Akikazu Ishihara

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.

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.

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.

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.

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.

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.

Inflammatory effects of autologous, genetically modified autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses

OBJECTIVES To compare the clinical and inflammatory joint responses to intra-articular injection of bone marrow-derived mesenchymal stem cells (MSC) including autologous, genetically modified autologous, allogeneic, or xenogeneic cells in horses. METHODS Six five-year-old Thoroughbred mares had one fetlock joint injected with Geys balanced salt solution as the vehicle control. Each fetlock joint of each horse was subsequently injected with 15 million MSC from the described MSC groups, and were assessed for 28 days for clinical and inflammatory parameters representing synovitis, joint swelling, and pain. RESULTS There were not any significant differences between autologous and genetically modified autologous MSC for synovial fluid total nucleated cell count, total protein, interleukin (IL)-6, IL-10, fetlock circumference, oedema score, pain-free range-of-motion, and soluble gene products that were detected for at least two days. Allogeneic and xenogeneic MSC produced a greater increase in peak of inflammation at 24 hours than either autologous MSC group. CLINICAL SIGNIFICANCE Genetically engineered MSC can act as vehicles to deliver gene products to the joint; further investigation into the therapeutic potential of this cell therapy is warranted. Intra-articular MSC injection resulted in a moderate acute inflammatory joint response that was greater for allogeneic and xenogeneic MSC than autologous MSC. Clinical management of this response may minimize this effect.

Comparative efficacy of dermal fibroblast-mediated and direct adenoviral bone morphogenetic protein-2 gene therapy for bone regeneration in an equine rib model.

Cell-mediated and direct adenoviral (Ad) vector gene therapies can induce bone regeneration, including dermal fibroblasts (DFbs). We compared two effective therapies, DFb-mediated and direct Ad vector delivery of bone morphogenetic protein-2 (BMP2), for relative efficacy in bone regeneration. Equine rib drill defects were treated by percutaneous injection of either DFb-BMP2 or an Ad-BMP2 vector. At week 6, both DFb-BMP2- and Ad-BMP2-treated rib defects had greater bone filling volume and mineral density, with DFb-BMP2 inducing greater bone volume and maturity in the cortical bone aspect of the defect than Ad-BMP2. The transplantation of DFb alone induced modest bone formation. Increased mineral density and bone turnover were evident in the cortical and cancellous bone directly adjacent to the healing drill defects treated with either DFb-BMP2 or Ad-BMP2. Using our cell/vector dosage and model, BMP2, whether delivered by the DFb vector or direct Ad vector, induced greater and robust bone regeneration. DFb-mediated BMP2 therapy promoted greater cortical bone regeneration than did direct gene delivery, possibly because of an increased cellularity of the bone healing site. BMP2 delivery, regardless of gene delivery method, increased the mineral density of the neighboring bone, which may be beneficial clinically in repairing or weak bone.

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.

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.