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- Author or Editor: Allison A. Stewart x
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Abstract
Objective—To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system.
Animals—6 healthy horses, 6 months to 5 years of age.
Procedures—Bone marrow–derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm2 and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days. Pellets were analyzed for collagen type II content by use of an ELISA, total glycosaminoglycan content by use of the dimethylmethylene blue dye–binding assay, and DNA content by use of fluorometric quantification. Semiquantitative PCR assay was performed to assess relative concentrations of collagen type II and aggrecan mRNAs.
Results—Use of 100 ng of FGF-2/mL significantly increased pellet DNA and glycosaminoglycan content. Collagen type II content of the pellet was also increased by use of 10 and 100 ng of FGF-2/mL. Collagen type II and aggrecan mRNA transcripts were increased by treatment with FGF-2. Some control samples had minimal evidence of collagen type II and aggrecan transcripts after 35 cycles of amplification.
Conclusions and Clinical Relevance—FGF-2 treatment of bone marrow–derived MSC monolayers enhanced subsequent chondrogenic differentiation in a 3-dimensional culture. This result is important for tissue engineering strategies dependent on MSC expansion for cartilage repair.
Abstract
Objective—To investigate in vitro effects of radial shock waves on membrane permeability, viability, and structure of chondrocytes and articular cartilage.
Sample Population—Cartilage explants obtained from the third metacarpal and metatarsal bones of 6 horses.
Procedure—Equine cartilage was subjected to radial shock waves and then maintained as explants in culture for 48 hours. Treatment groups consisted of a negative control group; application of 500, 2,000, and 4,000 impulses by use of a convex handpiece (group A); and application of 500, 2,000, and 4,000 impulses by use of a concave handpiece (group B). Effects on explant structure were evaluated by use of environmental scanning electron microscopy (ESEM). Membrane permeability was determined by release of lactate dehydrogenase (LDH). Chondrocyte viability was assessed by use of vital cell staining. Comparisons of LDH activity and nonviable cell percentages were performed by ANOVA.
Results—Cell membrane permeability increased significantly after application of 2,000 and 4,000 impulses in groups A and B. A significant decrease in cell viability was observed for application of 4,000 impulses in explants of group A. There was no detectable damage to integrity of cartilage explants observed in any treatment group by use of ESEM.
Conclusions and Clinical Relevance—Radial shock waves do not appear to structurally damage articular cartilage but do impact chondrocyte viability and membrane permeability. Caution should be exercised when extremely high periarticular pulse doses are used until additional studies can determine the longterm outcome of these effects and appropriate periarticular treatment regimens can be validated. (Am J Vet Res 2005;66:1757–1763)
Abstract
Objective—To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype.
Sample Population—MSCs obtained from 5 young horses.
Procedures—First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10−7M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1α.
Results—Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2–dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2–dexamethasone remained significantly increased.
Conclusions and Clinical Relevance—Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage.
Abstract
Objective—To evaluate the effects of glucosamine on equine articular chondrocytes and synoviocytes at concentrations clinically relevant to serum and synovial fluid concentrations.
Sample Population—Articular cartilage and synovium with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age).
Procedures—In vitro chondrocyte and synoviocyte cell cultures from 8 horses were treated with glucosamine (0.1 to 20 μg/mL) with or without interleukin-1 (IL-1; 10 ng/mL) for 48 hours. Negative control cultures received no glucosamine or IL-1, and positive control cultures received only IL-1. Cultures were assayed for production of proteoglycan (via media containing sulfur 35 (35S)-labeled sodium sulfate and Alcian blue precipitation), prostaglandin E2 (PGE2; via a colorimetric assay), cyclooxygenase-2 (via real-time reverse-transcriptase PCR assay), microsomal PGE2 synthase (mPGEs; via real-time reverse-transcriptase PCR assay), and matrix metalloproteinase (MMP)-13 (via a colorimetric assay).
Results—Glucosamine had no impact on proteoglycan production or MMP-13 production under noninflammatory (no IL-1) or inflammatory (with IL-1) conditions. Glucosamine at 0.1 and 0.5 μg/mL significantly decreased IL-1–stimulated production of mPGEs by chondrocytes, compared with that of positive control chondrocytes. Glucosamine at 0.1 and 5 μg/mL significantly decreased IL-1–stimulated production of mPGEs and PGE2, respectively, compared with that of positive control synoviocytes.
Conclusions and Clinical Relevance—Glucosamine had limited effects on chondrocyte and synoviocyte metabolism at clinically relevant concentrations, although it did have some anti-inflammatory activity on IL-1–stimulated articular cells. Glucosamine may have use at clinically relevant concentrations in the treatment of inflammatory joint disease.
Abstract
Objective—To evaluate the effects of methylprednisolone acetate (MPA) on proteoglycan production by equine chondrocytes and to investigate whether glucosamine hydrochloride modulates these effects at clinically relevant concentrations.
Sample Population—Articular cartilage with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age).
Procedures—In vitro chondrocyte pellets were pretreated with glucosamine (0, 1, 10, and 100 μg/mL) for 48 hours and exposed to MPA (0, 0.05, and 0.5 mg/mL) for 24 hours. Pellets and media were assayed for proteoglycan production (Alcian blue precipitation) and proteoglycan content (dimethylmethylene blue assay), and pellets were assayed for DNA content.
Results—Methylprednisolone decreased production of proteoglycan by equine chondrocytes at both concentrations studied. Glucosamine protected proteoglycan production at all 3 concentrations studied.
Conclusions and Clinical Relevance—Methylprednisolone, under noninflammatory conditions present in this study, decreased production of proteoglycan by equine chondrocytes. Glucosamine had a protective effect against inhibition of proteoglycan production at all 3 concentrations studied. This suggested that glucosamine may be useful as an adjunct treatment when an intra-articular injection of a corticosteroid is indicated and that it may be efficacious at concentrations relevant to clinical use.
Abstract
Objective—To determine the rate of development of septic arthritis after elective arthroscopy and evaluate associations between various factors and development of this complication in horses.
Design—Retrospective case series.
Animals—682 horses that underwent arthroscopic procedures at the University of Illinois Veterinary Teaching Hospital from 1994 to 2003.
Procedures—Information pertaining to signalment, joints treated, whether antimicrobials were administered, and development of postoperative septic arthritis was collected from medical records. Horses with a primary problem of septic arthritis or wounds involving joints were excluded. The following factors were evaluated to determine their roles in joint sepsis: breed, sex, joint, and preoperative and intra-articular administration of antimicrobials. Telephone interviews with clients were used to determine whether unreported septic arthritis had developed.
Results—8 of 932 (0.9%) joints in 7 of 682 (1.0%) horses that underwent arthroscopy developed postoperative septic arthritis. Follow-up information after discharge from the hospital was available for 461 of the 682 horses, and of those, 8 of 627 (1.3%) joints in 7 of 461 (1.5%) horses developed septic arthritis. Breed and joint treated were significant risk factors for development of postoperative septic arthritis, with draft breeds and tibiotarsal joints more likely than others to be affected. Sex, preoperatively administered antimicrobials, and intra-articularly administered antimicrobials were not associated with development of postoperative septic arthritis.
Conclusions and Clinical Relevance—Results can be used for comparison with data from other institutions and surgical facilities. Additional precautions should be undertaken when arthroscopic surgery involves draft breeds and tibiotarsal joints.
Abstract
Objective—To determine whether the effects of a high–molecular-weight sodium hyaluronate alone or in combination with triamcinolone acetonide can mitigate chondrocyte glyocosaminoglycan (GAG) catabolism caused by interleukin (IL)-1 administration.
Sample Population—Chondrocytes collected from metacarpophalangeal joints of 10 horses euthanized for reasons unrelated to joint disease.
Procedures—Chondrocyte pellets were treated with medium (negative control), medium containing IL-1 only (positive control), or medium containing IL-1 with hyaluronic acid only (0.5 or 2.0 mg/mL), triamcinolone acetonide only (0.06 or 0.6 mg/mL), or hyaluronic acid (0.5 or 2.0 mg/mL) and triamcinolone acetonide (0.06 or 0.6 mg/mL) in combination. Chondrocyte pellets were assayed for newly synthesized GAG, total GAG content, total DNA content, and mRNA for collagen type II, aggrecan, and cyclooxygenase (COX)-2.
Results—High-concentration hyaluronic acid increased GAG synthesis, whereas high-concentration triamcinolone acetonide decreased loss of GAG into the medium. High concentrations of hyaluronic acid and triamcinolone acetonide increased total GAG content. There was no change in DNA content with either treatment. Triamcinolone acetonide reduced COX-2 mRNA as well as aggrecan and collagen type II expression. Treatment with hyaluronic acid had no effect on mRNA for COX-2, aggrecan, or collagen type II.
Conclusions and Clinical Relevance—Results indicated that high concentrations of hyaluronic acid or triamcinolone acetonide alone or in combination mitigated effects of IL-1 administration on GAG catabolism of equine chondrocytes.
Abstract
Objective—To evaluate tendon injuries in horses over a 16-week period by use of ultrasonography and low-field magnetic resonance imaging (MRI).
Sample—Tendons of 8 young adult horses.
Procedures—The percentage of experimentally induced tendon injury was evaluated in cross section at the maximal area of injury by use of ultrasonography and MRI at 3, 4, 6, 8, and 16 weeks after collagenase injection. The MRI signal intensities and histologic characteristics of each tendon were determined at the same time points.
Results—At 4 weeks after collagenase injection, the area of maximal injury assessed on cross section was similar between ultrasonography and MRI. In lesions of > 4 weeks' duration, ultrasonography underestimated the area of maximal cross-sectional injury by approximately 18%, compared with results for MRI. Signal intensity of lesions on T1-weighted images was the most hyperintense of all the sequences, lesions on short tau inversion recovery images were slightly less hyperintense, and T2-weighted images were the most hypointense. Signal intensity of tendon lesions was significantly higher than the signal intensity for the unaltered deep digital flexor tendon. Histologically, there was a decrease in proteoglycan content, an increase in collagen content, and minimal change in fiber alignment during the 16 weeks of the study.
Conclusions and Clinical Relevance—Ultrasonography may underestimate the extent of tendon damage in tendons with long-term injury. Low-field MRI provided a more sensitive technique for evaluation of tendon injury and should be considered in horses with tendinitis of > 4 weeks' duration.
Abstract
Objective—To compare in vitro expansion, explant colonization, and matrix synthesis of equine tendon- and bone marrow–derived cells in response to insulin-like growth factor-I (IGF-I) supplementation.
Sample—Cells isolated from 7 young adult horses.
Procedures—Tendon- and bone marrow–derived progenitor cells were isolated, evaluated for yield, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability and expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein mRNAs. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period.
Results—Tendon- and bone marrow–derived cells required 17 to 19 days of monolayer culture to reach 2 passages. Mean ± SE number of monolayer cells isolated was higher for tendon-derived cells (7.9 ± 0.9 × 106) than for bone marrow–derived cells (1.2 ± 0.1 × 106). Cell numbers after culture for 7 days on acellular tendon matrix were 1.6- to 2.8-fold higher for tendon-derived cells than for bone marrow–derived cells and 0.8- to 1.7-fold higher for IGF-I supplementation than for untreated cells. New collagen and glycosaminoglycan syntheses were significantly greater in tendon-derived cell groups and in IGF-I–supplemented groups. The mRNA concentrations of collagen type I, collagen type III, and cartilage oligomeric matrix protein were not significantly different between tendon- and bone marrow–derived groups.
Conclusions and Clinical Relevance—In vitro results of this study suggested that tendon-derived cells supplemented with IGF-I may offer a useful resource for cell-based strategies in tendon healing.
