surgically exposed, but not injected, and the contralateral lateral palmar digital nerve was not treated. For each heel, severity of lameness in response to experimentally induced heel pain (lameness score and peak vertical force), thermal reaction time, and heel skin sensitivity scores were recorded. Heel pain was experimentally induced by advancing a threaded bolt through a custom-made horseshoe to apply pressure to the palmar horned aspect of the hoof. Horses were followed up for 112 days, when a subset of nerves was sampled for histologic analysis. RESULTS: Alcohol and formaldehyde significantly reduced all measures of heel pain, and analgesia was evident over the 112 days of the study. Pastern circumference was significantly greater for formaldehyde-treated than for alcohol-treated limbs. Histologic evaluation showed preservation of nerve fiber alignment with an intact epineurium, loss of axons, axon degeneration, fibrosis, and inflammation in alcohol-treated and formaldehyde-treated nerves. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that intraneural injection of either ethyl alcohol or formaldehyde in the palmar digital nerves of horses resulted in substantial, but partial, heel analgesia that persisted for at least 112 days. No advantage of formaldehyde over alcohol was found, and formaldehyde resulted in greater soft tissue inflammation.

Evaluation of a single intra-articular injection of autologous protein solution for treatment of osteoarthritis in horses.

OBJECTIVE: To evaluate intra-articular autologous protein solution (APS) for the treatment of osteoarthritis in horses. Animals-40 client-owned horses with naturally occuring osteoarthritis. PROCEDURES: APS was generated from a dual-device system that concentrated plasma and WBC proteins and enriched platelet growth factors. Horses were randomly assigned to receive an intra-articular injection of 5 mL of saline (0.9% NaCl) solution (n = 20) or APS (20), exercised on a treadmill, and evaluated on the basis of lameness grades, kinetic gait analysis, joint circumference, and range of motion for 14 days. Horses that received saline solution were administered APS at termination of the study, and clients scored horses for lameness and discomfort before, 12 weeks after, and 52 weeks after the APS injection. RESULTS: The APS group had significant improvements in lameness grade, asymmetry indices of vertical peak force, and range of joint motion by 14 days, compared with baseline or control group values. No adverse effects associated with APS treatment were evident. Clients assessed lameness and comfort as improved at 12 and 52 weeks. The APS had greater likelihood (OR, 4.3 to 30.0) of a therapeutic response in horses with a lameness score < 4, < 10% vertical force asymmetry, or absence of marked osteophyte formation, subchondral sclerosis, or joint space narrowing. Concentration of interleukin-1 receptor antagonist in APS was 5.8 times that in blood. CONCLUSIONS AND CLINICAL RELEVANCE: Intra-articular administration of APS can be considered an effective treatment option for equine osteoarthritis, with the potential for disease-modifying effects.

Investigation of the immune response to autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses.

Mesenchymal stem cells have demonstrated immunomodulatory capabilities as well as modest efficacy in animal models of joint injury, warranting further study as a potential treatment of joint disease. The goal of the study was to investigate the blood and synovial immune and histologic response to intra-articular injection of autologous, allogeneic, and xenogeneic bone marrow-derived mesenchymal stem cells (MSC) in horses. The study group consisted of 6 five-year-old Thoroughbred mares that had been injected previously with 15 million, genetically modified autologous, allogeneic, or xenogeneic MSC into the fetlock joints. One group of autologous cells was genetically modified to permit MSC biolocalization in the synovium. To assess response to the injection, synovial biopsies were obtained via arthroscopy 60 days after MSC injection for gross, histologic and molecular analyses. Peripheral blood mononuclear cells were isolated from each horse 120 days after MSC injection and co-cultured with a monolayer of each MSC group to permit quantification of activated CD4+ lymphocytes and cytokine release (ELISA) upon re-exposure to MSC. Arthroscopic examination revealed normal synovium with no grossly detrimental effect to the synovium or cartilage. Intra-articular MSC produced a persistent mononuclear infiltrate for at least 60 days, mostly perivascular, identified as CD3+ lymphocytes. An immune response (significant increase in CD4+ lymphocytes) was detected upon re-exposure to xenogeneic but not to allogeneic or autologous MSC. An inflammatory cytokine release from peripheral blood mononuclear cell/MSC co-cultures was present in all MSC groups but was significantly greater in the xenogeneic group. In conclusion, intra-articular injection of MSC, regardless of cell origin, incited a persistent mononuclear synovitis demonstrating a sustained biologic influence of these cells. Allogeneic cells did not elicit a detectable immune response upon re-exposure using our methods.

Performance of a gravitational marrow separator, multidirectional bone marrow aspiration needle, and repeated bone marrow collections on the production of concentrated bone marrow and separation of mesenchymal stem cells in horses.

Objective-To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses. Animals-8 healthy adult horses. Procedures-Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, MSCs, and cell viability. Results-Concentrated bone marrow samples obtained with the marrow separator had 5- to 19-fold bone marrow-derived MSC, WBC, and platelet counts, compared with original bone marrow samples. Use of a multidirectional needle increased the frequency of obtaining MSC-richer concentrated bone marrow. Repeating bone marrow aspiration at 1 month yielded greater MSC numbers but slightly lower cell viability after processing. Conclusions and Clinical Relevance-The gravitational bone marrow separator and multidirectional needle were used to effectively harvest bone marrow and improve the quality of concentrated bone marrow. Comparable, or even greater, numbers of bone marrow-derived MSCs were collected by repeated bone marrow aspiration after a 1-month interval from the same aspiration sites. Use of the marrow separator and multidirectional bone marrow aspiration needle can facilitate a 1-step, point-of-care, nonlaboratory method to obtain concentrated bone marrow as a mixture of bone marrow-derived MSCs and growth factors from platelets and plasma.

Cell-mediated and direct gene therapy for bone regeneration.

INTRODUCTION: Bone regeneration is required for the treatment of fracture non/delayed-unions and bone defects. However, most current treatment modalities have limited efficacy, and newer therapeutic strategies, such as gene therapy, have substantial benefit for bone repair and regeneration. AREAS COVERED: This review discusses experimental and clinical applications of cell-mediated and direct gene therapy for bone regeneration. The review covers literature on this subject from 2000 to February 2012. EXPERT OPINION: Direct gene therapy using various viral and non-viral vectors of cell-mediated genes has been demonstrated to induce bone regeneration, although use of such vectors has shown some risk in human application. Osteoinductive capability of a number of progenitor cells isolated from bone marrow, fat, muscle and skin tissues, has been demonstrated by genetic modification with osteogenic genes. Cell-mediated gene therapy using such osteogenic gene-expressing progenitor cells has shown promising results in promoting bone regeneration in extensive animal work in recent years.

Inflammation and immune response of intra-articular serotype 2 adeno-associated virus or adenovirus vectors in a large animal model.

Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad), serotype 2 adeno-associated virus vectors (rAAV2), or self-complementary (sc) AAV2 vectors carrying green fluorescent protein (GFP). Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb) titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.

Dermal fibroblast-mediated BMP2 therapy to accelerate bone healing in an equine osteotomy model.

This study evaluated healing of equine metacarpal/metatarsal osteotomies in response to percutaneous injection of autologous dermal fibroblasts (DFbs) genetically engineered to secrete bone morphogenetic protein-2 (BMP2) or demonstrate green fluorescent protein (GFP) gene expression administered 14 days after surgery. Radiographic assessment of bone formation indicated greater and earlier healing of bone defects treated with DFb with BMP2 gene augmentation. Quantitative computed tomography and biomechanical testing revealed greater mineralized callus and torsional strength of DFb-BMP2-treated bone defects. On the histologic evaluation, the bone defects with DFb-BMP2 implantation had greater formation of mature cartilage and bone nodules within the osteotomy gap and greater mineralization activity on osteotomy edges. Autologous DFbs were successfully isolated in high numbers by a skin biopsy, rapidly expanded without fastidious culture techniques, permissive to adenoviral vectors, and efficient at in vitro BMP2 protein production and BMP2-induced osteogenic differentiation. This study demonstrated an efficacy and feasibility of DFb-mediated BMP2 therapy to accelerate the healing of osteotomies. Skin cell-mediated BMP2 therapy may be considered as a potential treatment for various types of fractures and bone defects.

Radiofrequency energy on cortical bone and soft tissue: a pilot study.

BACKGROUND: Radiofrequency-generating energy devices have been used clinically in musculoskeletal procedures to provide hemostasis and capsular shrinkage (thermal capsulorrhaphy). However, the dose-effects are not well known. QUESTIONS/PURPOSES: We therefore determined dosage effects of radiofrequency energy on bone, skin incisions, and joint capsule in sheep. METHODS: Five mature sheep had six 2.5-cm(2) tibial periosteal defects and six 1.0-cm skin incisions assigned to six treatments varying by watts and fluence (f = watts . seconds/cm(2)): (1) untreated control, (2) 50 W for 9.5 seconds (190f; n = 5), (3) 110 W for 4.3 seconds (190f; n = 5), (4) 170 W for 2.8 seconds (190f; n = 5), (5) 170 W for 5.6 seconds (380f; n = 5), or (6) 170 W for 8.4 seconds (570f; n = 5). Outcomes included hemostasis, contraction, healing, and histomorphometry for inflammation and necrosis at 2 weeks. RESULTS: Radiofrequency energy application on skin at 190f or greater had more than 80% hemostasis and dose-dependent contraction, inflammation, and necrosis. Radiofrequency energy application on bone had good (70%) hemostasis at 190f and complete (> 95%) hemostasis at 380f and 570f, without histologic or clinically detectable necrosis. CONCLUSIONS: Hemostasis can be achieved with radiofrequency energy at 190f in skin and bone. Bone necrosis was not detected at up to 570f. Using fluence greater than 190f in skin achieved dose-dependent necrosis and incisional contraction. CLINICAL RELEVANCE: Radiofrequency energy can be used on bone and skin for hemostasis, but potential incisional complications, such as necrosis and an atypical firm and desiccated surface, should be expected.

Use of kinetic gait analysis for detection, quantification, and differentiation of hind limb lameness and spinal ataxia in horses.

OBJECTIVE: To evaluate use of kinetic gait analysis for detection, quantification, and differentiation of hind limb lameness and spinal ataxia in horses. DESIGN: Prospective clinical study. ANIMALS: 36 horses. Procedures-Kinetic gait analysis with a force plate was performed for 12 clinically normal horses, 12 horses with hind limb lameness, and 12 horses with spinal ataxia. Kinetic variables were compared among groups, correlated to subjective grading, and used to build predictive models to assess the accuracy of discrimination. RESULTS: Subsets of kinetic variables were characteristically altered in ataxic and lame gaits. Ataxic horses had significantly increased lateral force peak and variation in vertical force peaks in both hind limbs. Lame horses had significantly decreased vertical force peak and increased variation in vertical force peaks only in the lame hind limb. These variables were used to differentiate between spinal ataxia and hind limb lameness with excellent accuracy. There were significant correlations between a subset of kinetic variables and subjective lameness and neurologic grades. CONCLUSIONS AND CLINICAL RELEVANCE: Kinetic gait variables, specifically lateral force peak and the variation in vertical force, can be used to support the differential diagnosis between spinal ataxia and hind limb lameness in horses. Kinetic gait analysis may also be applied for quantification of equine hind limb gait abnormalities as well as confirming lack of lameness and ataxia in soundness examinations.

Anti-inflammatory and analgesic effects of intra-articular injection of triamcinolone acetonide, mepivacaine hydrochloride, or both on lipopolysaccharide-induced lameness in horses.

OBJECTIVE: To assess analgesia, inflammation, potency, and duration of action associated with intra-articular injection of triamcinolone acetonide (TA), mepivacaine hydrochloride, or both in metacarpophalangeal (MCP) joints of horses with experimentally induced acute synovitis. ANIMALS: 18 horses. PROCEDURES: Both forelimbs of each horse were injected with lipopolysaccharide (LPS) 3 times. After the first LPS injection, 1 forelimb of each horse was treated with intra-articular injection of mepivacaine (80 mg; n=6), TA (9 mg; 6), or mepivacaine with TA (same doses of each; 6) 12 hours after the initial LPS injection. Contralateral limbs served as control limbs. Joint pain was assessed via lameness score and measurements of vertical force peak and pain-free range of motion of the MCP joint. Periarticular edema was evaluated. Degree of synovial inflammation was determined via synovial fluid analysis for WBC count and total protein concentration. Samples of plasma and synovial fluid were analyzed for TA and mepivacaine concentrations. RESULTS: Each injection of LPS induced lameness and joint inflammation. Mepivacaine effectively eliminated lameness within 45 minutes after injection, regardless of whether TA was also administered, whereas TA reduced lameness, edema, and concentration of synovial fluid protein after the second LPS injection, regardless of whether mepivacaine was also injected. Treatment with TA also induced higher WBC counts and mepivacaine concentrations in synovial fluid, compared with results for mepivacaine alone. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested TA is a potent analgesic and anti-inflammatory medication for acute synovitis in horses and that simultaneous administration of mepivacaine does not alter the potency or duration of action of TA.

Effects of triamcinolone acetonide, sodium hyaluronate, amikacin sulfate, and mepivacaine hydrochloride, alone and in combination, on morphology and matrix composition of lipopolysaccharide-challenged and unchallenged equine articular cartilage explants.

OBJECTIVE: To evaluate the effects of triamcinolone acetonide (TA), sodium hyaluronate (HA), amikacin sulfate (AS), and mepivacaine hydrochloride (MC) on articular cartilage morphology and matrix composition in lipopolysaccharide (LPS)-challenged and unchallenged equine articular cartilage explants. SAMPLE POPULATION: 96 articular cartilage explants from 4 femoropatellar joints of 2 adult horses. PROCEDURES: Articular cartilage explants were challenged with LPS (100 ng/mL) or unchallenged for 48 hours, then treated with TA, HA, AS, and MC alone or in combination for 96 hours or left untreated. Cartilage extracts were analyzed for glycosaminoglycan (GAG) content by dimethyl-methylene blue assay (ng/mg of dry wt). Histomorphometric quantification of total lacunae, empty lacunae, and lacunae with pyknotic nuclei was recorded for superficial, middle, and deep cartilage zones. RESULTS: LPS induced a significant increase in pyknotic nuclei and empty lacunae. Treatment with TA or HA significantly decreased empty lacunae (TA and HA), compared with groups without TA or HA, and significantly decreased empty lacunae of LPS-challenged explants, compared with untreated explants. Treatment with AS or MC significantly increased empty lacunae in unchallenged explants, and these effects were attenuated by TA. Treatment with MC significantly increased empty lacunae and pyknotic nuclei and, in combination with LPS, could not be attenuated by TA. Content of GAG did not differ between unchallenged and LPS-challenged explants or among treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with TA or HA supported chondrocyte morphology in culture and protected chondrocytes from toxic effects exerted by LPS, AS, and MC.

Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models.

This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites.

Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery.

OBJECTIVE: To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow-derived mesenchymal stem cells (BMD-MSCs). SAMPLE POPULATION: Articular cartilage, synovium, and bone marrow from 15 adult horses. PROCEDURES: Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1-deficient adenovirus vector encoding the beta-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1-deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection. RESULTS: -Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors. CONCLUSION AND CLINICAL RELEVANCE: Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.

Association between subjective lameness grade and kinetic gait parameters in horses with experimentally induced forelimb lameness.

OBJECTIVE: To evaluate the association between subjective lameness grades and kinetic gait parameters and assess the variability in kinetic parameters in horses with experimentally induced forelimb lameness. ANIMALS: 32 horses. PROCEDURES: Forelimb lameness was induced in each horse via injection of lipopolysaccharide into 1 metacarpophalangeal joint (40 experimental trials). Subjective lameness grading and 13 kinetic gait parameters (force plate analysis) were assessed before (baseline) and at 12, 18, and 24 hours after lipopolysaccharide injection. While horses were trotting, kinetic gait analysis was performed for 8 valid repetitions at each time point. Repeated-measures analyses were performed with 8 repetitions for each kinetic parameter as the outcome, and lameness grades, time points after lipopolysaccharide injection, and repetition order as explanatory variables. Sensitivity and specificity of kinetic parameters for classification of horses as sound or lame (in relation to subjective lameness scores) were calculated. Between- and within-horse variabilities of the 13 kinetic parameters were assessed by calculation of coefficients of variation. RESULTS: Subjective lameness grades were significantly associated with most of the kinetic parameters. Vertical force peak and impulse had the lowest between- and within-horse coefficients of variation and the highest correlations with subjective lameness grade. Vertical force peak had the highest sensitivity and specificity for lameness classification. Vertical force peak and impulse were significantly decreased even in horses with mild or unobservable lameness. CONCLUSIONS AND CLINICAL RELEVANCE: Among the kinetic gait parameters, vertical force peak and impulse had the best potential to reflect lameness severity and identify subclinical forelimb gait abnormalities.