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  • Author or Editor: Alicia L Bertone x
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Abstract

Objective—To determine whether human parathyroid hormone (hPTH) gene in collagen matrix could safely promote bone formation in diaphyseal or subchondral bones of horses.

Animals—8 clinically normal adult horses.

Procedure—Amount, rate, and quality of bone healing for 13 weeks were determined by use of radiography, quantitative computed tomography, and histomorphometric analysis. Diaphyseal cortex and subchondral bone defects of metacarpi were filled with hPTH1-34 gene-activated matrix (GAM) or remained untreated. Joints were assessed on the basis of circumference, synovial fluid analysis, pain on flexion, lameness, and gross and histologic examination.

Results—Bone volume index was greater for cortical defects treated with hPTH1-34 GAM, compared with untreated defects. Bone production in cortical defects treated with hPTH1-34 GAM positively correlated with native bone formation in untreated defects. In contrast, less bone was detected in hPTH1-34 GAM-treated subchondral bone defects, compared with untreated defects, and histology confirmed poorer healing and residual collagen sponge.

Conclusions and Clinical Relevance—Use of hPTH1-34 GAM induced greater total bone, specifically periosteal bone, after 13 weeks of healing in cortical defects of horses. The hPTH1-34 GAM impeded healing of subchondral bone but was biocompatible with joint tissues. Promotion of periosteal bone formation may be beneficial for healing of cortical fractures in horses, but the delay in onset of bone formation may negate benefits. The hPTH1-34 GAM used in this study should not be placed in articular subchondral bone defects, but contact with articular surfaces is unlikely to cause short-term adverse effects. (Am J Vet Res 2004;65:1223–1232)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses.

Sample—Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses.

Procedures—Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested amount of a proprietary siRNA molecule. Full-thickness cartilage explants were treated with 1× (1μM), 2× (2μM), and 4× (4μM) or 1× (0.13μM), 4× (0.52μM), and 8× (1.04μM) the recommended concentrations of the proprietary siRNA and the cationic liposome siRNA, respectively, in equivalent media volumes. Use of fluorescent siRNA duplexes allowed quantification of transfected cells via flow cytometry and direct visualization of the depth and distribution of in situ transfection via fluorescent microscopy.

Results—With both transfection reagents, > 90% of monolayer chondrocytes were transfected. In explants, only use of the proprietary molecule achieved > 50% transfection efficiency, whereas use of the cationic liposome achieved < 20%. Only the proprietary molecule-treated cartilage consistently contained fluorescent cells throughout all zones; the cationic liposome-transfected chondrocytes were restricted to explant surfaces.

Conclusions and Clinical Relevance—Robust transfection of chondrocytes in monolayer was achieved with both reagents, but only use of the proprietary molecule attained effective full-thickness transfection of explants that may allow relevant transcript reduction via RNAi.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine effects of various concentrations of retinoic acid (RA) or a synthetic RA receptor antagonist (LE135) on equine chondrocytes or bone marrow—derived equine mesenchymal stem cells (BMDMSCs) in monolayer cultures.

Sample—Articular cartilage and BMDMSCs from 5 clinically normal horses.

Procedures—Monolayers of chondrocytes cultured in standard media and of BMDMSCs cultured in chondrogenic media were treated with RA at concentrations of 0, 0.1, 1, or 10μM or LE135 at concentrations of 0, 0.1, 1, or 10μM on day 0. On days 7 and 14, samples were analyzed for DNA concentration, chondrocyte morphology or features consistent with chondrogenesis (ie, chondral morphology [scored from 0 to 4]), and gene expression of collagen type Ia (CI), collagen type II (CII), and aggrecan.

Results—Chondrocytes treated with RA had more mature chondral morphology (range of median scores, 3.0 to 4.0) than did untreated controls (range of median scores, 0.5 to 0.5). Chondrocytes treated with LE135 did not sustain chondrocyte morphology. All BMDMSCs had evidence of chondral morphology or high CII:CI ratio. Retinoic acid (1 or 10μM) or LE135 (10μM) treatment decreased DNA content of BMDMSC cultures. At 0.1 and 1μM concentrations, LE135 weakly but significantly increased chondral morphology scores, compared with untreated controls, but lack of aggrecan expression and lack of increased CII:CI ratio, compared with that of controls, did not affect chondrogenesis.

Conclusions and Clinical Relevance—RA promoted maturation and hypertrophy in chondrocytes but not BMDMSCs in monolayer cultures. Deficiency or blockade of RA may prevent hypertrophy and maturation of differentiated chondrocytes.

Full access
in American Journal of Veterinary Research

Abstract

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.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow–derived mesenchymal stem cells (BMDMSCs).

Animals—9 adult clinically normal horses.

Procedures—BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase–quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic profiling for effects of BMP12 was evaluated by use of an equine gene expression microarray on day 6.

Results—BMDMSCs and SDFTNs had high BMP12 gene expression and remained viable and healthy for at least 6 days. Type l collagen immunocytochemical staining for SDFTNs and tenocyte-like morphology for SDFTNs and BMDMSCs were greatest in BMP12 cells. Cartilage oligomeric matrix protein, as determined via RT-qPCR assay, and chondroitin sulfate, as determined via gene expression microarray analysis, were upregulated relative to control groups in SDFTN-BMP12 cells. The BMDMSCs and SDFTNs became mineralized with BMP2, but not BMP12. Superficial digital flexor tenocytes responded to BMP12 with upregulation of genes relevant to tendon healing and without mineralization as seen with BMP2.

Conclusions and Clinical Relevance—Targeted equine SDFTNs may respond to BMP12 with improved tenocyte morphology and without mineralization, as seen with BMP2. Bone marrow–derived mesenchymal stem cells may be able to serve as a cell delivery method for BMP12.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To identify patterns and correlations of gross, histologic, and gene expression characteristics of articular cartilage from horses with osteoarthritis.

Animals—10 clinically normal horses and 11 horses with osteoarthritis of the metacarpal condyles.

Procedures—Metacarpophalangeal joints were opened and digitally photographed, and gross lesions were scored and quantified. Representative cartilage specimens were stained for histologic scoring. Total RNA from dorsal and palmar articular surfaces was processed on an equine gene expression microarray.

Results—Histologic scores were greater in both regions of osteoarthritic joints, compared with corresponding regions in control joints. Cartilage from the palmar aspect of diseased joints had the highest histologic scores of osteoarthritic sites or of either region in control joints. A different set of genes for dorsal and palmar osteoarthritis was identified for high and low gene expression. Articular cartilage from the dorsal region had surface fraying and greater expression of genes coding for collagen matrix components and proteins with anti-apoptotic function, compared with control specimens. Articular cartilage from the palmar region had greater fraying, deep fissures, and less expression of genes coding for glycosaminoglycan matrix formation and proteins with anti-apoptotic function, compared with cartilage from disease-free joints and the dorsal aspect of affected joints.

Conclusions and Clinical Relevance—Metacarpal condyles of horses with naturally occurring osteoarthritis had an identifiable and regional gene expression signature with typical morphologic features.

Full access
in American Journal of Veterinary Research

Abstract

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 β-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.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of pretreatment with α-linolenic acid, an omega-3 polyunsaturated fatty acid, on equine synovial explants challenged with lipopolysaccharide (LPS).

Animals—8 mature mixed-breed horses (4 mares and 4 geldings).

Procedure—Synovial explants were assigned to receive 1 of 7 concentrations of α-linolenic acid, ranging from 0 to 300 µg/mL. At each concentration, half of the explants were controls and half were challenged with 0.003 µg of LPS as a model of synovial inflammation. Cell inflammatory response was evaluated by measurement of prostaglandin E2 production via an ELISA. Synovial cell viability, function, histomorphologic characteristics, and cell membrane composition were evaluated by use of trypan blue dye exclusion, hexuronic acid assay for hyaluronic acid, objective microscopic scoring, and high-performance liquid chromatography, respectively.

Results—Challenge with LPS significantly increased production of prostaglandin E2 and decreased production of hyaluronic acid. Treatment with α-linolenic acid at the highest dose inhibited prostaglandin E2 production. Cell viability and histomorphologic characteristics were not altered by treatment with α-linolenic acid or LPS challenge. Treatment with α-linolenic acid increased the percentage of this fatty acid in the explant cell membranes.

Conclusions and Clinical Relevance—Results suggest that investigation of α-linolenic acid as an anti-inflammatory medication for equine synovitis is warranted. (Am J Vet Res 2005;66:1503–1508)

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate 4 methods for generating decellularized equine synovial extracellular matrix.

SAMPLE Villous synovium harvested from the femoropatellar and medial femorotibial joints of 4 healthy adult horses < 7 years of age. Synovial samples were frozen (−80°C) until used.

PROCEDURES Synovial samples were thawed and left untreated (control) or decellularized with 1 of 4 methods (15 samples/horse/method): incubation in 0.1% peracetic acid (PAA), incubation in 0.1% PAA twice, incubation in 1% Triton X-100 followed by incubation in DNase, and incubation in 2M NaCl followed by incubation in DNase. Control and decellularized samples were examined for residual cells, villous integrity, and collagen structure and integrity by means of histologic examination and scanning electron microscopy; cell viability was evaluated by means of culture and exclusion staining. Decellularization efficiency was assessed by testing for DNA content and DNA fragment size.

RESULTS Incubation in PAA once preserved the synovial villous architecture, but resulted in high DNA content and retention of large (> 25,000 base pair) DNA fragments. Incubation in Triton and incubation in NaCl resulted in low DNA content and short (< 200 base pair) DNA fragments, but destroyed the synovial villous architecture. Incubation in PAA twice resulted in low DNA content and short DNA fragments while retaining the synovial villous architecture.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that of the methods evaluated, incubation in 0.1% PAA twice was the best method for generating decellularized equine synovial extracellular matrix.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the pharmacokinetics of methylprednisolone (MP) and the relationship between MP and hydrocortisone (HYD) concentrations in plasma and urine after intra-articular (IA) administration of 100 or 200 mg of MP acetate (MPA) to horses.

Animals—Five 3-year-old Thoroughbred mares.

Procedures—Horses exercised on a treadmill 3 times/wk during the study. Horses received 100 mg of MPA IA, then 8 weeks later received 200 mg of MPA IA. Plasma and urine samples were obtained at various times for 8 weeks after horses received each dose of MPA; concentrations of MP and HYD were determined. Pharmacokinetic-pharmacodynamic estimates for noncompartmental and compartmental parameters were determined.

Results—Maximum concentration of MP in plasma was similar for each MPA dose; concentrations remained greater than the lower limit of quantitation for 18 and 7 days after IA administration of 200 and 100 mg of MPA, respectively. Maximum concentration and area under the observed concentration-time curve for MP in urine were significantly higher (approximately 10-and 17-fold, respectively) after administration of 200 versus 100 mg of MPA. Hydrocortisone concentration was below quantifiable limits for ≥ 48 hours in plasma and urine of all horses after administration of each MPA dose.

Conclusions and Clinical Relevance—Pharmacokinetics of MP may differ among IA MPA dosing protocols, and MP may be detected in plasma and urine for a longer time than previously reported. This information may aid veterinarians treating sport horses. Further research is warranted to determine whether plasma HYD concentration can aid identification of horses that received exogenous glucocorticoids.

Full access
in American Journal of Veterinary Research