Objective—To assess the effects of supraphysiologic
concentrations of insulin-like growth factor-I (IGF-1) on
morphologic and phenotypic responses of chondrocytes.
Sample Population—Articular cartilage obtained
from 2 young horses.
Procedure—Chondrocytes were suspended in fibrin
cultures and supplemented with 25, 12.5, or 0 mg of
IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic
expression were assessed histologically,
using H&E and Alcian blue stains, immunoreaction to
collagen type I and II, and in situ hybridization.
Proteoglycan content, synthesis, and monomer size
were analyzed. The DNA content was determined by
bisbenzimide-fluorometric assay, and elution of IGF-1
into medium was determined by IGF-1 radioimmunoassay.
Results—Both 12.5 and 25 µg of IGF-1/ml enhanced
phenotypic expression of chondrocytes without
inducing detrimental cellular or metabolic effects.
Highest concentration of IGF-1 (25 µg/ml) significantly
increased total DNA content, glycosaminoglycan
(GAG) content, GAG synthesis, and size of proteoglycan
monomers produced, compared with cultures
supplemented with 12.5 µg of IGF-1/ml or untreated
cultures. Histologic examination confirmed these biochemical
effects. Matrix metachromasia, type-II collagen
in situ hybridization and immunoreaction were
increased in cultures treated with 25 µg of IGF-1/ml,
compared with cultures supplemented with 12.5 µg
of IGF-1/ml or untreated cultures.
Conclusions and Clinical Relevance—Chondrocytes
exposed to high concentrations of IGF-1 maintained
differentiated chondrocyte morphology and had
enhanced synthesis of matrix molecules without
inducing apparent detrimental effects on chondrocyte
metabolism. These results suggest that application of
such composites for in vivo use during cartilage grafting
procedures should provide an anabolic effect on
the grafted cells. (Am J Vet Res 2002;63:301–305)
OBJECTIVE To determine whether major histocompatability complex (MHC) class II expression in equine mesenchymal stem cells (MSCs) changes with exposure to a proinflammatory environment reflective of an inflamed joint.
SAMPLE Cryopreserved bone marrow-derived MSCs from 12 horses and cartilage and synovium samples from 1 horse euthanized for reasons other than lameness.
PROCEDURES In part 1 of a 3-part study, the suitability of a quantitative reverse transcriptase PCR (qRT-PCR) assay for measurement of MHC class II expression in MSCs following stimulation with interferon (IFN)-γ was assessed. In part 2, synoviocyte-cartilage cocultures were or were not stimulated with interleukin (IL)-1β (10 ng/mL) to generate conditioned media that did and did not (control) mimic an inflamed joint environment. In part 3, a qRT-PCR assay was used to measure MSC MHC class II expression after 96 hours of incubation with 1 of 6 treatments (control-conditioned medium, IL-1β-conditioned medium, and MSC medium alone [untreated control] or with IL-1β [10 ng/mL], tumor necrosis factor-α [10 ng/mL], or IFN-γ [100 ng/mL]).
RESULTS The qRT-PCR assay accurately measured MHC class II expression. Compared with MHC class II expression for MSCs exposed to the untreated control medium, that for MSCs exposed to IL-1β was decreased, whereas that for MSCs exposed to IFN-γ was increased. Neither the control-conditioned nor tumor necrosis factor-α medium altered MHC class II expression.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSC exposure to proinflammatory cytokine IL-1β decreased MHC class II expression and antigenicity. Treatment of inflamed joints with allogeneic MSCs might not be contraindicated, but further investigation is warranted.
Objective—To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1α, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes.
Sample Population—Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years.
Procedures—Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times.
Results—Cultures treated with MMP-13 or IL-1α had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1α. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1α. Expression of MMP-13 mRNA in cartilage was increased by IL-1α, but decreased in synoviocytes by MMP-13 treatment.
Conclusions and Clinical Relevance—Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.
Objective—To determine the concentration of doxycycline compounded from doxycycline hyclate tablets into liquid formulations for oral administration in veterinary species and stored for 28 days.
Sample—Doxycycline hyclate tablets (100 mg) crushed and mixed with a 50:50 mixture of syrup and suspension vehicles for oral administration to produce 3 batches each of 2 doxycycline formulations: 33.3 and 166.7 mg/mL.
Procedures—Formulations were stored, protected from light, at room temperature (22° to 26°C [71.6° to 78.8°F]) and at a controlled cold temperature (refrigerated 2° to 8°C [35.6° to 46.4°F]). Doxycycline was extracted from the formulations, and concentration was measured by high-pressure liquid chromatography on days 0 (date of preparation), 1, 4, 7, 14, 21, and 28. Concentrations were compared with those of a US Pharmacopeial Convention reference standard. Formulation quality at each point was also assessed through color change, formulation consistency, and suspension uniformity.
Results—On days 0, 1, 4, and 7, the concentration of each formulation was within 90% to 110% of the reference standard (range, 93% to 109%), which was deemed acceptable. However, doxycycline concentrations had decreased dramatically by day 14 and remained low for the duration of the study period. Doxycycline concentrations on days 14, 21, and 28 were all < 20% (range, 14% to 18%) of the reference standard, and the quality of the formulations decreased as well. No effect of storage temperatures on doxycycline concentration was identified.
Conclusions and Clinical Relevance—The concentration of doxycycline, compounded from commercial tablets in the vehicles evaluated to yield doses of 33.3 and 166.7 mg/mL, cannot be assured beyond 7 days.
Objective—To evaluate whether administering a tart cherry juice blend (TCJB) prior to exercise would reduce skeletal and cardiac muscle damage by decreasing the inflammatory and oxidative stress response to exercise in horses.
Procedures—Horses were randomly allocated into 2 groups in a crossover study with a 2-week washout period and orally administered either TCJB or a placebo solution (1.42 L, twice daily) in a double-masked protocol for 2 weeks prior to a stepwise incremental exercise protocol. Horses were tested for serum activities of creatine kinase and aspartate aminotransferase (AST) and concentrations of cardiac troponin I (cTnI), thiobarbituric acid reactive substances (TBARS; an indicator of oxidative stress), and serum amyloid A (SAA; an indicator of inflammation). To ensure that treatment would not result in positive results of an equine drug-screening protocol, serum samples obtained from each horse prior to and after 2 weeks of administration of TCJB or the placebo solution were tested.
Results—All horses had negative results of drug screening at both sample times. The exercise protocol resulted in a significant increase in TBARS concentration, SAA concentration, and serum AST activity in all horses. Administration of TCJB or placebo solution was not associated with an effect on malondialdehyde or SAA concentrations. However, administration of TCJB was associated with less serum activity of AST, compared with administration of placebo solution.
Conclusions and Clinical Relevance—Administration of TCJB may diminish muscle damage induced by exercise.
To identify chondroprotective factors as potential disease-modifying osteoarthritis treatments using an unbiased, bottom-up proteomics approach.
Paired equine cartilage explants and synovial membrane were collected postmortem from 4 horses with no history of lameness and grossly normal joints at necropsy.
Six groups were established: cartilage, synoviocytes, and cartilage + synoviocytes (coculture), all with or without interleukin (IL)-1β. The catabolic effect of IL-1β was verified by glycosaminoglycan (GAG) released from cartilage into media by 1,9-dimethyl-methylene blue assay and cartilage toluidine blue histochemistry. Conditioned media from cocultures with or with IL-1β were submitted for bottom-up proteomic analysis. Synoviocyte gene expression was evaluated using reverse transcription–quantitative PCR (RT-qPCR) for proteins of interest identified in the proteomics scan.
GAG content was retained in cartilage when in cocultures treated with IL-1β. Fourteen proteins of interest were selected from the proteomic analysis. From these 14 proteins, metalloproteinase inhibitor 3 precursor (TIMP3), tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), insulin-like growth factor-binding protein 2 (IGFBP2), and alpha-2 macroglobulin (A2M) were selected for synoviocyte gene expression analysis by RT-qPCR. Gene expression of TIMP3 (P = .02) and TNFRSF11B (P = .04) were significantly increased in synoviocytes from cocultures treated with IL-1β compared to controls. Contrary to expectations based on protein expression, IGFBP2 gene expression (P = .04) was significantly decreased in IL-1β-stimulated coculture synoviocytes compared to control coculture synoviocytes. A2M gene expression in synoviocytes was not different between coculture groups.
The secretome from synoviocytes could provide a milieu of bioactive factors to restore joint homeostasis in osteoarthritis.
Objective—To evaluate the effects of interleukin (IL)-1β on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes.
Sample Population—Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses.
Procedures—3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1β (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectrophotometric assay; RNA was isolated from synoviocytes and cartilage and analyzed for expression of matrix metalloproteinases (MMP)-3 and -13 (cartilage and synoviocytes), aggrecan (cartilage), collagen type IIB (cartilage), and 18S as a control (cartilage and synoviocytes) by use of quantitative PCR assays. Cartilage matrix metachromasia was assessed histochemically.
Results—IL-1β–induced GAG loss from cartilage was significantly less in cocultures than in cartilage-only cultures. Cartilage aggrecan gene expression was also significantly less downregulated and synoviocyte MMP-3 expression was less upregulated by IL-1β in cocultures, compared with cartilage- and synoviocyteonly cultures. Histochemical findings supported the molecular and biochemical results and revealed maintenance of matrix metachromasia in cocultured cartilage treated with IL-1β.
Conclusions and Clinical Relevance—Results suggest that synoviocytes secrete 1 or more mediators that preferentially protect matrix GAG metabolism from the degradative effects of IL-1β. Further studies involving proteomic and microarray approaches in similar coculture systems may elucidate novel therapeutic targets for the treatment of osteoarthritis.
OBJECTIVE To determine morphological characteristics of subchondral bone cysts (SBCs) in medial femoral condyles (MFCs) of adult horses with orthopedic disease.
SAMPLE CT scans of 7 MFCs with SBCs from 6 adult horses.
PROCEDURES CT was used to determine the volume, surface area, and centers of the articular cyst opening and SBC in each MFC. Cysts were ordered from smallest to largest on the basis of volume. Osseous pathological characteristics of the MFC were assessed in the frontal plane. Three-dimensional distance of displacement between the center of the articular cyst opening and center of the cyst was determined for each SBC. Cyst surface area-to-volume ratio was evaluated and compared with that of a true sphere.
RESULTS All SBCs had a defect in the subchondral bone plate at the cranial 15% to 20% of the MFC. Cyst center was located in a caudal, proximal, and abaxial direction with respect to the center of the articular cyst opening for each horse. Small- and intermediate-volume SBCs were irregular and multilobulated, whereas large-volume SBCs were smooth and discrete with a surface area-to-volume ratio approaching that of a sphere.
CONCLUSIONS AND CLINICAL RELEVANCE Consistency in morphological characteristics suggested a common etiopathogenesis for SBCs in MFCs of adult horses. Cyst enlargement may have been attributable to a biomechanical predisposition to decrease the surface area-to-volume ratio, resulting in a spherical cyst.