Effect of oral glucosamine on cartilage degradation in a rabbit model of osteoarthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 4, April 2005, pp 1118–1128 DOI 10.1002/art.20951 © 2005, American College of Rheumatology Effect of Oral Glucosamine on Cartilage Degradation in a Rabbit Model of Osteoarthritis Gabrielle Tiraloche,1 Christiane Girard,1 Luc Chouinard,2 John Sampalis,3 Luc Moquin,1 Mirela Ionescu,4 Agnes Reiner,4 A. Robin Poole,4 and Sheila Laverty1 Objective. To determine whether oral glucosamine alleviates cartilage degradation in an animal model of osteoarthritis (OA). Methods. The effect of 8 weeks of daily oral glucosamine hydrochloride on degeneration of articular cartilage was evaluated in rabbits in which anterior cruciate ligament transection (ACLT) was performed to induce OA. Animals were treated with glucosamine (n ⴝ 16) or a placebo (n ⴝ 16) and necropsied at 11 weeks. Seven unoperated rabbits served as controls. The articular cartilage was evaluated macroscopically and histologically and analyzed for total type II collagen and glycosaminoglycan (GAG) content. Results. Histologic analysis revealed that loss of proteoglycan, based on Safranin O–fast green staining, was significantly reduced in the lateral tibial plateau cartilage of ACL-transected limbs in the glucosamine group compared with ACL-transected limbs in the placebo group, with a similar, but not significant, trend for the lateral femoral condylar cartilage. Likewise, macroscopic analysis of cartilage showed that the lateral tibial plateau alone had a significantly lower rate of disease in the glucosamine group, which was consistent with the results of the independent histologic assessment. However, no significant treatment effect was detected when composite histologic scores were analyzed. A significant reduction in GAG content was observed in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACLtransected joints, compared with their respective contralateral unoperated joints. Conclusion. Oral administration of glucosamine had a detectable, site-specific, partial disease-modifying effect in this model of OA. From a clinical perspective, the administration of glucosamine did not prevent fibrillation and/or erosions of the articular cartilage in all of the treated animals, and no effects were detected in the medial joint compartments. Ms Tiraloche’s work was supported by the Canadian Arthritis Network and Clintrials Bioresearch. Dr. Poole’s work was supported by the Shriners Hospitals for Children, the NIH (National Institute on Aging), the Canadian Institute for Health Research, and the Canadian Arthritis Network. Dr. Laverty’s work was supported by the Groupe de Recherche en Médecine Équine du Québec, the Fond du Centenaire, the Faculté de Médecine Vétérinaire of the Université de Montréal, and the Canadian Arthritis Network. 1 Gabrielle Tiraloche, Christiane Girard, DMV, MSc, Dipl ACVP, Luc Moquin, Sheila Laverty, MVB, Dipl ACVS, Dipl ECVS: Faculté de Médecine Vétérinaire, Université de Montréal, St. Hyacinthe, Quebec, Canada; 2Luc Chouinard, DMV, Dipl ACVP: Clintrials Bioresearch, Senneville, Quebec, Canada; 3John Sampalis, MSc, PhD: JSS Medical Research, Montreal, Quebec, Canada; 4Mirela Ionescu, MSc, Agnes Reiner, MSc, A. Robin Poole, PhD: Shriners Hospitals for Children, McGill University, Montreal, Quebec, Canada. Dr. Poole has received consulting fees of more than $10,000 per year from Ibex Technologies, Montreal, Quebec, Canada. Address correspondence and reprint requests to Sheila Laverty, MVB, Dipl ACVS, Dipl ECVS, Département des Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, C. P. 5000, St. Hyacinthe, Quebec J2S 7C6, Canada. E-mail: sheila. firstname.lastname@example.org. Submitted for publication May 13, 2004; accepted in revised form December 21, 2004. In addition to decreasing pain, an ideal therapeutic agent for osteoarthritis (OA) would also prevent or retard the progression of established OA by reducing or, preferably, reversing the underlying pathologic processes (structure modifying), resulting in the retention or restoration of more normal articular cartilage function. The most common pharmacologic therapeutic agents currently used for OA are primarily palliative and include acetaminophen, nonsteroidal antiinflammatory drugs, corticosteroids, and hyaluronic acid (1,2). None of these drugs for OA are disease modifying. Analyses of many randomized clinical trials of oral glucosamine in OA patients support the contention that glucosamine is a symptom-modifying agent in OA (3–5), while investigators in many other trials have 1118 ORAL GLUCOSAMINE IN RABBIT OA detected no effect (6–8). It remains a subject of controversy whether glucosamine modifies cartilage structure in OA. Reports of recent clinical trials and metaanalyses concluded that, based on joint space narrowing, oral glucosamine slowed the radiographic progression of OA. This suggests that glucosamine has a structuremodifying effect (5,9,10). Although it remains the current method of reference (11), the validity of the use of radiographic assessment of joint space width as a measure of disease modification has been questioned (12). The apparent discrepancy between the various clinical trial results could, in part, be explained by the use of different formulations of glucosamine. The majority of the clinical trials have evaluated glucosamine sulfate, which is a prescription medicine in Europe. However, in the US and Canada, glucosamine is considered a dietary supplement (13), and content and purity have been shown to vary markedly between preparations (14,15). Animal models of OA offer an important opportunity to resolve the current controversy as to whether oral glucosamine administration has a structuremodifying effect in OA at the currently recommended standard dose. To date, there have been few reports of studies in animal models of OA examining this crucial question. In an instability model in rabbits, using a modification of the technique of Hulth et al (16), glucosamine administration (at 20 times the equivalent standard dose) had no identifiable effects on histologic parameters used to evaluate the severity of OA (17). This model of OA was severe, in that both cruciate ligaments were sectioned and the medial meniscus was removed. It is possible that the model was too severe (18) for a drug effect to be identified. A more recent study of a model of articular insult in the rabbit using chymopapain, which induces proteoglycan loss, revealed that glucosamine increased the glycosaminoglycan (GAG) content in the injured knees compared with that in controls (19). One of the most widely used experimental models of OA is the anterior cruciate ligament transection (ACLT) model. The use of experimental ACLT models is of particular clinical relevance, since rupture of the ACL occurs in humans and also leads to the development of OA (20). Rabbit ACLT is increasingly being used in OA studies (21–23) because disease onset is rapid. Furthermore, cartilage-specific analyses that measure proteoglycan content and the degradation of type II collagen, an important structural macromolecule of the matrix, are now available (24) and provide important 1119 additional quantitative information about matrix degradative events in animal models (25). The objective of our study was to determine whether oral administration of a clinically relevant dose of glucosamine had a detectable structure-modifying or metabolic effect on articular cartilage in a standard rabbit ACLT model of OA. To this end, we comprehensively evaluated combined macroscopic, histologic, and compositional parameters relating to cartilage damage, GAG content, and type II collagen degradation. MATERIALS AND METHODS Animals. We used 39 skeletally mature (9-month-old) male NZW rabbits (Charles River, St. Constant, Quebec, Canada) weighing a mean ⫾ SD of 3.69 ⫾ 1.4 kg. Radiographs of both femorotibial joints were taken to exclude animals with joint pathology. Thirty-two rabbits underwent unilateral ACLT under general anesthesia. The experimental animals were randomly assigned to 3 groups. Rabbits in the glucosamine group (n ⫽ 16) were administered 100 mg glucosamine hydrochloride (Sigma, St. Louis, MO) in a 5-gm wafer (Bio-serv, Frenchtown, NJ) daily for a period of 8 weeks starting 3 weeks after surgery. These doses are close to the recommended daily dose for humans with OA (⬃20 mg/kg). The wafers were given in the morning before the rabbits were fed. Rabbits in the placebo group (n ⫽ 16) were administered the same wafer without glucosamine. There were 7 unoperated and untreated control rabbits (control group). All experiments were conducted with the approval of the Institutional Animal Care and Use Committee. ACLT surgery. An analgesic (buprenorphine hydrochloride, 0.03 mg/kg injected intramuscularly every 6 hours) was administered preoperatively and for 3 days following surgery. Antibiotics (sulfadiazine and trimethoprim [24%], 15 mg/kg injected subcutaneously twice a day) were also administered preoperatively and for 2 days postoperatively. Anesthesia for the surgical procedure was maintained with a mixture of isoflurane (Technilab, Mirabel, Quebec, Canada) in oxygen. The limb was clipped and prepared for surgery in a standard manner. A medial arthrotomy was performed on the left femoropatellar joint to permit transection of the ACL. Routine skin incision closure was performed. Tissues were harvested following euthanasia, 11 weeks after surgery. Macroscopic cartilage assessment. All femorotibial joint compartments (medial femoral condyle [MFC], lateral femoral condyle [LFC], medial tibial plateau [MTP], and lateral tibial plateau [LTP]) in all animals were examined for gross morphologic changes of the articular cartilage and immediately graded, following application of India ink, as described previously (26). Briefly, compartments were graded from 1 to 4 (grade 1 ⫽ no uptake of India ink, indicating an intact surface; grade 2 ⫽ minimal focal uptake of India ink, indicating mild surface irregularity; grade 3 ⫽ evident large focal dark patches of ink uptake, showing overt fibrillation; grade 4a ⫽ erosion of cartilage [whiter area demonstrating visible bone] ⱕ2 mm; grade 4b ⫽ erosion of cartilage ⬎2 mm but ⱕ5 mm; and grade 4c ⫽ erosion of cartilage ⬎5 mm). The 1120 examination was performed by 2 independent observers (SL and GT) who were blinded to the treatment groups. The lesions were documented digitally using a D1 camera (Nikon, Tokyo, Japan). Histologic assessment of cartilage. Histologic assessment was performed on the femorotibial joints of rabbits in the glucosamine (n ⫽ 8), placebo (n ⫽ 8), and control (n ⫽ 7) groups. The femoral and tibial articular surfaces were fixed in 10% neutral buffered formalin. Tissue blocks were decalcified with 14% EDTA (Fisher Scientific, Nepean, Ontario, Canada) in 10 mM phosphate buffer (pH 7.4), dehydrated through graded alcohols, cleared with toluol, and embedded in paraffin. Six-micrometer sections were cut at a standard site centrally in the MFC, LFC, MTP, and LTP. The sections were stained with Safranin O–fast green and hematoxylin– eosin–saffron. Two histologic sections from each site were evaluated using a modified Mankin grading system (27–29) (Table 1) by 2 independent, board-certified veterinary pathologists (CG and LC) who were blinded to the treatment groups. Cartilage markers of type II collagen degradation (Col2-3/4C and Col2-3/4m neoepitopes) and type II collagen content. Full-depth articular cartilage was removed by careful dissection with a scalpel from the tibial plateau and femoral condyles (either operated or unoperated), avoiding the periarticular margins and osteophytes, and was analyzed for each of the rabbits in the glucosamine (n ⫽ 8), placebo (n ⫽ 8), and control (n ⫽ 7) groups. Wet weights were determined and recorded for each region after blotting to remove any excess moisture. For solubilization, cartilage was incubated overnight at 37°C, first with ␣-chymotrypsin and then with proteinase K as described previously (30,31). The samples were assayed for both the Col2-3/4C neoepitope (using the ␣-chymotrypsin extract) (30) and the Col2-3/4m hidden epitope (using the ␣-chymotrypsin and proteinase K extracts) (31) to determine denatured and total type II collagen content. The Col2-3/4C neoepitope assay was a modification of the reported assay (30). The peptide CGPP(OH)GPQG (the Col2-3/4C neoepitope generated by collagenase) was conjugated to ovalbumin, and a polyclonal antibody was prepared as described (30). Binding of the biotinylated antibody was detected using dissociationenhanced lanthanide fluoroimmunoassay (DELFIA) europium-labeled streptavidin (EG&G Wallac, Turku, Finland) diluted 1:1,000 in DELFIA assay buffer (EG&G Wallac). Fluorescence at an excitation of 340 nm and with an emission at 615 nm was measured by a 1412 plate reader (EG&G Wallac). A commercial assay for the Col2-3/4C neoepitope, called the C1,2C assay, is available from Ibex Technologies (Montreal, Quebec, Canada). Total type II collagen content of each sample was determined from the total amount of Col2-3/4m epitope in the ␣-chymotrypsin extract and in the proteinase K digest (31). The amount of denatured collagen (Col2-3/4m epitope) found in the ␣-chymotrypsin digest was recorded. Cartilage GAG analysis. The ␣-chymotrypsin and proteinase K cartilage digests were assayed to determine total GAG content (primarily a measure of the proteoglycan aggrecan content) using a modification of the colorimetric dimethylmethylene blue dye assay (32). All results were expressed per mg wet weight of cartilage. TIRALOCHE ET AL Table 1. Criteria (grading) for histologic evaluation Safranin O–fast green staining 0 ⫽ uniform staining throughout articular cartilage 1 ⫽ loss of staining in the superficial zone for less than one-half of the length of the condyle or plateau 2 ⫽ loss of staining in the superficial zone for one-half or more of the length of the condyle or plateau 3 ⫽ loss of staining in the superficial and middle zones for less than one-half of the length of the condyle or plateau 4 ⫽ loss of staining in the superficial and middle zones for onehalf or more of the length of the condyle or plateau 5 ⫽ loss of staining in all 3 zones for less than one-half of the length of the condyle or plateau 6 ⫽ loss of staining in all 3 zones for one-half or more of the length of the condyle or plateau Chondrocyte loss 0 ⫽ no decrease in cells 1 ⫽ minimal decrease in cells 2 ⫽ moderate decrease in cells 3 ⫽ marked decrease in cells 4 ⫽ very extensive decrease in cells Structure 0 ⫽ normal 1 ⫽ surface irregularities 2 ⫽ 1–3 superficial clefts 3 ⫽ ⬎3 superficial clefts 4 ⫽ 1–3 clefts extending into the middle zone 5 ⫽ ⬎3 clefts extending into the middle zone 6 ⫽ 1–3 clefts extending into the deep zone 7 ⫽ ⬎3 clefts extending into the deep zone 8 ⫽ clefts extending to calcified cartilage Clone formation 0 ⫽ normal 1 ⫽ minimal (ⱕ4) 2 ⫽ moderate (⬎4 but ⱕ8) 3 ⫽ marked (⬎8) Tidemark integrity 0 ⫽ intact 1 ⫽ crossed by blood vessels Statistical analysis. Macroscopic grades. Since the macroscopic grades were not continuous, the chi-square test was used to compare the glucosamine and placebo groups. In addition, the 6 grades were divided into 2 categories for statistical analysis (diseased joints [evident fibrillation or erosion] with grades 3 or 4 and nondiseased joints with grades 1 or 2), since the 6 grades of classification defined disease and normal states. The odds ratio was used to estimate the relative risk for having a diseased joint in the placebo group compared with the glucosamine group. Histologic scores. Student’s t-test was used to assess differences between the glucosamine and placebo groups with respect to histologic parameters (Safranin O–fast green staining, structure, chondrocyte loss, chondrocyte clusters, tidemark integrity) of the operated limbs. These analyses were done for the total joint and for each individual compartment (MFC, LFC, MTP, LTP) separately, for each of the histologic parameters. When a statistically significant difference between the 2 groups was detected, analysis of variance (ANOVA) was used to adjust for the confounding effects of animal and pathologist. Spearman’s rank correlation coefficient was used to evaluate the association between macroscopic grades and ORAL GLUCOSAMINE IN RABBIT OA 1121 Figure 1. Macroscopic grades of the articular surfaces of the femoral condyles and tibial plateaus. Four compartments were graded (medial femoral condyle [MFC], lateral femoral condyle [LFC], medial tibial plateau [MTP], and lateral tibial plateau [LTP]). Data are presented as the mean percentage distribution of grades. Grades 1 and 2 represent normal cartilage with no or minor focal uptake, respectively, of India ink; grades 3 and 4 represent overt fibrillation and erosion, respectively (see Materials and Methods). Control ⫽ joints from unoperated untreated animals; placebo ACLT ⫽ anterior cruciate ligament–transected joints from placebo-treated animals; placebo unop ⫽ contralateral unoperated control joints from placebo-treated animals; GlcN ACLT ⫽ ACL-transected joints from glucosamine-treated animals; GlcN unop ⫽ contralateral unoperated control joints from glucosamine-treated animals. histologic scores for each of the different joint compartments. Interobserver agreement between the 2 pathologists (CG and LC) was measured with an interclass correlation coefficient. Cartilage type II collagen and GAG analyses. A KruskalWallis test was used to evaluate differences in cartilage wet weight, in results of type II collagen analyses, and in GAG levels among control unoperated, glucosamine-treated ACLtransected, placebo-treated ACL-transected, and contralateral unoperated TP and FC regions. When significant differences occurred among treatments, post hoc tests with adjusted P values were used to determine which pairs of treatments actually differed. Differences between glucosamine- and placebo-treated ACL-transected regions and their respective contralateral unoperated control regions were examined with the Wilcoxon signed rank test. The 5% significance level was used. RESULTS Experimental animals. The rabbits rapidly ingested the wafers, usually within 5 minutes of administration, thus effectively mimicking a bolus dose of glucosamine. Postmortem inspection revealed that ACLT was complete in all rabbits included in the study. Two animals with some persisting ACL fibers were excluded from the study. These had been assigned to the biochemical analysis section of the glucosamine group. Therefore, 6 animals remained for biochemical analysis of articular cartilage. Effect of glucosamine on macroscopic parameters. In the unoperated right femorotibial joints (n ⫽ 32) and control rabbit joints (n ⫽ 7), the articular Table 2. Proportion of animals with disease (cartilage fibrillation or erosion) on macroscopic evaluation using India ink* Group Compartment Glucosamine (n ⫽ 14) Placebo (n ⫽ 16) OR P† MFC LFC MTP LTP 43 43 21 14 56 56 19 44 0.42 0.42 1.18 0.21 0.450 0.450 0.780 0.046 * Values are percentages of animals with disease in a given compartment. OR ⫽ odds ratio; MFC ⫽ medial femoral condyle; LFC ⫽ lateral femoral condyle; MTP ⫽ medial tibial plateau; LTP ⫽ lateral tibial plateau. † By chi-square test. 1122 TIRALOCHE ET AL Figure 2. Representative Safranin O–fast green–stained histologic sections illustrating cartilage lesions. a, Normal articular cartilage with intact surface stained with Safranin O–fast green. b, Superficial diffuse loss of Safranin O–fast green staining, with clusters of cells (arrowhead) present in the superficial layer and surface irregularity and fibrillation. c, Severe loss of Safranin O–fast green staining and loss of cartilage, with cleft extending to the middle zone (arrowhead). d, Erosion of cartilage matrix (solid arrowhead) to calcified cartilage (open arrowheads). (Original magnification ⫻ 40.) cartilage macroscopic grades were 1 (normal) or 2 (scarcely perceptible ink uptake), with the exception of 1 animal in each group that had grade 3 lesions (overt fibrillation with distinct focal patches of ink uptake). No cartilage erosions (grade 4) were observed in any of the joint compartments (results not shown). The unoperated control group was included for comparative purposes to demonstrate that the ACLT surgery performed by us induced articular cartilage changes. The induced OA lesions (fibrillation and erosion of articular cartilage) were focal and occurred at the same sites in all ACL-transected joints. The glucosamine group (n ⫽ 14) tended to have a lower severity grade of macroscopic articular cartilage lesions in all 4 joint compartments compared with the placebo group (n ⫽ 16) (Figure 1), but this difference was not significant. The macroscopic grade data, categorized as disease (overt fibrillation and cartilage erosion) or nondisease, ORAL GLUCOSAMINE IN RABBIT OA 1123 Figure 3. Histologic scores at 11 weeks. Shown are scores for the histologic parameters with comparisons between groups. Values are the mean and SD. Histologic analysis revealed that loss of proteoglycan, based on Safranin O–fast green staining, was significantly reduced in the LTP cartilage of ACL-transected limbs in the glucosamine group compared with ACL-transected limbs in the placebo group, with a similar, but not significant, trend for the LFC cartilage. See Figure 1 for definitions. showed 44% of the placebo group with disease in the LTP compartment, compared with 14% in the glucosamine group (P ⫽ 0.046). This indicated a statistically significantly lower rate of disease for the glucosamine group compared with the placebo group at this site (Table 2). Effect of glucosamine on histologic parameters. Representative histologic sections are shown in Figure 2. We observed a normal integrity of the articular surface. This was lost with early mild fibrillation, and a loss of Safranin O–fast green staining was observed along with chondrocyte clustering. With increased degeneration, there was a loss of Safranin O–fast green staining accompanying cleft formation that extended into the middle and deep zones. In severe cases, calcified cartilage sometimes remained, but often, there was eburnation of bone. Unoperated limbs (including contralateral unoperated joints in glucosamine and placebo groups [n ⫽ 8 in each group] and joints of control unoperated rabbits [n ⫽ 8]) exhibited some mild degenerative histologic changes, such as changes in Safranin O–fast green staining, changes in structure, or chondrocyte loss, in the different joint compartments. These were most evident in the MTP compartment (Figure 3). Interestingly, ACLT did not change the histologic score for these parameters at the MTP site to the extent seen in the other compartments. There was a significantly lower mean score for Safranin O–fast green staining in the LTP compartment of the ACL-transected limbs in the glucosamine group compared with the ACL-transected limbs in the placebo group (P ⫽ 0.049). There was a trend (P ⫽ 0.11) toward lower Safranin O–fast green staining scores in the LFC 1124 TIRALOCHE ET AL Figure 4. Biochemical analysis of cartilage from the femoral condyles (FC) and tibial plateaus (TP). Shown are concentrations of glycosaminoglycan (GAG), type II collagen, collagenase-cleaved type II collagen, and denatured type II collagen. Values are the mean and SD. A significant loss of GAG occurred in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACL-transected joints, compared with their respective contralateral unoperated joints. No other significant changes were detected. See Figure 1 for other definitions. compartment of the ACL-transected limbs in the glucosamine group. There were no statistically significant differences in the Safranin O–fast green staining scores in the MTP and MFC compartments between ACLtransected limbs in the glucosamine group and those in the placebo group. No significant differences in structural scores, chondrocyte loss, or amount of chondrocyte clusters were detected between the glucosamine and placebo groups in any of the ACL-transected joint compartments (Figure 3). Chondrocyte cluster formation (cloning) was only seen in ACL-transected joints, never in unoperated joints. Alterations in the tidemark integrity were not observed in this study. When all histologic parameters were combined from each site to obtain a total joint score, no statistically significant differences were detected between ACL-transected joints in the glucosamine group and those in the placebo group. Results of the multivariate ANOVA with treatment and pathologist entered as covariates indicated that the largest component of observed variation in histologic measurements was due to treatment, followed by variation due to the pathologist, indicating a high interrater agreement. There was no significant variation attributed to animal effect. Statistical correlations. In ACL-transected joints in both the glucosamine and placebo groups, a high correlation was observed between the macroscopic grades and the histologic scores in the MFC (rs ⫽ 0.9, P ⫽ 0.006 for the glucosamine group; rs ⫽ 0.9, P ⫽ 0.0001 for the placebo group), LFC (rs ⫽ 0.9, P ⫽ 0.001 for the glucosamine group; rs ⫽ 0.9, P ⫽ 0.007 for the ORAL GLUCOSAMINE IN RABBIT OA placebo group), and LTP (rs ⫽ 0.7, P ⫽ 0.04 for the glucosamine group; rs ⫽ 0.8, P ⫽ 0.03 for the placebo group) compartments. In the MTP compartment, the values were rs ⫽ 0.5, P ⫽ 0.19 for the glucosamine group and rs ⫽ ⫺0.1, P ⫽ 0.8 for the placebo group. Effect of glucosamine on cartilage type II collagen and proteoglycan. There were significant effects of experimental group on the amount of cartilage harvested from the femoral condyles (P ⫽ 0.003) and tibial plateaus (P ⫽ 0.044). Post hoc tests revealed that significantly more cartilage was harvested from the femoral condyles of ACL-transected joints in both the glucosamine and placebo groups than from the femoral condyles of control unoperated joints (data not shown). Similar changes were observed in the tibial plateau, with the exception that no significant differences were detected between glucosamine-treated ACL-transected joints and control unoperated joints. In addition, significantly more cartilage was harvested from the femoral condyles (P ⫽ 0.008) and the tibial plateaus (P ⫽ 0.023) of placebo-treated ACL-transected joints than from the corresponding regions of their contralateral unoperated joints. This was not observed in the glucosamine group. No significant differences in type II collagen content, denaturation, or cleavage were detected in the biochemical analysis of cartilage in any of the regions (TP or FC) among the glucosamine (n ⫽ 6), placebo (n ⫽ 8), or control (n ⫽ 7) groups (Figure 4). In contrast, a significant reduction (P ⫽ 0.02) in GAG content was observed in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACL-transected joints, compared with their respective contralateral unoperated joints (Figure 4). The intraassay variation for all of the assays for cartilage analysis ranged from 5% to 10%. The interassay variation ranged from 7% to 15%. DISCUSSION This study provides in vivo experimental evidence indicating that the oral administration of glucosamine hydrochloride can alter the degeneration of articular cartilage in OA. Safranin O–fast green staining revealed that loss of proteoglycan can be prevented in the lateral compartment within the operated joint. It is important to note that the histologic sections were scored by 2 independent, board-certified veterinary pathologists who were blinded to the treatment groups. The interrater agreement between the 2 pathologists was high, suggesting that the modified Mankin assessment we 1125 used was reliable and provided consistency in grading the changes observed on the histologic slides. Interestingly, when macroscopic findings, reflective of the extent of articular cartilage surface damage and severity of lesions, were evaluated by 2 other independent assessors and statistically analyzed as dichotomous variables (disease or nondisease), there was also significantly less disease in the LTP compartment in the glucosamine group, which was consistent with the histologic assessment. However, it is also important to note from a clinically relevant perspective that, based on both macroscopic and histologic analysis, the administration of glucosamine did not prevent fibrillation and/or erosions of the articular cartilage in this model, although there was an overall trend toward a reduction in severity of the disease in the operated joints of the glucosamine group. Furthermore, statistical analysis of the histologic results revealed that any variation we observed was primarily due to treatment effects. Taken together, the results indicate that glucosamine had a positive effect on proteoglycan retention in the lateral compartment of the ACL-transected joint (statistically significant in the LTP compartment and a trend in the LFC compartment). However, no effects were detected in the MFC and MTP compartments of the joint. A separate group of animals was analyzed independently for biochemical changes in cartilage GAG content and type II collagen degradation. In this case, for reasons related to tissue requirements for the analyses, we were unable to examine lateral and medial compartments separately. In spite of this, we found that glucosamine treatment prevented the loss of proteoglycan observed in the femoral condylar cartilage of the placebo group compared with unoperated contralateral joint cartilage from the same region. No significant changes were detected for the collagen parameters. Since OA lesions are usually focal, there would have been less lesional cartilage relative to distant nonlesional cartilage, and effects of treatment on the lesion could have been missed. This effect of cartilage sampling has also been highlighted by others (19). We are confident, however, that when a significant effect was detected, it was relevant, since these quantitative analyses involved a small series of animals in each group. We unexpectedly harvested a greater amount of cartilage from the femoral condyles of the ACLtransected limbs (in both the glucosamine and placebo groups) compared with the unoperated limbs. Intuitively, we would have expected less cartilage in the ACL-transected limbs because of erosion of the cartilage, particularly at the femoral condyles. It is possible 1126 that this increased yield could reflect the recognized hypertrophic response of cartilage in OA (33). However, this finding could also be due to a sampling bias. The operator may have unknowingly shaved cartilage from these regions more painstakingly because lesions were present. Our biochemical results were expressed per mg wet weight to avoid this confounding issue. The observed site-specific effects of glucosamine could be explained by differences in disease development and/or rapidity of progression in the different joint compartments. Regional differences in the development of OA lesions in the femorotibial joint in the ACLT rabbit model have already been highlighted by others (34). Discrepancies between results of our study and those of Lippiello et al (17), who reported that 20 times the typical oral dose of glucosamine had no chondroprotective effect in a rabbit model of OA, may be explained by differences in the models examined. First, only the cranial cruciate ligament was transected in the present study, whereas both cruciate ligaments and the medial meniscus were removed in the study by Lippiello et al, which would have led to greater instability and more rapid progression of disease. Second, the femoral condyles alone were evaluated by Lippiello et al, whereas the femorotibial sites were also evaluated histologically in our study. Third, we administered glucosamine in a wafer, which was rapidly consumed so as to simulate bolus dosing, whereas Lippiello et al added glucosamine to the feed, which the animals eat progressively throughout the day, and this would have resulted in different doses and different peak serum glucosamine concentrations. Taken together, it is easy to understand that divergent results could have been obtained with these different studies, and this highlights the difficulties of comparing compound efficacy results in studies using different animal models of OA conducted with different dosing regimens. Oral glucosamine, although at a higher dose, has been reported to restore GAG in the damaged cartilage in a chymopapain-injected rabbit model of joint injury, and the investigators in that study proposed that it may have been effective because it represented an exogenous source of glucosamine for GAG synthesis in a tissue undergoing active repair (19). Interestingly, the data in the present study revealed no changes in collagen content or turnover following ACLT and no effects of glucosamine. Similarly, in the chymopapain-injected rabbit model, Oegema et al (19) reported no significant changes in collagen content and type II collagen messenger RNA with glucosamine therapy. TIRALOCHE ET AL Although an extensive body of in vitro studies to elucidate the effects of glucosamine on articular cartilage has been published (35–46), the majority of these studies have identified effects of the monosaccharide at higher concentrations than those recently reported to be attainable in vivo in the synovial fluid (0.3–0.7 M) following the administration of a clinically relevant dose (20 mg/kg) in an equine model (47). Therefore, to conclude that these in vitro effects occur in vivo may be erroneous, since cartilage would not be exposed to these high levels. It has been proposed that glucosamine may be concentrated in articular cartilage and that this could explain its beneficial effects (48), but concrete evidence for this is currently not available. Components of the hexosamine pathway (glucosamine or biosynthetic derivatives) have been shown to be capable of modulating several genes in different tissues (49–51), and glucosamine effects in articular tissues could also be induced by the regulation of various genes. It is also possible that the observed in vivo effects may be due to an influence of glucosamine on other tissues that subsequently release factors that modify the chondrocyte response secondarily (19). Support for the concept of an indirect effect of glucosamine administration on cartilage is provided by the recent finding that it affects subchondral bone remodeling in rabbit OA (52). A weakness of the ACLT model is that the persistent joint instability may counteract attempts at cartilage repair, and a drug with less potency may not have detectable effects. As a result, pathology in the ACLT model may develop rapidly compared with OA in humans, the progression of which is usually slow and may take place over a period of 15–30 years. A strength of this study is that we elected to use glucosamine hydrochloride from a chemical company to avoid any commercial bias, which has been a concern in previous studies of glucosamine (53). We also based the oral dose on a typical human dose described in the literature (53), and we chose to avoid the use of glucosamine sulfate, since sulfation may influence the outcome of therapy, being another building block of proteoglycans (54). Taken together, these results indicate that glucosamine hydrochloride can produce partial disease modification at specific sites in a classic model of articular trauma–induced OA at a dosage relevant to that used in humans. Ideally, a disease/structuremodifying drug should prevent or alleviate articular cartilage degeneration generally throughout the joint. 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