The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples.

Middle carpal cartilage explants from 4 horses with mild osteoarthritis involving that joint were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan (PSGAG) on proteoglycan synthesis and degradation. Cultures were exposed to 0.025 or 25 mg of PSGAG/ml for 48 hours, after which the medium was replaced with medium containing similar doses of PSGAG and 35S. Subsequently, the sulfated proteoglycan content of the medium and extracts of the explants was measured. Gel filtration chromatography was used to estimate the size and to purify the principal, large proteoglycan monomer, which was further characterized by digestion, using glycosidic enzymes. In a second experiment, explants were incubated with 35S for 48 hours, and were subsequently exposed to the same concentrations of the PSGAG for an additional 48 hours. The amount of remaining labeled proteoglycan was determined for culture medium and cartilage extracts. Gel filtration chromatography was used to assess the hydrodynamic size of the large proteoglycan monomer. Aliquots of proteoglycans from the second experiment were incubated in high-molecular weight hyaluronate and chromatographed to assess reaggregation. Polysulfated glycosaminoglycan caused a significant (P < 0.04) decrease in sulfated proteoglycan synthesis by cartilage explants. Radioactive proteoglycan content in explants labeled prior to exposure to PSGAG were similar. Large proteoglycan monomer size was similar in both experiments (median partition coefficient [K(AV)] = 0.40), and was not influenced by PSGAG treatment. Prelabeled explants exposed to hyaluronate and chromatographed under associative conditions had similar proportions of the radiolabel eluting as proteoglycan aggregate. Enzymatic digestion of newly synthesized large monomer revealed a mild dose-dependent increase in the proportion of keratan sulfate substitution on core protein. It was concluded that PSGAG in vitro, at the dosages evaluated, caused a decre.

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.

Objective: To determine concentration-dependent effects of tiludronate on cartilage explants incubated with or without recombinant equine interleukin-1β (rEq IL-1). Sample: Articular cartilage explants from the femorotibial joints of 3 young adult horses. Procedures: Cartilage explants were incubated with 1 of 6 concentrations (0, 0.19, 1.9, 19, 190, or 1,900 mg/L) of tiludronate and with or without rEq IL-1 (0.01 ng/mL) for 96 hours. Prostaglandin E2 (PGE2) concentrations in culture medium and explant digests were analyzed via PGE2 enzyme immunoassay. Sulfated glycosaminoglycan (sGAG) concentrations in culture medium were quantified via 1,9-dimethylmethylene blue assay. Chondrocyte apoptosis in paraffin embedded explant sections was measured via terminal deoxynucleotidyl transferase-mediated dUTP nick end–labeling assay. Relative gene expression of matrix metalloproteinases (MMPs), interleukin (IL)-6, and IL-8 was determined via the comparative cycle threshold method. Results: rEq IL-1 increased PGE2 concentration, sGAG release from explants, chondrocyte apoptosis, and MMP gene expression. Lower tiludronate concentrations reduced rEq IL-1–induced sGAG release and chondrocyte apoptosis, whereas the higher tiludronate concentrations increased sGAG release and chondrocyte apoptosis. At the highest tiludronate concentration evaluated, IL-8 gene expression was increased independent of whether rEq IL-1 was present. Conclusions and Clinical Relevance: Tiludronate had biphasic concentration-dependent effects on cartilage explants that were independent of PGE2 secretion or MMP gene expression. Low tiludronate concentrations had some chondroprotective effects, whereas high tiludronate concentrations were detrimental to equine articular cartilage. Administration of tiludronate intra-articularly to horses may be detrimental, dependent on the dose used. In vivo studies are needed before intra-articular tiludronate administration to horses can be recommended.

Objective: To determine whether incubation of cruciate ligament cells with acetylsalicylic acid, carprofen, meloxicam, or robenacoxib provides protection against apoptosis induced by sodium nitroprusside (SNP). Sample: Explants of cranial (CCL) and caudal (CaCL) cruciate ligaments from eight 1-day-old Beagles. Procedures: Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of carprofen/mL; 0.1, 1, or 10 μg of meloxicam/mL; or 0.1, 1, or 10 μg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E2 concentrations were measured via an ELISA. Results: Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 μg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE2 production from SNP-damaged cells. Conclusions and Clinical Relevance: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments.

Objective-To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. Sample Population-Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. Procedure-The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interluekin-1, tumor necrosis factor-α, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP. Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). Results-All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and nonstimulated cultures. Conclusion and Clinical Relevance—Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples. However, similar analysis of data on GAG release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of LPS derived from different bacterial sources made a significant difference in the response of explants to incubation with LPS.

Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis. Cultures were exposed to concentrations of 0, 50, or 200 microgram of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage. In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days. Subsequently, the amount of remaining labeled proteoglycan was determined. Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment. Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure. Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug. Proteoglycan monomer size was similar in all treatment groups. It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture.

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 (1micromolar [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested amount of a proprietary siRNA molecule. Full-thickness cartilage explants were treated with 1× (1micromolar), 2× (2micromolar), and 4× (4micromolar) or 1× (0.13micromolar), 4× (0.52micromolar), and 8× (1.04micromolar) 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 RelevanceRobust 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.