Protective effects of corticosteroids on cartilage lesions and osteophyte formation in the Pond-Nuki dog model of osteoarthritis

The in vivo effects of corticosteroids on osteoarthritic (OA) lesions were examined in 12 dogs in which the anterior cruciate ligament had been sectioned. Six were treated with oral prednisone and 6 were treated with intraarticular (IA) injections of triamcinolone hexacetonide (TH), at surgery and 4 weeks later. Twelve other operated dogs received no treatment. All dogs were killed 8 weeks postsurgery. Four of 15 normal control dogs received IA TH injections. Operated untreated dogs developed significant cartilage lesions on the femoral condyles and tibial plateaus with prominent osteophytes. Operated dogs treated orally or with IA injections had a significant reduction in osteophyte size. Cartilage erosions on femoral condyles were observed in 25% of the untreated dogs, 8% of the dogs receiving oral prednisone, and none of the dogs receiving IA TH. In both groups of treated dogs, the size of the tibial plateau lesions was significantly reduced compared with the operated untreated dogs. Histologically, corticosteroids significantly reduced the severity of OA structural changes of the cartilage on both medial and lateral femoral condyles and tibial plateaus in operated animals, with the exception of the lateral plateaus of those treated orally. In electron microscopy studies, we found no evidence of increased cell degeneration or death associated with steroids. IA corticosteroids had no deleterious effects on normal articular cartilage. These results indicate that glucocorticoids administered orally or intraarticularly are effective against the development of OA lesions in this model.     

Effects of tenidap on canine experimental osteoarthritis i. morphologic and metalloprotease analysis

Objective. To examine the effects of tenidap and diclofenac on osteoarthritic lesions and metalloprotease activity in experimental osteoarthritis (OA). Methods. The anterior cruciate ligament of the right stifle joint of 25 mongrel dogs was sectioned by a stab wound. Seven dogs received no treatment, 6 were treated with oral omeprazole (20 mg/day), another 6 were treated with diclofenac (0.25 mg/kg/twice daily) plus omeprazole (20 mg/day), and 6 received oral tenidap (3 mg/kg/twice daily) plus omeprazole (20 mg/day). The dogs received medication for 8 weeks; all dogs were killed at the end of this period. Eight normal dogs were used as controls. Lesions were evaluated macroscopically for the incidence and size of osteophytes and the area and grade of cartilage erosions on the condyles and plateaus, along with histologic evaluation of the severity of the cartilage lesions and synovial inflammation. Stromelysin and collagenase activities and the collagenase messenger RNA (mRNA) level were measured in cartilage and synovial membrane. Results. Compared with the untreated or omeprazole-treated OA groups, the dogs treated with tenidap exhibited significant reduction in the incidence (P < 0.001) and size (P < 0.0001) of osteophytes. Tenidap also significantly decreased the size and grade of cartilage macroscopic lesions, as well as the histologic severity of cartilage lesions on both condyles and plateaus. The histologic severity of synovial inflammatory reaction was also significantly reduced (P < 0.003) in the tenidap group. Tenidap markedly decreased stromelysin and collagenase activity in both cartilage (stromelysin P < 0.003; collagenase P < 0.01) and synovial membrane (stromelysin P < 0.003; collagenase P < 0.005). Moreover, tenidap also decreased the collagenase mRNA level in cartilage (P < 0.005) and synovial membrane (P < 0.002). Diclofenac slightly reduced the incidence and size of osteophytes and cartilage lesions, but these changes were not statistically significant. Diclofenac had no effect on the severity of synovial inflammation, metalloprotease activity, or collagenase expression. Conclusion. This study showed that tenidap had a more potent anti-osteoarthritic effect than diclofenac in this model. The effect of the drug in suppressing metalloprotease synthesis, a process known to play a major role in the pathophysiology of osteoarthritic lesions, may explain its mechanism of action.     

Effects of tenidap on the progression of osteoarthritic lesions in a canine experimental model. Suppression of metalloprotease and interleukin-1 activity

Objective. To study, in vivo, the therapeutic effectiveness of tenidap, an antirheumatic drug, on the progression of lesions in an experimental osteoarthritis (OA) dog model. The action of tenidap on the activity and expression of metalloproteases in cartilage, as well as on the bioactivity of interleukin-1 (IL-1) in synovial fluid, was determined. Methods. The anterior cruciate ligament of the right stifle joint of 20 mongrel dogs was sectioned through a stab wound. Dogs were divided into 3 groups: group I (n = 7) received no treatment, group II (n = 6) was treated with oral omeprazole (20 mg/day), and group III (n = 7) received oral omeprazole (20 mg/day) and a therapeutic dosage of oral tenidap (3 mg/kg twice daily). Four weeks following surgery, the untreated dogs (group I) were killed, and drug treatments were begun for the other dogs (groups II and III). These dogs received medication for 8 weeks (weeks 4–12) and then were killed. Evaluations were made of the incidence and size of osteophytes as well as of the size and grade of cartilage erosions on both the condyles and plateaus. Histologic examination of the severity of the cartilage lesions and synovial inflammation was also performed. Activity levels of collagenase, stromelysin, and gelatinase as well as collagenase-1, collagenase-3, and stromelysin-1 messenger RNA were determined in the cartilage. The level of IL-1 activity in the synovial fluid was also measured. Results. Among the dogs with OA, lesions were more severe at 12 weeks than at 4 weeks. Group III (tenidap-treated) dogs had a slightly reduced incidence of osteophytes compared with the group II (12-week OA) dogs (71% versus 100%), and the size of the osteophytes was significantly diminished (mean ± SEM 1.75 ± 0.69 mm versus 4.38 ± 0.64 mm). Macroscopically, tenidap decreased the size (condyles 6.00 ± 2.18 mm² versus 21.08 ± 6.70 mm², plateaus 15.50 ± 4.77 mm² versus 35.0 ± 3.64 mm²) and the grade (condyles 0.57 ± 0.20 versus 1.17 ± 0.21, plateaus 1.07 ± 0.22 versus 2.00 ± 0.25) of the cartilage lesions compared with the 12-week OA dogs. At the histologic level, the severity of cartilage lesions was also decreased in the tenidap-treated dogs versus the 12-week OA dogs, both on the condyles (3.43 ± 0.54 versus 5.55 ± 0.38) and on the plateaus (3.39 ± 0.35 versus 5.54 ± 0.60). All 3 OA groups showed a significant and similar level of synovial inflammation. Tenidap markedly decreased collagenase, stromelysin, and gelatinase activity, as well as the level of expression of collagenase-3 in the cartilage. Interestingly, the activity level of IL-1 in synovial fluid was also significantly reduced in the tenidap-treated dogs. Conclusion. Tenidap markedly reduced the severity of OA lesions, indicating the effect of this drug in decreasing the progression of disease. It appears that the drug acts by reducing the activity and/or expression of metalloproteases in cartilage, a process known to play a major role in the pathophysiology of OA lesions. This effect could be mediated by the suppressive effect of tenidap on IL-1 activity.     

Licofelone (ML-3000), a dual inhibitor of 5-lipoxygenase and cyclooxygenase,reduces the level of cartilage chondrocyte death in vivo in experimental dog osteoarthritis: inhibition of pro-apoptotic factors

OBJECTIVE: To evaluate in vivo therapeutic efficacy of licofelone, a novel competitive dual inhibitor of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) in chondrocyte death in the canine ligament transection model of osteoarthritis (OA), and to explore its effect on factors involved in the apoptotic phenomenon, i.e., caspase-3, COX-2, and inducible nitric oxide synthase (iNOS). METHODS: Cartilage specimens were obtained from 3 experimental groups of dogs: Group 1, dogs subjected to sectioning of the anterior cruciate ligament of the right knee and given placebo treatment; Groups 2 and 3, operated dogs that received oral treatment with licofelone (2.5 or 5.0 mg/kg/day, respectively) for 8 weeks starting immediately after surgery. All dogs were killed 8 weeks postsurgery. The cartilage level of chondrocyte death was detected by TUNEL reaction. Cartilage distribution of caspase-3, COX-2, and iNOS was documented by immunohistochemistry using specific antibodies, and other levels were quantified by morphometric analysis. RESULTS: In cartilage specimens from placebo treated dogs a large number of chondrocytes in the superficial layers stained positive for TUNEL reaction. Treatment with therapeutic concentrations of licofelone (2.5 and 5.0 mg/kg/day) markedly reduced the level of chondrocyte apoptosis to the same extent in both therapeutic groups (p < 0.0001, p < 0.002, respectively). In these groups, the levels of caspase-3, COX-2, and iNOS in cartilage from both condyles and plateaus were also significantly decreased (p < 0.0001, p < 0.0001, p < 0.0002, respectively) compared to the control (placebo) group. CONCLUSION: Licofelone is an effective treatment in vivo, capable of reducing the level of OA chondrocyte death. This effect is likely mediated by a decrease in the level of caspase-3 activity, which may be related to the reduced production of 2 major factors involved in chondrocyte apoptosis, NO and prostaglandin E2. These findings may explain some of the mechanisms by which licofelone reduces the progression of experimental OA.     

The in situ up-regulation of chondrocyte interleukin-1-converting enzyme and interleukin-18 levels in experimental osteoarthritis is mediated by nitric oxide

OBJECTIVE: To investigate in situ the relationship between 2 key mediators implicated in osteoarthritic (OA) cartilage: nitric oxide (NO) and interleukin-1-converting enzyme (ICE). Interleukin-18 (IL-18) was also studied and served as reference for the effects of ICE. METHODS: An OA model was created in dogs by sectioning (stab wound) the anterior cruciate ligament of the right stifle joint. Three experimental groups were studied: unoperated untreated dogs, operated untreated dogs (OA), and OA dogs treated with oral N-iminoethyl-L-lysine (L-NIL), a specific inhibitor of inducible nitric oxide synthase (iNOS) (10 mg/kg twice a day starting immediately after surgery). At 12 weeks after surgery, cartilage from the femoral condyles and tibial plateaus were processed for immunohistochemistry for ICE, IL-18, and protease inhibitor 9 (PI-9), a natural inhibitor of ICE, followed by morphometric analysis. Cartilage specimens from the femoral condyles of untreated OA dogs were dissected and incubated with specific inhibitors of different signaling pathways likely to be involved in the OA process: SB 202190 (10 microM; a p38 mitogen-activated protein kinase [MAPK] inhibitor), PD 98059 (100 microM; a MAPK kinase 1/2 [MEK-1/2] inhibitor), NS-398 (10 ng/ml; a specific cyclooxygenase 2 [COX-2] inhibitor), and L-NIL (50 microM). RESULTS: Both ICE and IL-18 were present in situ in the canine cartilage, with a significant increase in the level of these 2 proteins in OA cartilage. In contrast, the level of PI-9 was lower in OA than in normal cartilage (difference not statistically significant). Compared with untreated OA cartilage, oral treatment with L-NIL significantly decreased ICE and IL-18 levels in cartilage from the femoral condyles and tibial plateaus, to values similar to those in normal dogs. L-NIL also increased the PI-9 level in normal dogs compared with OA dogs, reaching statistical significance for femoral condyle cartilage. Interestingly, in vitro experiments demonstrated significant inhibition of ICE levels by p38, MEK-1/2, and COX-2 inhibitors, but not by the iNOS inhibitor. CONCLUSION: This study demonstrated that in situ in OA cartilage, the stimulation of chondrocytes by NO is at least partly responsible for the up-regulation of ICE and IL-18 synthesis while decreasing the level of the ICE inhibitor PI-9. The ICE level is controlled by the activation of at least 2 MAPK pathways, p38 and MEK-1/2. Interestingly, it appears that ICE synthesis is not regulated by the endogenous production of NO. These data highlight the role played by iNOS in regulating the synthesis of major catabolic factors involved in OA cartilage degradation.     

The peroxisome proliferator-activated receptor gamma agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: in vivo protective effects mediated through the inhibition of key signaling and catabolic pathways

OBJECTIVE: Emerging evidence indicates that peroxisome proliferator-activated receptor gamma (PPARgamma) may have protective effects in osteoarthritis (OA). The aim of this study was to evaluate the in vivo effect of a PPARgamma agonist, pioglitazone, on the development of lesions in a canine model of OA, and to explore the influence of pioglitazone on the major signaling and metabolic pathways involved in OA pathophysiologic changes. METHODS: OA was surgically induced in dogs by sectioning of the anterior cruciate ligament. The dogs were then randomly divided into 3 treatment groups in which they were administered either placebo, 15 mg/day pioglitazone, or 30 mg/day pioglitazone orally for 8 weeks. Following treatment, the severity of cartilage lesions was scored. Cartilage specimens were processed for histologic and immunohistochemical evaluations; specific antibodies were used to study the levels of matrix metalloproteinase 1 (MMP-1), ADAMTS-5, and inducible nitric oxide synthase (iNOS), as well as phosphorylated MAPKs ERK-1/2, p38, JNK, and NF-kappaB p65. RESULTS: Pioglitazone reduced the development of cartilage lesions in a dose-dependent manner, with the highest dosage producing a statistically significant change (P < 0.05). This decrease in lesions correlated with lower cartilage histologic scores. In addition, pioglitazone significantly reduced the synthesis of the key OA mediators MMP-1, ADAMTS-5, and iNOS and, at the same time, inhibited the activation of the signaling pathways for MAPKs ERK-1/2, p38, and NF-kappaB. CONCLUSION: These results indicate the efficacy of pioglitazone in reducing cartilage lesions in vivo. The results also provide new and interesting insights into a therapeutic intervention for OA in which PPARgamma activation can inhibit major signaling pathways of inflammation and reduce the synthesis of cartilage catabolic factors responsible for articular cartilage degradation.     

Hypertrophic repair of canine articular cartilage in osteoarthritis after anterior cruciate ligament transection

Transection of the anterior cruciate ligament in the dog is used frequently to produce a model of osteoarthritis (OA). Although it results in metabolic, biochemical, biomechanical and morphological changes in the articular cartilage of the unstable knee which resemble those in human OA, it rarely results in full thickness cartilage loss. The lack of progression of the cartilage lesions in this model has generally been attributed to stabilization of the joint by osteophytes and capsular fibrosis which develop following transection of the ligament. In our study transection of the anterior cruciate ligament of adult foxhounds and mongrels resulted in a progressive increase in the amount of articular cartilage in the unstable knee. In articular cartilage from the medial femoral condyle of the unstable knee the total amount of proteoglycan, as assessed by hexuronic acid analysis, and, often, the proteoglycan concentration were increased. Furthermore, the rate of proteoglycan synthesis was commonly elevated, in comparison with that from the contralateral knee. Thus, in these animals knee instability did not lead to loss of cartilage but to an active synthetic response by the chondrocytes resulting in hypertrophic cartilage repair which was sustained for up to 64 weeks of observation.     

Selective inhibition of inducible nitric oxide synthase reduces progression of experimental osteoarthritis in vivo: possible link with the reduction in chondrocyte apoptosis and caspase 3 level

OBJECTIVE: To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase, on the progression of structural lesions in the experimental canine model of osteoarthritis (OA), and to explore the effect of L-NIL on the level of chondrocyte apoptosis and of important proteins involved in the apoptotic phenomenon, i.e., caspase 3 (inducer) and Bcl-2 (inhibitor). METHODS: The OA model was created by sectioning the anterior cruciate ligament. Dogs were placed into 4 experimental groups: unoperated dogs that received no treatment (controls), operated (OA) dogs that received placebo treatment, OA dogs that received oral L-NIL at 10 mg/kg/day, and OA dogs that received oral L-NIL at 1.0 mg/kg/day. In both L-NIL groups, treatment started immediately after surgery. The OA dogs were killed at 12 weeks after surgery. RESULTS: OA dogs treated with L-NIL showed a reduction in the size of osteophytes and a significant decrease in the severity of macroscopic and histologic cartilage lesions on both condyles and plateaus, compared with untreated OA dogs. L-NIL treatment also significantly decreased metalloprotease activity in cartilage. Immunohistochemical analysis revealed that the levels of chondrocyte apoptosis, caspase 3, and Bcl-2 were markedly increased in OA cartilage (P < 0.0001). A positive correlation between the levels of chondrocyte apoptosis and levels of caspase 3 was found (r = 0.54, P < 0.0001). OA dogs treated with the higher dosage L-NIL showed significantly reduced levels of chondrocyte apoptosis (P < 0.003) and caspase 3 (P < 0.04), but no effect on the increased level of Bcl-2 was demonstrated. CONCLUSION: This study shows that L-NIL reduces the progression of experimental OA. This effect could be related to a reduced level of chondrocyte apoptosis and is likely mediated by a decrease in the level of caspase 3 activity. A sparing effect of L-NIL on the increased level of Bcl-2 may also be a contributing factor.     

Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expression

Objective. To investigate the in vivo effect of recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) on the development of lesions and the expression of metalloproteases in the canine experimental osteoarthritis (OA) model. Methods. The right anterior cruciate ligament was sectioned percutaneously in 3 groups of dogs. The control group (n = 5) received an intraarticular injection of sterile physiologic saline (1 ml) twice weekly for 4 weeks starting on the day of surgery. The remaining 2 groups received intraarticular injections of either 2 mg (n = 6) or 4 mg (n = 5) rHuIL-1Ra in 1 ml of physiologic saline according to the same schedule as the first group. All dogs were killed 4 weeks after surgery. The macroscopic appearance of femoral condyle osteophytes and the size and severity of cartilage lesions on femoral condyles and tibial plateaus were evaluated, as were the histologic features of cartilage and synovial membrane. Levels of collagenase-1 and stromelysin-1 messenger RNA expression in cartilage and synovium were determined by Northern blotting. Results. Recombinant human IL-1Ra exerted a dose-dependent protective effect on the development of osteophytes and cartilage lesions in vivo. Treatment with rHuIL-1Ra reduced the incidence (saline-treated group 70%, 2 mg rHuIL-1Ra–treated group 42%, 4 mg rHuIL-1Ra–treated group 20%) and size (saline-treated group 2.3 ± 0.7 mm [mean ± SEM], 2 mg rHuIL-1Ra–treated group 0.7 ± 0.3 mm, 4 mg rHuIL-1Ra–treated group 0.5 ± 0.3 mm) of femoral condyle osteophytes. In addition, a dose-dependent decrease in the size (saline-treated group 24.40 ± 8.17 mm², 2 mg rHuIL-1Ra–treated group 20.90 ± 8.01 mm², 4 mg rHuIL-1Ra–treated group 7.70 ± 5.16 mm²) and the grade (0–4 scale; saline-treated group 1.20 ± 0.29, 2 mg rHuIL-1Ra–treated group 1.00 ± 0.26, 4 mg rHuIL-1Ra–treated group 0.30 ± 0.21) of the tibial plateau cartilage lesions was found, with a significant difference (P < 0.04) reached only with 4 mg rHuIL-1Ra. Similarly, the histologic lesions in dogs treated with 4 mg rHuIL-1Ra (Mankin scale; mean ± SEM 2.95 ± 0.53) were significantly less severe (P < 0.002) compared with those in the saline-treated group (4.95 ± 0.54). Importantly, rHuIL-1Ra treatment led to a significant reduction (P < 0.005) of collagenase-1 expression in OA cartilage. Conclusion. This study demonstrated that intraarticular injections of rHuIL-1Ra can protect against the development of experimentally induced OA lesions. This effect could result, at least in part, from a reduction of collagenase-1 expression. However, other catabolic processes involved in the degradation of OA cartilage may also be affected.     

Intraarticular Injections with Methylprednisolone Acetate Reduce Osteoarthritic Lesions in Parallel with Chondrocyte Stromelysin Synthesis in Experimental Osteoarthritis

Objective. To examine the effect of intraarticular injections of methylprednisolone acetate (MA) on osteoarthritic lesions and chondrocyte stromelysin synthesis in experimental osteoarthritis (OA). Methods. In 15 mongrel dogs, the anterior cruciate ligament of the right knee was sectioned by a stab wound. Eight dogs received intraarticular injections of MA (20 mg) at the time of surgery and 4 weeks later; 7 had no treatment. The dogs were killed 8 weeks after surgery. Five normal dogs were used as controls. Macroscopic evaluation of the lesions, including measurements of osteophytes and areas of surface lesions on the condyles and plateaus, was conducted, along with histologic evaluation of the severity of lesions. Immunohistochemical analysis was carried out using a rabbit polyclonal antibody against stromelysin, followed by evaluation of matrix and chondrocyte staining using morphometric analysis. Results. Treatment with MA significantly reduced the incidence (P < 0.0004) and size (P < 0.0001) of osteophytes. The histologic grading of cartilage lesions was also significantly reduced both on condyles (P < 0.01) and on plateaus (P < 0.002). Immunohistochemical studies revealed, for OA cartilage, a marked increase (P < 0.002) in the percentage of chondrocytes positive for stromelysin and in the intensity of staining throughout all the layers of the cartilage, as well as specific matrix staining (P < 0.005). Treatment with MA reduced staining at both the chondrocyte (P < 0.002) and the matrix (P < 0.01) levels toward normal. Conclusion. These findings provide additional evidence for the protective effect of corticosteroid injections on OA lesions, and indicate that the effect of this drug may be mediated through the suppression of stromelysin synthesis.     

The in situ up‐regulation of chondrocyte interleukin‐1–converting enzyme and interleukin‐18 levels in experimental osteoarthritis is mediated by nitric oxide

Objective

To investigate in situ the relationship between 2 key mediators implicated in osteoarthritic (OA) cartilage: nitric oxide (NO) and interleukin‐1–converting enzyme (ICE). Interleukin‐18 (IL‐18) was also studied and served as reference for the effects of ICE.

Methods

An OA model was created in dogs by sectioning (stab wound) the anterior cruciate ligament of the right stifle joint. Three experimental groups were studied: unoperated untreated dogs, operated untreated dogs (OA), and OA dogs treated with oral N‐iminoethyl‐L ‐lysine (L‐NIL), a specific inhibitor of inducible nitric oxide synthase (iNOS) (10 mg/kg twice a day starting immediately after surgery). At 12 weeks after surgery, cartilage from the femoral condyles and tibial plateaus were processed for immunohistochemistry for ICE, IL‐18, and protease inhibitor 9 (PI‐9), a natural inhibitor of ICE, followed by morphometric analysis. Cartilage specimens from the femoral condyles of untreated OA dogs were dissected and incubated with specific inhibitors of different signaling pathways likely to be involved in the OA process: SB 202190 (10 μM; a p38 mitogen‐activated protein kinase [MAPK] inhibitor), PD 98059 (100 μM; a MAPK kinase 1/2 [MEK‐1/2] inhibitor), NS‐398 (10 ng/ml; a specific cyclooxygenase 2 [COX‐2] inhibitor), and L‐NIL (50 μM).

Results

Both ICE and IL‐18 were present in situ in the canine cartilage, with a significant increase in the level of these 2 proteins in OA cartilage. In contrast, the level of PI‐9 was lower in OA than in normal cartilage (difference not statistically significant). Compared with untreated OA cartilage, oral treatment with L‐NIL significantly decreased ICE and IL‐18 levels in cartilage from the femoral condyles and tibial plateaus, to values similar to those in normal dogs. L‐NIL also increased the PI‐9 level in normal dogs compared with OA dogs, reaching statistical significance for femoral condyle cartilage. Interestingly, in vitro experiments demonstrated significant inhibition of ICE levels by p38, MEK‐1/2, and COX‐2 inhibitors, but not by the iNOS inhibitor.

Conclusion

This study demonstrated that in situ in OA cartilage, the stimulation of chondrocytes by NO is at least partly responsible for the up‐regulation of ICE and IL‐18 synthesis while decreasing the level of the ICE inhibitor PI‐9. The ICE level is controlled by the activation of at least 2 MAPK pathways, p38 and MEK‐1/2. Interestingly, it appears that ICE synthesis is not regulated by the endogenous production of NO. These data highlight the role played by iNOS in regulating the synthesis of major catabolic factors involved in OA cartilage degradation.

     

In vivo dual inhibition of cyclooxygenase and lipoxygenase by ML-3000 reduces the progression of experimental osteoarthritis: suppression of collagenase 1 and interleukin-1beta synthesis

OBJECTIVE: To study the therapeutic effectiveness of ML-3000, a new antiinflammatory drug that has balanced dual inhibitory activity against 5-lipoxygenase and cyclooxygenase, on the development of lesions in the experimental osteoarthritis (OA) dog model, and to determine the action of ML-3000 on the synthesis of collagenase 1 in cartilage and interleukin-1beta (IL-1beta) in synovial membrane. METHODS: The anterior cruciate ligament of the right stifle joint of 21 mongrel dogs was sectioned with a stab wound. Dogs were divided into 3 groups: group 1 (n = 7) received placebo; groups 2 (n = 7) and 3 (n = 7) were treated with therapeutic dosages of oral ML-3000 at 2.5 mg/kg/day and 5 mg/kg/day, respectively. The dogs began receiving medication the day after surgery and were killed 8 weeks later. The size and grade of cartilage erosions on both the condyles and plateaus were evaluated, and the severity of the cartilage lesions and synovial inflammation was examined histologically. Levels of collagenase 1 in cartilage and IL-1beta in the synovial membrane were measured by immunohistochemistry. In addition, levels of prostaglandin E2 (PGE2) in the synovial fluid and leukotriene B4 (LTB4) in cultured synovial membrane explants were determined using specific enzyme immunoassays. RESULTS: Serum levels of ML-3000 in treated dogs were within the therapeutic range. ML-3000 significantly decreased the size and grade of the cartilage lesions in tibials and plateaus, compared with placebo. At the histologic level, the severity of cartilage lesions was also decreased in the ML-3000-treated dogs versus the placebo-treated dogs in both the condyles and the plateaus. All 3 OA groups exhibited a notable and similar level of synovial inflammation. ML-3000 significantly decreased the level of PGE2 in synovial fluid and LTB4 production by synovium. It also markedly reduced the levels of collagenase 1 in cartilage and IL-1beta in synovial membrane. CONCLUSION: ML-3000 significantly reduced the development of lesions in experimental dog OA. The drug acts by reducing the synthesis of the inflammation mediators PGE2 and LTB4 and catabolic factors such as collagenase 1 and IL-1beta, which are known to play an important role in the pathophysiology of OA lesions. The effect of the drug on catabolic factors could possibly be related to its inhibitory action on LTB4 synthesis.     

Reduction of the severity of canine osteoarthritis by prophylactic treatment with oral doxycycline.

OBJECTIVE. In vitro studies have indicated that levels of neutral metalloproteinases in osteoarthritic (OA) cartilage are elevated and that doxycycline (doxy) inhibits collagenolytic and gelatinolytic activity in extracts of OA cartilage. The purpose of the present study was to test the effect of oral doxy administration on the severity of cartilage degeneration in OA. METHODS. OA was induced in 12 adult mongrel dogs by transection of the anterior cruciate ligament (ACL) 2 weeks after dorsal root ganglionectomy. Six dogs received doxy orally from the day after ACL transection until they were killed 8 weeks later; the other 6 served as untreated OA controls. RESULTS. The unstable knee of each untreated dog exhibited extensive full-thickness cartilage ulceration of the medial femoral condyle. In sharp contrast, cartilage on the distal aspect of the femoral condyle of the unstable knee was grossly normal in 2 doxy-treated dogs, and exhibited only thinning and/or surface irregularity in the others. Degenerative cartilage lesions on the medial trochlear ridge, superficial fibrillation of the medial tibial plateau, and osteophytosis were, however, unaffected by doxy treatment. Collagenolytic activity and gelatinolytic activity in cartilage extracts from OA knees of untreated dogs were 5-fold and 4-fold greater, respectively, than in extracts from dogs given doxy. CONCLUSION. Prophylactic administration of doxy markedly reduced the severity of OA in weight-bearing regions of the medial femoral condyle. It remains to be determined whether administration of doxy after OA changes have developed is also effective.     

The role of subchondral bone remodeling in osteoarthritis: reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model

OBJECTIVE: It has been suggested that subchondral bone remodeling plays a role in the progression of osteoarthritis (OA). To test this hypothesis, we characterized the changes in the rat anterior cruciate ligament transection (ACLT) model of OA and evaluated the effects of alendronate (ALN), a potent inhibitor of bone resorption, on cartilage degradation and on osteophyte formation. METHODS: Male Sprague-Dawley rats underwent ACLT or sham operation of the right knee. Animals were then treated with ALN (0.03 and 0.24 microg/kg/week subcutaneously) and necropsied at 2 or 10 weeks postsurgery. OA changes were evaluated. Subchondral bone volume and osteophyte area were measured by histomorphometric analysis. Coimmunostaining for transforming growth factor beta (TGF beta), matrix metalloproteinase 9 (MMP-9), and MMP-13 was performed to investigate the effect of ALN on local activation of TGF beta. RESULTS: ALN was chondroprotective at both dosages, as determined by histologic criteria and collagen degradation markers. ALN suppressed subchondral bone resorption, which was markedly increased 2 weeks postsurgery, and prevented the subsequent increase in bone formation 10 weeks postsurgery, in the untreated tibial plateau of ACLT joints. Furthermore, ALN reduced the incidence and area of osteophytes in a dose-dependent manner. ALN also inhibited vascular invasion into the calcified cartilage in rats with OA and blocked osteoclast recruitment to subchondral bone and osteophytes. ALN treatment reduced the local release of active TGF beta, possibly via inhibition of MMP-13 expression in articular cartilage and MMP-9 expression in subchondral bone. CONCLUSION: Subchondral bone remodeling plays an important role in the pathogenesis of OA. ALN or other inhibitors of bone resorption could potentially be used as disease-modifying agents in the treatment of OA.     

Reduction of the severity of canine osteoarthritis by prophylactic treatment with oral doxycycline

Objective. In vitro studies have indicated that levels of neutral metalloproteinases in osteoarthritic (OA) cartilage are elevated and that doxycycline (doxy) inhibits collagenolytic and gelatinolytic activity in extracts of OA cartilage. The purpose of the present study was to test the effect of oral doxy administration on the severity of cartilage degeneration in OA. Methods. OA was induced in 12 adult mongrel dogs by transection of the anterior cruciate ligament (ACL) 2 weeks after dorsal root ganglionectomy. Six dogs received doxy orally from the day after ACL transection until they were killed 8 weeks later; the other 6 served as untreated OA controls. Results. The unstable knee of each untreated dog exhibited extensive full-thickness cartilage ulceration of the medial femoral condyle. In sharp contrast, cartilage on the distal aspect of the femoral condyle of the unstable knee was grossly normal in 2 doxy-treated dogs, and exhibited only thinning and/or surface irregularity in the others. Degenerative cartilage lesions on the medial trochlear ridge, superficial fibrillation of the medial tibial plateau, and osteophytosis were, however, unaffected by doxy treatment. Collagenolytic activity and gelatinolytic activity in cartilage extracts from OA knees of untreated dogs were 5-fold and 4-fold greater, respectively, than in extracts from dogs given doxy. Conclusion. Prophylactic administration of doxy markedly reduced the severity of OA in weight-bearing regions of the medial femoral condyle. It remains to be determined whether administration of doxy after OA changes have developed is also effective.     

Osteoarthritic changes in canine articular cartilage, subchondral bone, and synovium fifty-four months after transection of the anterior cruciate ligament.

Anterior cruciate ligament transection (ACLT) in the dog results in osteophyte formation and in morphologic, metabolic, biochemical, and biomechanical changes in the articular cartilage of the unstable knee that mimic those of human osteoarthritis (OA). However, in dogs studied up to 2 years after ACLT, the changes have appeared to be self-limiting, which has led to the suggestion that this is a model of cartilage damage and repair, rather than of OA. To ascertain whether changes in articular cartilage and subchondral bone of dogs subjected to ACLT lead to progressive changes of OA, we studied 3 dogs for 54 months after ACLT. Arthrotomy was performed in the dogs to visualize and then transect the anterior cruciate ligament. When the dogs were killed, full-thickness ulceration of the articular cartilage was seen on the medial femoral condyle and tibial plateau of the unstable knee, while cartilage in other regions was thicker than that of the contralateral knee, consistent with hypertrophic cartilage repair. Synovial infiltration by mononuclear cells was not more severe than that seen in dogs killed at earlier intervals after ACLT, although gross fibrotic thickening of the capsule was apparent in each dog. Histomorphometric studies revealed a marked increase in subchondral bone volume and active bone formation. These findings show that the changes that develop in the canine knee joint after ACLT are progressive and are unambiguously those of OA.     

壳聚糖膝关节腔内注射疗法对兔骨关节炎关节软骨的影响

目的观察关节内注射羧甲基壳聚糖(CMCTS)对兔骨关节炎(OA)关节软骨退变及软骨基质金属蛋白酶(MMP)-1,-3mRNA表达的影响。方法24只大耳白兔行单侧膝关节前交叉韧带切断术,术后5周将动物随机分为3组,A组关节内注射2%CMCTS0.3ml,每2周1次;B组关节内注射1%透明质酸钠(HA)0.3ml,每周1次;C组关节内不注射药物。术后11周处死动物,观察各组股骨内髁关节软骨的大体变化,采用反转录-聚合酶链反应(RT-PCR)方法检测股骨内髁退变软骨中MMP-1和MMP-3的mRNA表达。结果CMCTS和HA注射组软骨退变程度较不用药组明显减轻,CMCTS注射组软骨MMP-1、MMP-3的mRNA表达明显低于HA注射组和不用药组。HA注射组软骨MMP-1和MMP-3的mRNA表达与不用药组比较,差异没有显著性意义。结论CMCTS能够明显抑制OA软骨MMP-1和MMP-3的mRNA表达,明显减轻软骨退变的程度,CMCTS对早期OA软骨有修复保护作用。