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Evaluation of equine synovial-derived extracellular matrix scaffolds seeded with equine synovial-derived mesenchymal stem cells Texto completo
2018
Reisbig, Nathalie A. | Hussein, Hayam A. | Pinnell, Erin | Bertone, Alicia L.
OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs). SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers. PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 10(6) cells/0.5 mL) by use of a 30% serum gradient. Samples were evaluated on days 0, 3, 7, and 14. Cell migration, differentiation, and distribution into the synECMs were determined by cell surface marker CD90, viability, histologic morphology, and fluorescence microscopy results and expression of GFP, BMP-2, hyaluronan (HA), and proteoglycan (PG). RESULTS At day 14, synMSCs were viable and had multiplied 2.5-fold in the synECMs. The synECMs seeded with synMSCs had a significant decrease in CD90 expression and significant increases in HA and PG expression. The synECMs seeded with synMSCs cotransduced with GFP, or BMP-2 had a significant increase in BMP-2 expression. CONCLUSIONS AND CLINICAL RELEVANCE The synECM seeded with synMSCs or synMSCs cotransduced with GFP, or BMP-2 yielded a bioactive synovial scaffold. Expression of BMP-2 by synMSCs cotransduced to enhance expression of BMP-2 or GFP and an accompanying increase in both HA and PG expression indicated production of anabolic agents and synoviocyte differentiation in the scaffold. Because BMP-2 can promote repair of damaged cartilage, such a bioactive scaffold could be useful for treatment of injured cartilage.
Mostrar más [+] Menos [-]Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors Texto completo
2018
Kyostio-Moore, Sirkka | Berthelette, Patricia | Cornell, Cathleen Sookdeo | Nambiar, Bindu | Figueiredo, Monica Dias
OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.
Mostrar más [+] Menos [-]Evaluation of hyaluronic acid, procollagen type III N-terminal peptide, and tissue inhibitor of matrix metalloproteinase-1 as serum markers of canine hepatic fibrosis Texto completo
2016
Lidbury, Jonathan A. | Hoffmann, Aline Rodrigues | Fry, Joanna K. | Suchodolski, Jan S. | Steiner, Jörg M.
The only way to diagnose hepatic fibrosis in dogs is by histological assessment of a liver biopsy specimen. As this technique is invasive and susceptible to sampling variation, serum biomarkers are used to detect hepatic fibrosis in humans. The objective of this study was to assess the utility of hyaluronic acid (HA), procollagen type III N-terminal peptide (PIIINP), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as serum markers of canine hepatic fibrosis. Serum samples were collected from 47 dogs with histologically confirmed hepatobiliary disease and 24 healthy dogs in order to measure concentrations of HA, PIIINP, and TIMP-1. Hepatic fibrosis was staged using a 5-point scoring scheme. There was no correlation between serum concentrations of HA or PIIINP and the severity of hepatic fibrosis. There was a negative correlation between serum concentration of TIMP-1 and the severity of hepatic fibrosis (r s = −0.33; P = 0.036). It was not possible to use serum concentrations of HA, PIIINP, or TIMP-1 to discriminate between dogs with absent-to-moderate hepatic fibrosis and those with marked-to-very-marked fibrosis. The results of this study do not support the utility of measuring serum concentrations of HA, PIIINP, or TIMP-1 for diagnosing canine hepatic fibrosis. Further studies are needed to support this finding.
Mostrar más [+] Menos [-]Efficacy of intravenous administration of hyaluronan, sodium chondroitin sulfate, and N-acetyl-d-glucosamine for prevention or treatment of osteoarthritis in horses Texto completo
2016
Frisbie, David D. | Mcllwraith, Wayne | Kawcak, Christopher E. | Werpy, Natasha M.
OBJECTIVE To evaluate the efficacy of IV administration of a product containing hyaluronan, sodium chondroitin sulfate, and N-acetyl-d-glucosamine for prevention or treatment of osteoarthritis in horses. ANIMALS 32 healthy 2- to 5-year-old horses. PROCEDURES The study involved 2 portions. To evaluate prophylactic efficacy of the test product, horses received 5 mL of the product (n = 8) or saline (0.9% NaCl) solution (8; placebo) IV every fifth day, starting on day 0 (when osteoarthritis was induced in the middle carpal joint of 1 forelimb) and ending on day 70. To evaluate treatment efficacy, horses received either the product or placebo (n = 8/treatment) on days 16, 23, 30, 37, and 44 after osteoarthritis induction. Clinical, diagnostic imaging, synovial fluid, gross anatomic, and histologic evaluations and other tests were performed. Results of each study portion were compared between treatment groups. RESULTS Limb flexion and radiographic findings were significantly worse for horses that received the test product in the prophylactic efficacy portion than for placebo-treated horses or product-treated horses in the treatment efficacy portion. In the prophylactic efficacy portion, significantly less articular cartilage erosion was identified in product-treated versus placebo-treated horses. In the treatment efficacy portion, joints of product-treated horses had a greater degree of bone edema identified via MRI than did joints of placebo-treated horses but fewer microscopic articular cartilage abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that caution should be used when administering the evaluated product IV to horses, particularly when administering it prophylactically, as it may have no benefit or may even cause harm.
Mostrar más [+] Menos [-]Concentration and molecular weight distribution of hyaluronate in synovial fluid from clinically normal horses and horses with diseased joints
1994
Tulamo, R.M. | Heiskanen, T. | Salonen, M.
High molecular weight (MW) hyaluronate (HA) is an integral part of synovial fluid (SF), regulating many important physiologic and pathophysiologic mechanisms. Many of its effects depend on, or are reflected in, the concentration and MW of HA. High-performance liquid chromatography was used to assess simultaneously the concentration and MW of HA in SF obtained from horses with various arthritides: acute traumatic arthritis; chronic traumatic arthritis, including degenerative joint disease (DJD); and infectious arthritis. The size-exclusion column was calibrated, using appropriate HA concentration and MW standards, before the high-performance liquid chromatographic assays of the SF samples. Calibration of the column disclosed that the maximal limit for MW estimation of HA was around 3 million. In control joints, MW of HA ranged from 2 to 3 X 10(6) (mean 2.5 X 10(6)) and did not differ significantly from MW of HA in SF from horses with acute or chronic traumatic arthritis (mean 2 x 10(6); range 1.5 to 3 x 10(6)). Interestingly, a small amount of HA of moderately high MW (approx 1 to 1.5 x 10(6)) was detected in chromatograms of SF from infected joints. This degree of polymerization of SF HA was significantly (P < 0.01) lower, compared with that for control joints. There was no difference in mean (+/- SD) concentration of HA between control joints and joints with acute or chronic traumatic arthritis (0.33 +/- 0.12 g/L vs 0.18 +/- 0.03 g/L or 0.23 +/- 0.12 g/L), indicating that SF HA concentration probably should not be used as a diagnostic marker for the condition. However, the SF HA concentration was significantly (P < 0.01) lower in joints with infectious arthritis (0.07 +/- 0.03 g/L) and in the joints with radiographic evidence of DJD (0.12 +/- 0.01 g/L), compared with control joints.
Mostrar más [+] Menos [-]Quantitative characterization of viscoelastic properties of synovial fluid from forelimb joints of orthopedically normal Thoroughbreds and warmblood horses Texto completo
2019
Tyrnenopoulou, Panagiota C. | Rizos, Eleftheiros D. | Kristepi-Konstantinou, Maria | Papadopoulou, Paraskevi L. | Patsikas, Michail N. | Papazoglou, Lysimachos G. | Aggeli, Amalia | Diakakis, Nikolaos E.
OBJECTIVE To determine whether differences existed in the viscoelastic properties of synovial fluid samples from the metacarpophalangeal, intercarpal, and distal interphalangeal joints of orthopedically normal athletic horses. ANIMALS 45 warmblood horses and 30 Thoroughbreds (age range, 4 to 16 years). PROCEDURES Synovial fluid samples were aseptically obtained via arthrocentesis from 1 metacarpophalangeal, intercarpal, and distal interphalangeal joint of each horse, and nucleated cell counts were performed. A commercial ELISA was used to measure sample hyaluronic acid concentrations, and full rheological characterization of samples was performed to measure the elastic or storage modulus G' and viscous or loss modulus G“ at 37.5°C (representing the body temperature of horses). Findings were compared among joints and between breed groups by means of ANOVA. RESULTS Significant differences in synovial fluid G' and G“ values were identified between Thoroughbreds and warmblood horses for the metacarpophalangeal joint, between the metacarpophalangeal and intercarpal joints of Thoroughbreds, and between the metacarpophalangeal and distal interphalangeal joints and intercarpal and distal interphalangeal joints of warmblood horses. No significant differences were identified between breed groups or among joints in synovial fluid hyaluronic concentrations or nucleated cell counts. CONCLUSIONS AND CLINICAL RELEVANCE Viscoelastic properties of the forelimb joints of orthopedically normal Thoroughbreds and warmblood horses differed within and between these 2 groups, mainly as a function of the evaluated joint. To the authors' knowledge, this was the first study of its kind, and additional research is warranted to better understand the viscoelastic properties of synovial fluid in horses to optimize their locomotive function.
Mostrar más [+] Menos [-]Characterization and therapeutic application of canine adipose mesenchymal stem cells to treat elbow osteoarthritis Texto completo
2017
Kriston-Pal, Eva | Czibula, Agnes | Gyuris, Zoltán | Balka, Gyula | Seregi, Antal | Sukosd, Farkas | Suth, Mikló | Kiss-Toth, Endre | Haracska, Lajos | Uher, Ferenc | Monostori, Eva
Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia.
Mostrar más [+] Menos [-]Effects of anti-arthritis preparations on gene expression and enzyme activity of cyclooxygenase-2 in cultured equine chondrocytes Texto completo
2002
Tung, Jayne T. | Venta, Patrick J. | Eberhart, Susan W. | Yuzbasiyan-Gurkan, Vilma | Alexander, Lee | Caron, John P.
Objective-To determine the effects of recombinant equine interleukin -1beta (reIL-1beta) and 4 anti-inflammatory compounds on the expression and activity of cyclooxygenase (COX)-2 in cultured equine chondrocytes. Sample Population-Articular cartilage from 9 young adult horses. Procedure-Reverse transcriptase-polymerase chain reaction methods were used to amplify a portion of equine COX-2 to prepare a cDNA probe. Northern blot analysis was used to quantify the expression of COX-2 in first-passage cultures of equine articular chondrocytes propagated in media containing dexamethasone (DEX), phenylbutazone (PBZ), polysulfated glycosaminoglycan, and hyaluronan, each at concentrations of 10 and 100 micrograms/ml and each with or without reIL-1beta. A commercial immunoassay was used to determine prostaglandin E2 (PGE2) concentrations in conditioned medium of similarly treated cells to quantify COX-2 activity. Results-Addition of reIL-1beta increased the expression of COX-2 in a dose-dependent manner, which was paralleled by an increased concentration of PGE2 in culture medium. Concentration of PGE2 in spent medium from reIL-1beta-treated chondrocytes was significantly reduced by DEX and PBZ; however, only DEX significantly reduced gene expression of COX-2. Conclusions and Clinical Relevance-Prostaglandin E2 is considered to be an important mediator in the pathophysiologic processes of arthritis, and cultured chondrocytes respond to interleukin-1 with enhanced expression and activity of COX-2. Palliative relief in affected horses is probably attributable, in part, to inhibition of PGE2 synthesis; however, analysis of these data suggests that of the 4 compounds tested, only DEX affects pretranslational regulation of the COX-2 gene in cultured equine chondrocytes.
Mostrar más [+] Menos [-]Effect of sodium hyaluronate in collagenase-induced superficial digital flexor tendinitis in horses
1992
Foland, J.W. | Trotter, G.W. | Powers, B.E. | Wrigley, R.H. | Smith, F.W.
Superficial digital flexor tendinitis was induced in each forelimb of 8 horses by injecting 4,000 U of collagenase into the midmetacarpal region of the tendon. In each horse, each tendon was treated 24 and 96 hours after the collagenase injection with sc injections of sodium hyaluronate (treated limbs) or an equal volume of 0.9% NaCl solution (control limbs). Exercise was restricted for the first 3 weeks of the study, and a controlled exercise program was instituted for the remainder of the study. Horses were evaluated clinically for lameness, tendon swelling, and midmetacarpal limb circumference. Ultrasonographic examinations were performed regularly (11 examinations/horse) throughout the study, and all horses were euthanatized 12 weeks after collagenase injections. Tendons from 4 horses were harvested for biomechanical testing, and samples were obtained from tendons from the remaining 4 horses for biochemical analysis of collagen. Samples were obtained from all tendons for microscopic evaluation. Significant differences between treated and control tendons were not noticed in any of the variables examined in live horses, although trends toward less lameness in treated limbs and toward better healing on ultrasonographic examination in control limbs were recorded. Significant differences were not noticed in biomechanical or biochemical evaluations, and the only significant (P < 0.05) microscopic finding was more severe inflammation in tendons from treated limbs. This study did not reveal significant benefits of treatment with sodium hyaluronate outside a synovial sheath on tendon repair in collagenase-induced tendinitis.
Mostrar más [+] Menos [-]Characteristics of digital flexor tendon sheath fluid from clinically normal horses
1991
Malark, J.A. | Nixon, A.J. | Skinner, K.L. | Mohammed, H.
Physical, biochemical, and cytologic properties of synovial fluid from digital flexor tendon sheaths of clinically normal horses were investigated. Tendon sheath fluid was pale yellow, clear, and did not clot. Volume of fluid within a tendon sheath varied minimally, with a mean of 2.11 ml. Total erythrocyte counts were higher than values observed in normal equine joint fluid, whereas values for total leukocyte count (770 +/- 73 cells/mm3), viscosity (6.05 +/- 0.58 cs), and protein concentration (7.87 +/- 0.03 mg/ml) were similar to those in joint fluid. Large mononuclear cells were the predominant synovial fluid cell type. Mean hyaluronic acid concentration (0.74 +/- 0.02 mg/ml) and mucinous precipitate quality were lower than values in joint fluid.
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