Osteoarthritis is one of the most common performance-limiting maladies of athletic horses.1,2 Traditional osteoarthritis treatment regimens have focused on the use of NSAIDs. Although these drugs have been effective in reducing arthritis-related pain, NSAIDs do not reliably arrest progression of joint disease and have been implicated in the development of clinically important adverse effects such as gastrointestinal ulceration.3 They may also speed progression of osteoarthritis by allowing increased use of damaged joints, thereby increasing cartilage destruction.4 Intra-articular injection of corticosteroids has been an economical and efficacious treatment; however, certain preparations and doses may cause undesirable morphologic changes in articular cartilage, leading to progression of osteoarthritis.5–8 Other promising treatments have included polysulfated glycosaminoglycans and hyaluronic acid; however, rigorous randomized controlled clinical trials proving efficacy are scant.9
Interest in alternative treatments for osteoarthritis to improve patient mobility, reduce clinical pain, relieve financial burden of treatment, improve ease and convenience of treatment, and reduce adverse effects of traditional treatments has been growing. The 6-carbon amino sugar glucosamine has been used as an alternative treatment for arthritis. Some support is found within the peer-reviewed literature, suggesting that glucosamine may possess benefits as a palliative reliever of osteoarthritis pain and may confer beneficial metabolic effects on articular chondrocytes.10–12 In vitro research has elucidated several mechanisms of glucosamine that lend support to its role in osteoarthritis treatment. Proteoglycans are a major component of cartilage matrix, and their degradation is an important contributor to the pathophysiologic processes of osteoarthritis. Glucosamine prevents degradation of cartilage proteoglycans in vitro13,14 and upregulates production of proteoglycan by chondrocytes.15 These effects are likely secondary to downregulation of aggrecanase enzymes and upregulation of aggrecan gene expression.16–18 Inflammation is an important contributor to osteoarthritis, and glucosamine downregulates the inflammatory agents nitric oxide and PGE2.17,19 Additionally, MMPs, a family of degradative enzymes responsible for cartilage extracellular matrix damage, are also downregulated by glucosamine.11,16,17 Taken together, these findings support a role for glucosamine in the protection of cartilage matrix and chondrocyte metabolism, suggesting a possible mechanism whereby glucosamine may help alleviate clinical signs and retard progression of osteoarthritis.
Pharmacokinetic evidence has recently been presented that suggests the majority of in vitro research has been conducted with glucosamine concentrations well in excess of those attained in vivo after oral administration.20–22 Serum glucosamine concentrations reach 2 μg/mL in humans after oral administration.20 These data agree well with findings in horses, with serum glucosamine concentrations reaching between approximately 1 and 10 μg/mL after oral administration, depending on the dose given.21,22 Some existing evidence suggests that glucosamine retains beneficial effects at similar concentrations.16,17,19 However, synovial fluid glucosamine concentrations in horses reach only 10% of those found in serum.22 If the beneficial actions of glucosamine are attributed to local effects within the joint environment, then support should be found for metabolic actions at these low concentrations.
Synoviocytes are important moderators of articular cartilage damage.23 Synoviocytes are likely exposed to plasma glucosamine concentrations 10-fold higher than the synovial fluid concentrations that chondrocytes are exposed to because of their proximity to the periarticular blood supply. Given the importance of synoviocytes in the pathogenesis of osteoarthritis and their exposure to potentially higher concentrations of glucosamine than chondrocytes, synoviocytes may be a target for therapeutic activity.
The objective of the study reported here was to evaluate the effects of glucosamine on equine articular chondrocytes and synoviocytes at concentrations clinically relevant to plasma and synovial fluid concentrations. Our hypothesis was that glucosamine, at clinically achievable tissue concentrations, would reduce inflammatory mediator release, reduce degradative enzyme release, and improve production of proteoglycan by cultured equine chondrocytes and synoviocytes.
Materials and Methods
Tissue sources—Articular cartilage and synovium with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints were obtained from 8 horses (1 to 10 years of age) that had died or had been euthanatized with an overdose of pentobarbital for reasons other than joint disease. Cartilage was dissected free of underlying subchondral bone, and synovium was dissected free from the outer layer (or subintima). Both tissues were incubated separately at 25°C for 1 hour in physiologic saline (0.9% NaCl) solution containing 1% penicillin and streptomycin.a Chondrocytes and synoviocytes were isolated separately by 16-hour digestion at 37°C with 0.15% collagenaseb in Dulbecco modified Eagle medium.c Following digestion, cells were counted by use of a hemocytometer and viability was determined via trypan blue stain exclusion.d Chondrocytes and synoviocytes were separated by centrifugation (300 × g for 5 minutes), washed, and resuspended in Dulbecco modified Eagle medium supplemented with 1% penicillin and streptomycin, 4.5 g of glucose/L, 1% L-glutamine,e 50 μg of ascorbic acid/mL,f and 10% fetal bovine serum.g
Cell cultures—Chondrocyte pellets (5 × 106 cells) were formed by centrifugation (300 × g for 5 minutes) in 15-mL polypropylene centrifuge tubes and incubated under standard cell culture conditions (37°C in 95% relative humidity with 5% carbon dioxide). Synoviocytes were seeded at a density of 5 × 104 cells in culture flasks and grown as monolayers to confluence in identical conditions. Medium was renewed every 2 to 3 days. Cells were deprived of serum for 3 days prior to the start of an experiment. On day 7, cultures were supplemented with glucosamineh (0 to 20 μg/mL) with or without IL-1 (10 ng/mL)i and incubated for an additional 48 hours. Twenty-four hours prior to the end of the experiment, chondrocyte pellets were radiolabeled with media containing sulfur 35 (35S) labeled sodium sulfate (10 μCi/mL).j Cells and media were collected and stored at −80°C.
Proteoglycan—Radiolabeled pellets were papain digested (150 μg of papaink/mL) at 65°C for 24 hours. Precipitation with Alcian blue dyek and scintillation of the explant digests and media were used to determine new proteoglycan production as a result of incorporation of 35S into the cartilage matrix.24 Results were expressed as counts per minute per chondrocyte pellet.
MMP-13—Stored media from chondrocyte and synoviocyte cultures were assayed for MMP-13 and proMMP-13 by means of a commercially available kitl (in accordance with the instructions of the manufacturer). Briefly, 100 μL of prepared standard and test media (diluted 1:4) was incubated at 25°C for 2 hours in assay wells coated with anti-MMP-13 antibody. Wells were washed with the supplied phosphate buffer and incubated at 25°C for 1 hour with 100 μL of anti-MMP-13 peroxidase conjugate. Wells were washed and incubated at 25°C for precisely 30 minutes with 100 μL of 3,3′5,5′-tetramethylbenzidine-hydrogen peroxide in 20% (wt/vol) dimethylformamide. The reaction was stopped by the addition of 1.0M sulfuric acid, and optical density was measured at 450 nm.m
PGE2—The concentration of PGE2 in spent media from chondrocyte and synoviocyte cultures was determined by use of a commercial colorimetric assay,n following the directions of the manufacturers. Briefly, 10-fold dilutions of the stored media were incubated in assay buffer containing a fixed amount of competitive alkaline phosphatase–labeled PGE2. Assay wells were washed, and p-nitrophenyl phosphate was added as a substrate for alkaline phosphatase. Absorbance was measured at 405 nm,m and PGE2 concentrations were determined by comparison to a standard curve.
Quantitative real-time reverse-transcriptase PCR assay—Total RNA was isolated from chondrocytes and synoviocytes by Trizolo extraction, following the instructions of the manufacturer. First-strand cDNA was synthesized by use of dNTP mixp and reverse transcriptaseq at 42°C for 50 minutes. Quantitative real-time reverse-transcriptase PCR assay was performed for cyclooxygenase-2 and mPGEs by use of elongation factor-1α as a normalization standard in an automated PCR detection system.r
Statistical analysis—Data were compared by use of repeated-measures ANOVA with a statistical software program.s Values of P < 0.05 were considered significant. Nonparametric data were compared by use of repeated-measures ANOVA on ranks. Post hoc analysis was accomplished by use of the Student-Newman-Keuls method.
Results
Proteoglycan and MMP-13—Treatment with glucosamine at any concentration had no effect on production of proteoglycan by chondrocytes as measured in chondrocyte pellets or media under noninflammatory (no IL-1) or inflammatory (with IL-1) conditions. Treatment with glucosamine at any concentration had no effect on production of MMP-13 by chondrocytes or synoviocytes under noninflammatory or inflammatory conditions.
PGE2—Treatment with glucosamine had no effect on production of PGE2 by chondrocytes at any concentration studied under noninflammatory (no IL-1) or inflammatory (with IL-1) conditions. Treatment with glucosamine at 5 μg/mL significantly decreased production of PGE2 by synoviocytes under inflammatory (with IL-1) conditions, compared with that of positive control synoviocytes (ie, with IL-1 and without glucosamine; Figure 1).
Mean ± SE production of PGE2 by equine synoviocytes. Treatment included absence () and presence (+) of IL-1 with glucosamine. *Significant (P < 0.05) decrease in production under inflammatory (with IL-1) conditions, compared with that of positive control conditions (ie, with IL-1 and no glucosamine [0 μg/mL]). G0, G0.1, G0.5, G1, G5, G10, and G20 = Treatment with glucosamine at 0, 0.1, 0.5, 1, 5, 10, and 20 μg/mL, respectively. For all treatments, n = 8 horses.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE production of PGE2 by equine synoviocytes. Treatment included absence () and presence (+) of IL-1 with glucosamine. *Significant (P < 0.05) decrease in production under inflammatory (with IL-1) conditions, compared with that of positive control conditions (ie, with IL-1 and no glucosamine [0 μg/mL]). G0, G0.1, G0.5, G1, G5, G10, and G20 = Treatment with glucosamine at 0, 0.1, 0.5, 1, 5, 10, and 20 μg/mL, respectively. For all treatments, n = 8 horses.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE production of PGE2 by equine synoviocytes. Treatment included absence () and presence (+) of IL-1 with glucosamine. *Significant (P < 0.05) decrease in production under inflammatory (with IL-1) conditions, compared with that of positive control conditions (ie, with IL-1 and no glucosamine [0 μg/mL]). G0, G0.1, G0.5, G1, G5, G10, and G20 = Treatment with glucosamine at 0, 0.1, 0.5, 1, 5, 10, and 20 μg/mL, respectively. For all treatments, n = 8 horses.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Cyclooxygenase-2—Treatment with glucosamine had no effect on cyclooxygenase-2 production by chondrocytes or synoviocytes at any concentration studied under noninflammatory (no IL-1) or inflammatory (with IL-1) conditions. A general decrease in cyclooxygenase-2 production by chondrocytes was observed with increases in glucosamine concentrations under inflammatory conditions, although this effect was not significant (Figure 2).
Mean ± SE relative production of cyclooxygenase-2 (COX-2) by equine chondrocyte pellets. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE relative production of cyclooxygenase-2 (COX-2) by equine chondrocyte pellets. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE relative production of cyclooxygenase-2 (COX-2) by equine chondrocyte pellets. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
mPGEs—Treatment with glucosamine at 0.1 and 0.5 μg/mL significantly decreased production of mPGEs by chondrocytes under inflammatory (with IL-1) conditions, compared with that of positive control chondrocytes (ie, with IL-1 and without glucosamine; Figure 3). Treatment with glucosamine at 0.1 μg/mL significantly decreased production of mPGEs by synoviocytes under inflammatory (with IL-1) conditions, compared with that of positive control synoviocytes (Figure 4).
Mean ± SE relative production of mPGEs by equine chondrocyte pellets. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE relative production of mPGEs by equine chondrocyte pellets. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE relative production of mPGEs by equine chondrocyte pellets. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE relative production of mPGEs by equine synoviocytes. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE relative production of mPGEs by equine synoviocytes. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Mean ± SE relative production of mPGEs by equine synoviocytes. Treatment included absence () and presence (+) of IL-1 with glucosamine. See Figure 1 for key.
Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1129
Discussion
Results of this study indicate that glucosamine, at clinically relevant concentrations, does not impact production of proteoglycan by chondrocytes or the amount of MMP-13 released by chondrocytes and synoviocytes under noninflammatory or inflammatory conditions in this culture system. Glucosamine did have some limited effects on markers of inflammation at concentrations between 0.1 and 5 μg/mL. The hypothesis was therefore largely unsupported by these data.
Although glucosamine has been shown to increase the production of proteoglycan by chondrocytes,15 other researchers have failed to demonstrate any effect on production of proteoglycan or aggrecan at concentrations close to those achieved in vivo after oral administration.16,25 Evidence exists that beneficial effects of glucosamine on extracellular matrix gene expression may be enhanced by its use in combination with chondroitin sulfate and that these effects may not be observed in short-term culture.26 In addition, other researchers have shown that beneficial effects of glucosamine on matrix synthesis may occur within a narrow optimal concentration range.18,27 It is likely that the use of glucosamine alone at low concentrations under short-term culture conditions precluded discovery of any benefit to production of proteoglycan in the present study.
Glucosamine decreases the IL-1–induced upregulation of MMP-13 transcription and translation.11 Clinically relevant concentrations of glucosamine have been shown to reduce inflammatory-induced MMP-13 mRNA expression.16,17 The lack of an effect by glucosamine on MMP-13 production in the present study, as with the proteoglycan data, is likely attributable to the use of low concentrations and short-term culture conditions. It is possible that the single time point at which MMP-13 was assayed may have contributed to the negative findings and that positive effects may be evident at other time points. Additionally, lack of treatment effect on certain markers in equine tissue,17 compared with findings in studies19,26 on bovine tissue, may be attributed to greater variability in responses in tissues from horses. The inherent genetic homogeneity in the bovine population may lend lower variability to metabolic treatment responses from these tissues.
Glucosamine has been shown to suppress PGE2 production in chondrocytes and synoviocytes from osteoarthritic joints, although this effect was not evident at concentrations < 100 μg/mL.28 Other investigators have reported beneficial effects of glucosamine at clinically relevant concentrations on inflammatory mediators in IL-1–stimulated chondrocytes.19 Our findings lend some support to the contention that glucosamine is an effective mitigator of inflammatory mediator production and release at clinically relevant concentrations. Although cyclooxygenase-2 was not downregulated with any glucosamine treatment in either cell type, PGE2 was abrogated in synoviocytes in inflammatory conditions at a concentration of 5 μg/mL. This concentration is realistic for what synoviocytes are exposed to in vivo, according to recent pharmacologic data.20–22 Furthermore, mPGEs, the terminal enzyme in PGE2 synthesis, was downregulated under inflammatory conditions at concentrations of 0.1 and 0.5 μg/mL in chondrocytes and 0.1 μg/mL in synoviocytes. These data may be particularly germane because chondrocytes are likely exposed to glucosamine concentrations < 1 μg/mL in vivo after oral administration.22 To our knowledge, this is the first study to determine anti-inflammatory effects of glucosamine at concentrations encountered by chondrocytes in vivo. It is important to note, however, that these effects were not globally observed across the PGE2 biosynthetic pathway. It is likely that the degree of anti-inflammatory activity by glucosamine may temporally fluctuate, as other investigators have reported this phenomenon when measuring degradative enzymes and proinflammatory agents.16,19 It is possible that the present study did not detect anti-inflammatory effects taking place earlier in the experiment.
The use of glucosamine as the sole treatment in the present study was designed to isolate the effects of this single agent and to allow investigation at several concentrations within the anticipated achievable in vivo range. However, it is important to recognize that glucosamine is often administered in combination with other agents, most commonly chondroitin sulfate. Some support exists for this rationale because others have reported additive or synergistic effects when glucosamine and chondroitin sulfate are administered concurrently. Glucosamine and chondroitin sulfate in combination stimulate glycosaminoglycan synthesis in a synergistic manner, compared with administration of either agent alone.29 Likewise, combination treatment is more effective than each agent alone in mitigating IL-1–induced increases in proteolytic enzymes.16 Despite the support for combination treatment, we believe it is important to evaluate the effects of these agents individually to more fully determine the mechanism of action of each. Whether glucosamine and chondroitin sulfate are complementary through similar modes of action or through separate but interrelated systems remains to be determined.
Despite evidence that synoviocytes are an important contributor to the progression of osteoarthritis,23 few studies14,28 have investigated the effects of glucosamine on these cells. Our results support a role for synoviocytes in the progression of osteoarthritis through upregulation of MMP-13 and the PGE2 biosynthetic pathway. Higher concentrations of glucosamine than those used in the present study have been shown to suppress PGE2 and MMP production by synoviocytes.28 Synoviocyte gene transfer experiments aimed at upregulating the rate-limiting enzyme in endogenous cellular glucosamine synthesis, glutamine:fructose-6-phosphate amidotransferase, have similarly decreased IL-1–induced PGE2 production.14 To our knowledge, this is the first study that has investigated the response of synoviocytes to glucosamine treatment at clinically relevant concentrations.
Several treatments resulted in values with high variability in this study, particularly in the production of PGE2. We attribute this high variability to genetic diversity of the tissues as well as to the wide age range of horses. An additional possibility is that varying glucosamine concentrations had an impact on cell growth or viability. We feel this possibility is less likely because of the low concentrations of glucosamine used as well as the short duration of exposure. Although glucosamine can have an impact on cell growth kinetics and viability, these effects have only been observed at much higher concentrations than those in the present report.18,30
Results of the present study have supported a role for glucosamine in mitigating the proinflammatory effects of IL-1 on chondrocytes and synoviocytes, although this activity was not observed at all concentrations. Importantly, these effects occurred at glucosamine concentrations relevant to clinical usage for chondrocytes and synoviocytes. Glucosamine did not confer beneficial effects on proteoglycan or MMP-13 production in either cell type at the concentrations studied. It is likely that the observed effects are related to the ability of glucosamine to modulate transcription factors that are upregulated in osteoarthritis and in IL-1–stimulated cells. Transcription factors nuclear factor-KB and activator protein-1 have been implicated in osteoarthritis and are downregulated by glucosamine.12,17,31,32 It is becoming clearer, as the body of work on glucosamine accumulates, that there is some basis for its purported positive effects as a treatment for osteoarthritis.
ABBREVIATIONS
| IL-1 | Interleukin-1 |
| MMP | Matrix metalloproteinase |
| mPGEs | Microsomal prostaglandin E2 synthase |
| PGE2 | Prostaglandin E2 |
BioWhittaker, Cambrex BioScience, Walkersville, Md.
Collagenase type 2, Worthington Biochemical Corp, Lakewood, NJ.
Mediatech Inc, Herndon, Va.
Trypan blue stain 0.4%, Invitrogen, Carlsbad, Calif.
L-glutamine, 200mM, Invitrogen, Carlsbad, Calif.
WAKO, Richmond, Va.
Gemini Bioproducts, Woodland, Calif.
Glucosamine hydrochloride, Sigma-Aldrich, St Louis, Mo.
rhIL-1, R&D Systems, Minneapolis, Minn.
Na2 (35S) SO4, AP Biochemicals, Costa Mesa, Calif.
Sigma-Aldrich, St Louis, Mo.
MMP-13 ELISA, Amersham Biosciences Inc, Piscataway, NJ.
FLUOstar Optima, BMG Labtech, Durham, NC.
PGE2 ELISA, R&D Systems, Minneapolis, Minn.
Invitrogen Corp, Carlsbad, Calif.
Oligo d(T), Invitrogen Corp, Carlsbad, Calif.
SuperScript II RT, Invitrogen Corp, Carlsbad, Calif.
iCycler iQ, Bio-Rad Laboratories, Hercules, Calif.
Sigma Stat, Systat Software, Richmond, Calif.
References
- 1.
Jeffcott LB, Rossdale PD, Freestone J, et al. An assessment of wastage in Thoroughbred racing from conception to 4 years of age. Equine Vet J 1982;14:185–198.
- 2.
Rossdale PD, Hopes R, Digby NJ, et al. Epidemiological study of wastage among racehorses, 1982 and 1983. Vet Rec 1985;116:66–69.
- 3.↑
May SA, Lees P. Nonsteroidal anti-inflammatory drugs. In: McIlwraith CW, Trotter GW, eds. Joint disease in the horse. Philadelphia: WB Saunders Co, 1996;223–236.
- 4.↑
Blot L, Marcelis A, Devogelaer JP, et al. Effects of diclofenac, aceclofenac and meloxicam on the metabolism of proteoglycans and hyaluronan in osteoarthritic human cartilage. Br J Pharmacol 2000;131:1413–1421.
- 5.
Chunekamrai S, Krook LP, Lust G, et al. Changes in articular cartilage after intra-articular injections of methylprednisolone acetate in horses. Am J Vet Res 1989;50:1733–1741.
- 6.
Trotter GW, McIlwraith CW, Yovich JV, et al. Effects of intra-articular administration of methylprednisolone acetate on normal equine cartilage. Am J Vet Res 1991;52:83–87.
- 7.
Shoemaker RS, Bertone AL, Martin GS, et al. Effects of intra-articular administration of methylprednisolone acetate on normal articular cartilage and on healing of experimentally induced osteochondral defects in horses. Am J Vet Res 1992;53:1446–1453.
- 8.
Frisbie DD, Kawcak CE, Baxter GM, et al. Effects of 6A-methylprednisolone acetate on an equine osteochondral fragment exercise model. Am J Vet Res 1998;59:1619–1628.
- 9.↑
Richardson DW, Loinaz R. An evidence-based approach to selected joint therapies in horses. Vet Clin North Am Equine Pract 2007;23:443–460.
- 10.
McAlindon TE, LaValley MP, Gulin JP, et al. Glucosamine and chondroitin for treatment of osteoarthritis: a systematic quality assessment and meta-analysis. JAMA 2000;283:1469–1475.
- 11.↑
Byron CR, Orth MW, Venta PJ, et al. Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharide-stimulated equine chondrocytes. Am J Vet Res 2003;64:666–671.
- 12.
Largo R, Alvarez-Soria A, Diez-Ortego I, et al. Glucosamine inhibits IL-1B-induced NFkB activation in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2003;11:290–298.
- 13.
Fenton JI, Chlebek-Brown KA, Peters TL, et al. Glucosamine HCl reduces equine articular cartilage degradation in explant culture. Osteoarthritis Cartilage 2000;8:258–265.
- 14.↑
Gouze JN, Gouze E, Palmer GD, et al. Adenovirus-mediated gene transfer of glutamine:fructose-6-phosphate amidotransferase antagonizes the effects of interleukin-1B on rat chondrocytes. Osteoarthritis Cartilage 2004;12:217–224.
- 15.↑
Bassleer C, Rovati L, Franchimont P. Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro. Osteoarthritis Cartilage 1998;6:427–434.
- 16.↑
Chan PS, Caron JP, Orth MW. Effect of glucosamine and chondroitin sulfate on regulation of gene expression of proteolytic enzymes and their inhibitors in interleukin-1-challenged bovine articular cartilage explants. Am J Vet Res 2005;66:1870–1876.
- 17.↑
Neil KM, Orth MW, Coussens PM, et al. Effects of glucosamine and chondroitin sulfate on mediators of osteoarthritis in cultured equine chondrocytes stimulated by use of recombinant equine interleukin-1beta. Am J Vet Res 2005;66:1861–1869.
- 18.
Varghese S, Theprungsirikul P, Sahani S, et al. Glucosamine modulates chondrocyte proliferation, matrix synthesis, and gene expression. Osteoarthritis Cartilage 2007;15:59–68.
- 19.↑
Chan PS, Caron JP, Rosa GJ, et al. Glucosamine and chondroitin sulfate regulate gene expression and synthesis of nitric oxide and prostaglandin E2 in articular cartilage explants. Osteoarthritis Cartilage 2005;13:387–394.
- 20.↑
Biggee BA, Blinn CM, McAlindon TE, et al. Low levels of human serum glucosamine after ingestion of glucosamine sulphate relative to capability for peripheral effectiveness. Ann Rheum Dis 2006;65:222–226.
- 21.
Du J, White N, Eddington ND. The bioavailability and pharmacokinetics of glucosamine hydrochloride and chondroitin sulfate after oral and intravenous single dose administration in the horse. Biopharm Drug Dispos 2004;25:109–116.
- 22.↑
Laverty S, Sandy JD, Celeste C, et al. Synovial fluid levels and serum pharmacokinetics in a large animal model following treatment with oral glucosamine at clinically relevant doses. Arthritis Rheum 2005;52:181–191.
- 23.↑
Theiler R, Ghosh P, Brooks P. Clinical, biochemical and imaging methods of assessing osteoarthritis and clinical trials with agents claiming chondromodulating activity. Osteoarthritis Cartilage 1994;2:1–23.
- 24.↑
Masuda K, Shirota H, Thonar E. Quantification of 35S labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates. Anal Biochem 1994;217:167–175.
- 25.
Qu CJ, Karjalainen HM, Helminen HJ, et al. The lack of effect of glucosamine sulphate on aggrecan mRNA expression and 35S-sulphate incorporation in bovine primary chondrocytes. Biochim Biophys Acta 2006;1762:453–459.
- 26.↑
Chan PS, Caron JP, Orth MW. Effects of glucosamine and chondroitin sulfate on bovine cartilage explants under long-term culture conditions. Am J Vet Res 2007;68:709–714.
- 27.
Derfoul A, Miyoshi AD, Freeman DE, et al. Glucosamine promotes chondrogenic phenotype in both chondrocytes and mesenchymal stem cells and inhibits MMP-13 expression and matrix degradation. Osteoarthritis Cartilage 2007;15:646–655.
- 28.↑
Nakamura H, Shibakawa A, Tanaka M, et al. Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteinases by chondrocytes and synoviocytes in osteoarthritis. Clin Exp Rheumatol 2004;22:293–299.
- 29.↑
Lippiello L, Woodward J, Karpman R, et al. In vivo chondroprotection and metabolic synergy of glucosamine and chondroitin sulfate. Clin Orthop Relat Res 2000;381:229–240.
- 30.
De Mattei M, Pellati A, Pasello M, et al. High doses of glucosamine-HCl have detrimental effects on bovine articular cartilage explants cultured in vitro. Osteoarthritis Cartilage 2002;10:816–825.
- 31.
Gouze JN, Bianchi A, Bécuwe P, et al. Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-KB pathway. FEBS Lett 2002;510:166–170.
- 32.
Scotto d'Abusco A, Calamia V, Cicione C, et al. Glucosamine affects intracellular signaling through inhibition of map kinase phosphorylation in human chondrocytes. Arthritis Res Ther 2007;9:R104.
