Effects of enrofloxacin and ciprofloxacin hydrochloride on canine and equine chondrocytes in culture

OBJECTIVE: To study chondrotoxic effects of enrofloxacin (ENR) and ciprofloxacin hydrochloride (CFX) on canine and equine articular chondrocytes in culture and to compare the effects with that of cultivation in Mg2+-free medium. SAMPLE POPULATION: Chondrocytes from articular cartilage of 4- and 6 -month old dogs and 2- to 4- year-old horses. PROCEDURE: Chondrocytes were cultivated with 10, 40, 80, and 160 microg of CFX/ml, 10, 50, 100, and 150 microg of ENR/ml, or in Mg2+-free medium. A live-to-dead test was performed to test cytotoxic effects. Morphologic changes were evaluated by electron microscopy. An attachment assay was used to test the ability of chondrocytes to adhere to collagen type-II coated-chamber slides in the presence of CFX and with Mg2+-free medium. RESULTS: Chondrocytes cultivated in quinolone-supplemented medium or Mg2+-free medium had a decreased ability to adhere to culture dishes. Cell shape and the actin and vimentin cytoskeleton changed in a concentration-dependent manner. These effects were not species-specific and developed with both quinolones. On day 1 of culture, adhesion of chondrocytes to collagen type II was reduced to 70 and 45% of control values in the CFX treatment and Mg2+-free treatment groups, respectively. On day 5 of culture, adhesion of chondrocytes was reduced to 45 and 40% of control values in the CFX treatment and Mg2+-free treatment groups, respectively. CONCLUSION AND CLINICAL RELEVANCE: In vitro, chondrotoxic effects of quinolones appear to be the result of irregular integrin signaling and subsequent cellular changes. Drug concentrations leading to morphologic changes in vitro may be achieved in articular cartilage in vivo.     

In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional culture

OBJECTIVE: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture. SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities. PROCEDURE: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined. RESULTS: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected. CONCLUSIONS: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.     

Phenotypic expression of equine articular chondrocytes grown in three-dimensional cultures supplemented with supraphysiologic concentrations of insulin-like growth factor-1

OBJECTIVE: To assess the effects of supraphysiologic concentrations of insulin-like growth factor-1 (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 kg of IGF-1/ml enhanced phenotypic expression of chondrocytes without inducing detrimental cellular or metabolic effects. Highest concentration of IGF-1 (25 microg/ml) significantly increased total DNA content, glycosaminoglycan (GAG) content, GAG synthesis, and size of proteoglycan monomers produced, compared with cultures supplemented with 12.5 microg 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 microg of IGF-1/ml, compared with cultures supplemented with 12.5 microg 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.     

In vitro effects of beta-hydroxy-beta-methylbutyrate (HMB) on cell-mediated immunity in fish

beta-Hydroxy-beta-methyl butyrate(HMB) has been shown to counteract many of the negative effects of intensive animal production methods and results in increased growth and protection against diseases. In the present study, the effect of HMB on the immunocompetence cell activity in rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) was examined. Pronephric phagocytes and lymphocytes were isolated from the fish and grown in culture medium (RPMI-1640) containing either 0, 0.1, 1, 5, 10, 25, 50 or 100 microg HMB/ml of medium. The effects of HMB on the respiratory burst activity (RBA) stimulated by phorbol myristate acetate (PMA), the potential killing activity (PKA) and lymphocyte proliferation stimulated by either concanavalin A (Con-A) or lipopolysaccharide (LPS) were examined. The addition of HMB to the culture medium increased the RBA by up to 84% (p<0.01) over that of cells grown without HMB. Similarly, the PKA of the phagocytes was also increased with HMB addition to the medium by up to 140% (p<0.01) over that of cells grown without HMB. Lymphocyte proliferation stimulated by both ConA and LPS was also increased approximately two-fold (p<0.01) when HMB was added to the culture medium at concentrations between 10 and 100 microg HMB/ml in both rainbow trout and carp. The greatest effects of HMB on RBA and PKA activities were observed at a concentration >50 microg HMB/ml while lymphocyte proliferation was maximally stimulated at 25 microg HMB/ml. In conclusion, the current study shows that HMB could potentially improve immunocompetence cell activity in fish through increased cell proliferation and functionality.     

Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.     

Effects of carprofen and dexamethasone on canine chondrocytes in a three-dimensional culture model of osteoarthritis

OBJECTIVE: To determine effects of carprofen and dexamethasone on chondrocytes in a culture model of osteoarthritis (OA). SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of the humeral head of 5 adult dogs. PROCEDURE: Chondrocytes were harvested, cultured and subcultured in monolayer, and then cultured in a 3-dimensional (3-D) medium. Cells from each dog were distributed into 6 groups with differing content of liquid medium for each 3-D construct (agarose [AG], AG plus interleukin [IL]-1beta, AG plus carprofen [4 microg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1beta [20 ng/mL] plus carprofen [4 microg/mL], and AG plus IL-1beta (20 ng/mL) plus dexamethasone (1 mg/mL). On days 3, 6, 12, and 20 of culture, samples from all groups were collected. Liquid media were assayed for glycosaminoglycan, prostaglandin (PG)E2, matrix metalloprotease (MMP)-3, and MMP-13 concentrations. All 3-D constructs were evaluated for viability, cell morphology, proteoglycan staining, and collagen type-II concentration. Total glycosaminoglycan content in each 3-D construct was quantitated by spectrophotometric assay. RESULTS: Addition of IL-1beta caused a significant loss of cell viability and matrix production. Addition of carprofen or dexamethasone caused significant decreases in PGE2 in the liquid media, and each was minimally effective in protecting chondrocytes against negative effects of IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Human recombinant IL-1beta resulted in loss of cell viability, alterations in extracellular matrix components, and production of PG and MMP Carprofen and dexamethasone had little effect on cell and matrix variables but did decrease PGE2 concentrations and primarily affected the inflammatory pathway of osteoarthritis.     

Dexamethasone induces apoptosis in proliferative canine tendon cells and chondrocytes

Dexamethasone (Dexa) has been commonly used in humans and domestic animals, particularly in the treatment of tendon injuries and cartilage degeneration. However, it is often associated with tendon rupture and impaired tendon and cartilage healing. In the present study, we investigated Dexa's in vitro effects on the growth of cell proliferation and the induction of apoptosis in canine Achilles tendon cells and chondrocytes. Cell proliferation after treatment with Dexa for two to six days was quantified by a 2,3-bis{2-methoxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5-carboxyanilide inner salt assay (XTT). The results showed that Dexa could inhibit the proliferation of tendon cells and chondrocytes at increasing concentrations (0.1-50 microg/ml) compared with untreated cells. Cell apoptosis was induced by Dexa, as evidenced by the typical nuclear apoptosis using Hoechst 33258 staining. Dexa increased the apoptosis of canine tendon cells and chondrocytes in a time-dependent manner. In canine tendon cells and chondrocytes that were treated with 25 and 50 microg/ml concentration of Dexa, the number of condensed apoptotic nuclei was significantly increased. In addition, culturing with Dexa and the glucocorticoid receptor blocker, mifepristone, significantly arrested apoptosis of tendon cells and chondrocytes. Based on our in vitro data, we hypothesized that in vivo treatment with glucocorticoids may diminish the proliferation of tendon and cartilage cells by increasing apoptosis and suppressing the proliferation. Our findings suggest that Dexa could be used with caution in dogs with articular or tendon problems.     

Isolation, propagation, and cryopreservation of equine articular chondrocytes.

Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffered with HEPES, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at -196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.     

Effects of human recombinant interleukin-1beta on canine articular chondrocytes in three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1beta on matrix synthesis and degradation by chondrocytes cultured in a 3-dimensional (3-D) gel medium. SAMPLE POPULATION: Chondrocytes from 7 dogs. PROCEDURE: Articular chondrocytes were harvested and cultured in 3-D gel medium alone or with 10 or 20 ng IL-1beta/ml that was added beginning on day 0, 3, 6, or 9. On days 3, 6, 12, and 20 of 3-D culture, samples of the liquid medium were evaluated for glycosaminoglycan (GAG), prostaglandin E2 (PGE2), and matrix metalloprotease (MMP)-3 content. The 3-D plug in each well was evaluated for histologic characteristics of viability, cell morphology, and proteoglycan staining, immunohistochemically stained for collagen type II, and spectrophotometrically analyzed for GAG content. RESULTS: Significant differences for all variables were detected between controls and each IL-1beta group, among groups with different IL-1beta concentrations, and among groups with IL-1beta added at various time points. Chondrocytes exposed to IL-1beta had loss of GAG, increased PGE2 and MMP-3 concentrations, and lack of collagen type-II synthesis. These IL-1beta effects appeared to be time and concentration dependent. CONCLUSIONS: Addition of IL-1beta to chondrocytes in 3-D gel medium results in time- and concentration-dependent effects on matrix synthesis and degradation and provides an appropriate in vitro model for many of the pathophysiologic events associated with osteoarthritis.     

Three-dimensional culture of feline articular chondrocytes in alginate microspheres

Chondrocytes isolated from proximal femoral articular cartilage from 3 adult cat cadavers were expanded in monolayer culture and subsequently cultured in alginate microspheres for 24 days. Cell proliferation and production of proteoglycans in alginate microspheres were observed during day 18 and 24. Quantification of chondroitin sulfates (CS) by capillary electrophoresis revealed that cultured chondrocytes synthesized CS6 but not CS4. Three-dimensional culture using alginate microspheres is a useful in vitro technique to study proliferation and metabolism of chondrocytes; however, further modifications are needed to apply the technique to feline articular chondrocytes.     

Interleukin 4 increases CCR9 expression and homing of lymphocytes to gut-associated lymphoid tissue in chickens

The effects of in vitro and in vivo IL-4 supplementation on thymocyte and splenocyte CCR9 mRNA amount and migration were studied. Thymocytes, splenocytes, splenocytes+thymocytes (2:1), and splenocytes+bursocyte cells (2:1) were supplemented with either 0 or 5 ng/ml IL-4 for 5d. CCR9 mRNA was undetectable in all experimental groups supplemented with 0 ng/ml IL-4. IL-4 treatment (5 ng/ml) upregulated (P=0.01) CCR9 mRNA only in the splenocyte+thymocyte cell culture. IL-4-mediated CCR9 mRNA induction in the splenocyte+thymocyte cell culture was dependent on the in vitro dose of IL-4 supplementation. IL-4-treated splenocyte+thymocyte cells when injected in vivo preferentially migrated to cecal tonsils. In vivo supplementation of IL-4 was achieved through in ovo injection of recombinant chicken IL-4 plasmid. Cecal tonsils in chicks hatched from IL-4-plasmid-injected eggs weighed more, had a higher amount of CCR9 mRNA, and had a higher percentage of CD8(+) cells than cecal tonsils from chicks hatched from PBS-injected eggs. It could be concluded that IL-4 induces CCR9 mRNA in thymocytes and splenocytes and directs the migration of cells to gut-associated lymphoid tissue.     

铜对体外仔猪软骨细胞增殖和细胞骨架的影响

体外分离、培养仔猪关节软骨细胞,在细胞培养液中分别添加铜0、7.8、15.6、31.2、62.5μmol/L。结果表明,软骨细胞在4种铜浓度中可存活并增殖,但随铜浓度的增加,其存活率、增殖率、3H-TdR掺入率有明显的差异,且能破坏软骨细胞骨架。培养液中添加铜31.2μmol/L,对软骨细胞的增殖作用最强,增殖率、3H-TdR掺入数显著高于对照组(P<0.01),软骨细胞形态及骨架均正常。表明31.2μmol/L铜浓度是促进体外软骨细胞增殖的最适浓度。     

Effects of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1

OBJECTIVE: To determine the effects of various concentrations of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1 (FHV-1). SAMPLE POPULATION: Cultured Crandell-Reese feline kidney (CRFK) cells and FHV-1 strain 727. PROCEDURE: Uninfected CRFK cells or CRFK cells infected with FHV-1 were cultured in Dulbecco's modified Eagle's medium or in 1 of 7 test media containing various concentrations of lysine and arginine. Viral titer and CRFK growth rate were assessed in each medium. RESULTS: Media depleted of arginine almost completely inhibited viral replication, whereas 2.5 or 5.0 microg of arginine/ml of media was associated with a significant increase in FHV-1 replication. In media with 2.5 microg of arginine/ml, supplementation with 200 or 300 microg of lysine/ml reduced viral replication by 34.2 and 53.9%, respectively. This effect was not seen in media containing 5.0 microg of arginine/ml. Growth rates of CRFK cells also were suppressed in media containing these concentrations of amino acids, but they were not significantly different from each other. CONCLUSIONS AND CLINICAL RELEVANCE: Arginine exerts a substantial growth-promoting effect on FHV-1. Supplementation of viral culture medium with lysine attenuates this growth-promoting effect in media containing low concentrations of arginine. Analysis of data from this study indicates that high concentrations of lysine reduce in vitro replication of FHV-1 but only in media containing low concentrations of arginine. Clinical trials will be necessary to determine whether supplemental administration of lysine, with or without arginine restriction, will be useful in the management of cats with FHV-1 infections.     

Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.     

The effects of enrofloxacin on canine tendon cells and chondrocytes proliferation <Emphasis Type="Italic">in vitro</Emphasis>

Enrofloxacin, a fluoroquinolone antibiotic has been used widely in humans and domestic animals, including dogs, because of its broad-spectrum activity and relative safety. The side effects of fluoroquinolone, induced tendinopathy, tendonitis, spontaneous tendon rupture and cartilage damage, remain incompletely understood. In the present study, we investigated the in vitro effects of enrofloxacin on cell proliferation and induction of apoptosis in canine Achilles tendon cells and chondrocytes. Cell growth and proliferation after treating with enrofloxacin for 2–6 days was quantified by a colorimetric 2,3-bis{2-methoxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay. The results showed that enrofloxacin could inhibit the proliferation of canine tendon cells and chondrocytes at increasing concentrations (10–200 μg/ml). The inhibition of proliferation of canine tendon cells and chondrocytes after exposure to enrofloxacin were associated with induction of apoptosis, as evidenced by the typical nuclear apoptotic condensed nuclei found using Hoechst 33258 staining. It was demonstrated that canine tendon cells and chondrocytes treated with 200 μg/ml enrofloxacin for 4 days exhibited apoptotic features and fragmentation of DNA. Enrofloxacin also increased the apoptosis of canine tendon cells and chondrocytes in a dose and time-dependent manner. The results indicate that enrofloxacin inhibits cell proliferation, induces apoptosis and DNA fragmentation, which might explain enrofloxacin-induced tendinopathy and cartilage damage.     

Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes

Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-alpha (100 ng mL(-1)), IL-1beta (100 ng mL(-1)), IGF-I (20 ng mL(-1)), TGF-beta (20 ng mL(-1)) and insulin (12.5 microg mL(-1)) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-beta, IL-1beta and TNF-alpha resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors.     

Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharide-stimulated equine chondrocytes

OBJECTIVE: To characterize potential mechanisms of action of glucosamine inhibition of matrix metalloproteinase (MMP) expression and activity in lipopolysaccharide (LPS)-stimulated equine chondrocytes. SAMPLE POPULATION: Chondrocytes cultured from samples of metacarpophalangeal articular cartilage collected from cadaveric limbs of horses. PROCEDURE: The effect of glucosamine on MMP activity in conditioned medium from LPS-stimulated cartilage explants was determined by a colorimetric assay with azocoll substrate. Treatments consisted of negative and positive controls, glucose (50 mM), and glucosamine (50, 25, 6.25, 3, and 1.5 mM). The influence of glucosamine on MMP synthesis was determined in chondrocytes in pellet culture incubated with LPS (20 microg/mL). Concentration of MMP-13 was quantified in spent medium via ELISA; nonspecific MMP activity was determined via azocoll digestion in organomercurial-activated medium. Effects of glucosamine on MMP mRNA concentration in similarly treated chondrocytes were determined by northern blot hybridization with MMP-1, -3, and -13 probes. Statistical analyses were performed with 2-way ANOVA. RESULTS: Glucosamine had no effect on activated MMP activity but inhibited MMP protein expression, as determined by azocoll digestion (glucosamine, 3 to 50 mM) and MMP-13 ELISA (glucosamine, 1.5 to 50 mM). Resting mRNA concentrations for MMP-1, -3, and -13 mRNA were significantly lower in cultures exposed to glucosamine at concentrations of 50 and 25 mM than those of positive controls. CONCLUSIONS AND CLINICAL RELEVANCE: Glucosamine appears capable of pretranslational, and possibly also translational, regulation of MMP expression; data suggest a potential mechanism of action for chondroprotective effects of this aminomonosaccharide.