Alicia L. Bertone

Intralesional injection of platelet-rich plasma followed by controlled exercise for treatment of midbody suspensory ligament desmitis in Standardbred racehorses

OBJECTIVE To determine outcome of Standardbred racehorses with moderate to severe midbody suspensory ligament desmitis (MSD) treated by means of ultrasound-guided intralesional injection of a single dose of platelet-rich plasma (PRP) followed by a program of gradually increased exercise. DESIGN Nonrandomized clinical trial. ANIMALS 9 Standardbred racehorses. PROCEDURES Following injection of PRP, horses were allowed a controlled, gradual return to exercise. Race records for the year prior to injury and for 3 consecutive years after horses returned to racing were reviewed. For comparison purposes, race records of 9 Standardbred racehorses with no history of MSD racing at the same time were also reviewed. RESULTS All 9 horses with MSD returned to racing after treatment; median time to return to racing was 32 weeks. All 9 horses raced at least once during the first and second years after returning to racing, but only 5 raced during the third year. When number of starts, total earnings, and earnings per start were compared between case and comparison horses, the only significant differences were number of starts during the third year after case horses returned to racing and earnings per start during the first year after case horses returned to racing, with values being significantly lower for case horses than for comparison horses. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that horses with moderate to severe MSD treated by means of intralesional injection of a single dose of PRP followed by a program of gradually increased exercise had an excellent prognosis for returning to racing.

Comparison of efficacy and safety of paste formulations of firocoxib and phenylbutazone in horses with naturally occurring osteoarthritis

OBJECTIVE To compare efficacy and safety of paste formulations of firocoxib and phenylbutazone in horses with naturally occurring osteoarthritis. DESIGN Randomized controlled clinical trial. ANIMALS 253 client-owned horses with naturally occurring osteoarthritis. PROCEDURES Horses were treated with firocoxib (0.1 mg/kg [0.045 mg/lb], PO, q 24 h) or phenylbutazone (4.4 mg/kg [2 mg/lb], PO, q 24 h) for 14 days. Physical examinations and lameness evaluations were performed prior to treatment and after 7 and 14 days. Clinical improvement was defined as a reduction of at least 1 lameness grade or a combined reduction of at least 3 points in scores for pain during manipulation or palpation, joint swelling, joint circumference, and range of motion. RESULTS Proportion of horses clinically improved on day 14 for the firocoxib group (104/123 [84.6%]) was not significantly different from the proportion for the phenylbutazone group (103/119 [86.6%]). Proportion of horses that were improved on day 14 was significantly greater for horses treated with firocoxib than for horses treated with phenylbutazone with regard to score for pain on manipulation or palpation (P = 0.028), joint circumference score (P = 0.026), and range of motion score (P = 0.012), but not for overall lameness score or joint swelling score. No direct treatment-related adverse effects were detected during the study. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that overall clinical efficacy of a paste formulation of firocoxib in horses with naturally occurring osteoarthritis was comparable to efficacy of a paste formulation of phenylbutazone.

Nonsteroidal anti-inflammatory drugs

Nonsteroidal anti-inflammatory drugs (NSAIDs) are substances other than steroids that inhibit a component of the inflammatory cascade. This article is dedicated to those substances which specifically inhibit cyclooxygenase. NSAIDs are used extensively in the veterinary field. This article discusses their pharmacologic mechanism of action, indications, and toxicity. The two isoforms of cyclooxygenase (COX-1 and COX-2) are reviewed along with the newer NSAID which are more effective and less toxic due to more specific COX-2 inhibition. Specific effects on soft tissue, bone, cartilage, and synovium are summarized. Pain modulation is extensively reviewed as well as the antiendotoxic and antithrombotic uses.

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.

Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells

Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E2 (PGE2) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE2 production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE2, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE2, IL-6, or MMP3 production. Taken together, the anti-inflammatory and anticatabolic gene expression profiles of fibroblast-like synovial cells treated with HA and subsequently challenged with LPS support the pharmacologic benefits of treatment with HA regardless of molecular weight. The higher molecular weight HA product provided a cellular protective effect not seen with the lower molecular weight HA product.

Direct delayed human adenoviral BMP-2 or BMP-6 gene therapy for bone and cartilage regeneration in a pony osteochondral model

OBJECTIVE To evaluate healing of surgically created large osteochondral defects in a weight-bearing femoral condyle in response to delayed percutaneous direct injection of adenoviral (Ad) vectors containing coding regions for either human bone morphogenetic proteins 2 (BMP-2) or -6. METHODS Four 13mm diameter and 7mm depth circular osteochondral defects were drilled, 1/femoral condyle (n=20 defects in five ponies). At 2 weeks, Ad-BMP-2, Ad-BMP-6, Ad-green fluorescent protein (GFP), or saline was percutaneously injected into the central drill hole of the defect. Quantitative magnetic resonance imaging (qMRI) and computed tomography (CT) were serially performed at 12, 24, and 52 weeks. At 12 (one pony) or 52 weeks, histomorphometry and microtomographic analyses were performed to assess subchondral bone and cartilage repair tissue quality. RESULTS Direct delivery of Ad-BMP-6 demonstrated delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and histologic evidence of greater Glycosaminoglycan (GAG) content in repair tissue at 12 weeks, while Ad-BMP-2 had greater non-mineral cartilage at the surface at 52 weeks (p<0.04). Ad-BMP-2 demonstrated greater CT subchondral bone mineral density (BMD) by 12 weeks and both Ad-BMP-2 and -6 had greater subchondral BMD at 52 weeks (p<0.05). Despite earlier (Ad-BMP-6) and more persistent (Ad-BMP-2) chondral tissue and greater subchondral bone density (Ad-BMP-2 and -6), the tissue within the large weight-bearing defects at 52 weeks was suboptimal in all groups due to poor quality repair cartilage, central fibrocartilage retention, and central bone cavitation. Delivery of either BMP by this method had greater frequency of subchondral bone cystic formation (p<0.05). CONCLUSIONS Delivery of Ad-BMP-2 or Ad-BMP-6 via direct injection supported cartilage and subchondral bone regeneration but was insufficient to provide long-term quality osteochondral repair.

A randomized controlled trial of the efficacy of autologous platelet therapy for the treatment of osteoarthritis in dogs

OBJECTIVE To determine efficacy of a single intra-articular injection of an autologous platelet concentrate for treatment of osteoarthritis in dogs. DESIGN Randomized, controlled, 2-center clinical trial. ANIMALS 20 client-owned dogs with osteoarthritis involving a single joint. PROCEDURES Dogs were randomly assigned to a treatment or control group. In all dogs, severity of lameness and pain was scored by owners with the Hudson visual analog scale and the University of Pennsylvania Canine Brief Pain Inventory, respectively, and peak vertical force (PVF) was determined with a force platform. Dogs in the treatment group were then sedated, and a blood sample (55 mL) was obtained. Platelets were recovered by means of a point-of-use filter and injected intra-articularly within 30 minutes. Control dogs were sedated and given an intra-articular injection of saline (0.9% NaCl) solution. Assessments were repeated 12 weeks after injection of platelets or saline solution. RESULTS Dogs weighed between 18.3 and 63.9 kg (40.3 and 140.6 lb) and ranged from 1.5 to 8 years old. For control dogs, lameness scores, pain scores, and PVF at week 12 were not significantly different from pretreatment values. In contrast, for dogs that received platelet injections, lameness scores (55% decrease in median score), pain scores (53% decrease in median score), and PVF (12% increase in mean PVF) were significantly improved after 12 weeks, compared with pretreatment values. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that a single intra-articular injection of autologous platelets resulted in significant improvements at 12 weeks in dogs with osteoarthritis involving a single joint.

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.

Articular Cartilage Optical Properties in the Spectral Range 300-850 nm.

Measurements of absolute total reflectance were recorded from weight-bearing (n=9) and nonweight-bearing (n=9) equine articular cartilage specimens from 300 to 850 nm using a spectrophotometer with integrating sphere attachment. Following correction of measured spectra for interfacial reflections and edge losses, Kubelka-Munk theory was applied to estimate absorption and scattering coefficient, one-dimensional light intensity distribution, and light penetration depth. Kubelka-Munk absorption coefficients ranged from ∼7 cm-1 at 330 nm to ∼1 cm-1 at 850 nm. A localized absorption peak was noted at ∼340 nm. Above 510 nm, weight-bearing cartilage demonstrated significantly higher absorption coefficients than nonweight-bearing tissue (paired t-test, p<0.05). Kubelka-Munk scattering coefficients ranged from ∼40 cm-1 at 360 nm to ∼6 cm-1 at 850 nm. No statistical differences in scattering coefficient were noted between weight-bearing and nonweight-bearing tissue. Penetration depths predicted by Kubelka-Munk theory ranged from 0.6 mm at 350 nm to over 3 mm at 850 nm. Stronger absorption in weight-bearing cartilage compared to nonweight-bearing tissue resulted in lower light penetration depths in weight-bearing cartilage at all wavelengths longer than 510 nm.