Cartilage In Experimental Osteoarthritis Research Articles

Nanomechanical Properties of Articular Cartilage Due to the PRP Injection in Experimental Osteoarthritis in Rabbits.

The purpose of this study was twofold. Firstly, we proposed a measurement protocol for the atomic force microscopy (AFM) method to determine the nanomechanical properties of articular cartilage in experimental osteoarthritis in rabbits. Then, we verified if mechanical properties can be evaluated with AFM shortly after platelet-rich plasma (PRP) injection. We hypothesized that the modulus determined by AFM indentation experiments could be utilized as a progressive disease marker during the treatment of osteoarthritis. The rabbits were equally divided into three groups of six: control (group 1); injections of saline (0.5 mL) and 10% surgical talc (Talcum Pharmaceutical®, Minsk, Belarus) were delivered into the right knee under the patella (group 2 and 3); and PRP was injected into the right knee (group 3). In group 2, the arithmetic average of absolute values (Ra) change was a 25% increase; the maximum peak height (Rp) increased by over 102%, while the mean spacing between local peaks (S) increased by 28% (p < 0.05). In group 3, Ra increased by 14% and Rp increased by 32%, while S decreased by 75% (p < 0.05). The Young’s modulus of the surface layers decreased by 18% as a result of induced model of osteoarthritis (IMO) (p < 0.05), and it increased by 9% (p < 0.05) as a result of PRP therapy, which means that the mechanical properties of cartilage were partially recovered. This research demonstrates that Young’s modulus utilized on a nanometer scale has potential to be a progressive disease marker during the treatment of osteoarthritis.

Alleviation of Murine Osteoarthritis by Cartilage-Specific Deletion of IκBζ.

IκBζ, an atypical IκB family member, regulates gene expression in the nucleus as a transcriptional cofactor. Although IκBζ has been extensively studied in the immune system, its specific roles in osteoarthritis (OA) are currently unknown. The objective of this study was to investigate the potential role of IκBζ in chondrocyte catabolism and OA pathogenesis. We also determined the molecular mechanism underlying its relationship to the transcription factor NF-κB. We determined expression levels of IκBζ in mouse chondrocytes treated with interleukin-1β (IL-1β), in human OA cartilage, and in mouse experimental OA cartilage. Adenovirus-mediated overexpression and small interfering RNA knockdown of IκBζ were performed to determine the impact of IκBζ on catabolic gene expression in vitro. Cartilage-specific IκBζ-transgenic and -knockout mice were generated and used for in vivo studies. Experimental and spontaneous OA were induced by surgical destabilization of the medial meniscus and by aging, respectively. Coimmunoprecipitation assay was used to examine the association between IκBζ and NF-κB subunits. IκBζ was highly up-regulated in chondrocytes in response to IL-1β and in OA cartilage of human and mouse knee joints. Overexpression of IκBζ in chondrocytes promoted spontaneous OA development by activating chondrocyte catabolism. Genetic ablation of IκBζ in chondrocytes abolished catabolic gene induction by IL-1β and protected against the development of experimental OA. IκBζ formed complexes with NF-κB members to regulate catabolic factor expression. These findings demonstrate a critical role for IκBζ in OA pathogenesis. Inhibition of IκBζ function might be an effective therapeutic approach for OA treatment.

Role of autophagy in the progression of osteoarthritis: The autophagy inhibitor, 3-methyladenine, aggravates the severity of experimental osteoarthritis.

Accumulating evidence suggests that autophagy is closely related to the pathogenesis of osteoarthritis (OA). The aim of this study was to determine the changes in autophagy during the progression of OA and to elucidate the specific role of autophagy in OA. For this purpose, a cellular model of OA was generated by stimulating SW1353 cells with interleukin (IL)-1β and a rabbit model of OA was also established by an intra-articular injection of collagenase, followed by treatment with the autophagy specific inhibitor, 3-methyladenine (3-MA). Cell viability was analyzed by MTS assay, and the mRNA expression levels of matrix metalloproteinases (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 were determined by RT-qPCR. Cartilage degeneration was examined under a light microscope, and autophagosome and chondrocyte degeneration was observed by transmission electron microscopy. The protein expression of Beclin-1 and light chain 3 (LC3)B was evaluated by western blot analysis and immunofluorescence staining. We found that the autophagy was enhanced during the early stages and was weakened during the late stages of experimental OA. The inhibition of autophagy by 3-MA significantly aggravated the degeneration of chondrocytes and cartilage in experimental OA. Our results thus determine the changes in autophagy during different stages of OA, as well as the role of impaired autophagy in the development of OA. Our data suggest that the regulation of autophagy may be a potential therapeutic strategy with which to attenuate OA.

MORPHOGENESIS OF KNEE HYALINE CARTILAGE DURING INTRAARTICULAR INJECTION OF PLATELET-RICH AUTOLOGOUS PLASMA AND/OR HYALURONIC ACID PREPARATION IN RATS WITH EXPERIMENTAL OSTEOARTHRITIS

According to current concepts, the influence of autologous platelet-rich plasma (PRP) and high molecular hyaluronates (HA) on the repair of hyaline cartilage during its inflammatory and degenerative changes has been insufficiently studied yet. The objective of the work was to evaluate the morphological changes in the structure of hyaline cartilage in experimental osteoarthritis after intra-articular injection of PRP and/or HA. Material and methods. The authors used 50 adult rats of Wistar line, weighing 250±2,2 g., distributed into five groups of 10 animals (two control and three experimental groups). An experimental gonarthosis was simulated on four groups of animals. Animals of the first experimental group received intra-articular injection of PRP, the second group – HA, the third – both PRP and HA. Results. No morphological signs of degenerative and inflammatory changes in the first control group were identified. Following osteoarthritis simulation the articular cartilage thinned to 121±20,4 microns (p0,05) and the volume fraction of chondrocyte decreased to 1,2±0,6% (p0,05). The authors observed an uneven coloration of collagen fibers with severe tinctorial properties disorder of the articular cartilage matrix. After the RPR introduction the authors observed tickening of the articular cartilage up to 275±18,9 micron (p0,05) and the volume fraction of chondrocytes up to 18,4±2,0% (p0,05). The contour of the cartilage surface became smoother with the formation of a cell-free zone. Collagen fibers demonstrated a uniform distribution, tinctorial properties of cartilage matrix in all areas were preserved, no signs of inflammation were noted. After HA introduction the authors observed thickening of the cartilage plate up to 264±21,3 microns (p0,05) and the volume fraction of chondrocytes up to 11,6±1,2% (p0,05). The surface of the cartilage featured uneven contours due to multiple areas of pulping. Uneven tinctorial properties of cartilage matrix, thickness and orientation of collagen fibres were retained. No signes of inflammation were observed. After the combined consecutive application of PRP and HA cartilage thickness increased up to 268±15,3 microns (p0,05) and the volume fraction of chondrocytes increased up to 12,7±0,9% (p0,05). Individual portions of pulping on the cartilage surface were observed. Tinctorial properties of the cartilage matrix were preserved; the focal uneven staining of collagen fibers in the basal zone was observed. No signs of inflammation were noted. Conclusion. During osteoarthritis simulation in knee joints of experimental Wistar line rats the authors observed severe structural changes in articular hyaline cartilage including complete destruction accompanied by vascular proliferation and granulomatous inflammation. Intraarticular injections of PRP, HA as well as PRP in combination with HA during the simulation of osteoarthrosis were accompanied by a decrease in the severity of degenerative and dystrophic processes and improval of tinctorial properties of articular cartilage matrix. Sole application of PRP or consecutive application of PRP followed by HA were observed as having the more significant reparative effect on articular cartilage as compared to HA.

Inhibition of BATF/JUN transcriptional activity protects against osteoarthritic cartilage destruction

ObjectiveThe basic leucine zipper transcription factor, ATF-like (BATF), a member of the Activator protein-1 family, promotes transcriptional activation or repression, depending on the interacting partners (JUN-B or C-JUN). Here, we...

Reciprocal activation of hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis amplifies catabolic signaling in osteoarthritis

Hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis in chondrocytes serve as catabolic regulators of osteoarthritic cartilage destruction by regulating the expression of catabolic factor genes. We explored possible crosstalk between these signaling pathways and its biological significance in osteoarthritis (OA). Microarray analysis, various mRNA and protein assays were conducted using primary cultured mouse articular chondrocytes and experimental OA cartilage to reveal molecular mechanisms underlying the crosstalk between HIF-2α and the zinc-ZIP8-MTF1 axis. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or MTF1 (Ad-Mtf1) in wild-type mice or mice with cartilage-specific conditional knockout of HIF-2α (Epas1(fl/fl);Col2a1-Cre), ZIP8 (Zip8(fl/fl);Col2a1-Cre), or MTF1 (Mtf1(fl/fl);Col2a1-Cre). HIF-2α activated the zinc-ZIP8-MTF1 axis in chondrocytes by upregulating the Zn(2+) transporter ZIP8, thereby increasing Zn(2+) influx and activating the downstream transcription factor MTF1. The zinc-ZIP8-MTF1 axis, in turn, acted as a novel transcriptional regulator of HIF-2α. HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis amplified HIF-2α regulation of OA cartilage destruction by synergistically promoting expression of matrix-degrading enzymes. Thus, HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis, together with zinc-ZIP8-MTF1 regulation of HIF-2α, acted collectively to synergistically promote expression of matrix-degrading enzymes and OA cartilage destruction. Our findings identify a reciprocal activation mechanism involving HIF-2α and the zinc-ZIP8-MTF1 axis during OA pathogenesis that amplifies catabolic signaling and cartilage destruction.

Astaxanthin ameliorates cartilage damage in experimental osteoarthritis

Purpose. Astaxanthin is a red-pigment carotenoid found in certain marine animals and plants. Astaxanthin has been shown to inhibit matrix metalloproteinases (MMPs) expression in vitro. However, the effect of astaxanthin on cartilage is still unclear. The aim of this study was to investigate the effects of astaxanthin on cartilage in experimental osteoarthritis (OA).Methods. New Zealand rabbits underwent anterior cruciate ligament transection to induce OA in right knee. Rabbits received intra-articular injection containing 0.3 ml of vehicle (dimethyl sulfoxide) or astaxanthin (50 μM). Injection was started on the day of operation, and the injection were performed once weekly for six consecutive weeks. Then, rabbits were sacrificed and the right knees were harvested for study.Results. Cartilage degradation was reduced by astaxanthin, as assessed by morphological and histological examination. Astaxanthin inhibited the gene expression of MMP-1, MMP-3, and MMP-13 in cartilage as compared with the vehicle group.Conclusions. The results suggest that astaxanthin may be considered as pharmaceutical agent in OA treatment.

Low-density lipoprotein receptor–related protein 5 governs Wnt-mediated osteoarthritic cartilage destruction

IntroductionWnt ligands bind to low-density lipoprotein receptor–related protein (LRP) 5 or 6, triggering a cascade of downstream events that include β-catenin signaling. Here we explored the roles of LRP5 in interleukin 1β (IL-1β)- or Wnt-mediated osteoarthritic (OA) cartilage destruction in mice.MethodsThe expression levels of LRP5, type II collagen, and catabolic factors were determined in mouse articular chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild-type, Lrp5 total knockout (Lrp5-/-) and chondrocyte-specific knockout (Lrp5fl/fl;Col2a1-cre) mice was caused by aging, destabilization of the medial meniscus (DMM), or intra-articular injection of collagenase. The role of LRP5 was confirmed in vitro by small interfering RNA–mediated knockdown of Lrp5 or in Lrp5-/- cells treated with IL-1β or Wnt proteins.ResultsIL-1β treatment increased the expression of LRP5 (but not LRP6) via JNK and NF-κB signaling. LRP5 was upregulated in human and mouse OA cartilage, and Lrp5 deficiency in mice inhibited cartilage destruction. Treatment with IL-1β or Wnt decreased the level of Col2a1 and increased those of Mmp3 or Mmp13, whereas Lrp5 knockdown ameliorated these effects. In addition, we found that the functions of LRP5 in arthritic cartilage were subject to transcriptional activation by β-catenin. Moreover, Lrp5-/- and Lrp5fl/fl;Col2a1-cre mice exhibited decreased cartilage destruction (and related changes in gene expression) in response to experimental OA.ConclusionsOur findings indicate that LRP5 (but not LRP6) plays an essential role in Wnt/β-catenin-signaling-mediated OA cartilage destruction in part by regulating the expression levels of type II collagen, MMP3, and MMP13.

NAMPT (visfatin), a direct target of hypoxia-inducible factor-2α, is an essential catabolic regulator of osteoarthritis

ObjectiveHypoxia-inducible factor 2α (HIF-2α), encoded by Epas1, causes osteoarthritic cartilage destruction by regulating the expression of matrix-degrading enzymes. We undertook this study to explore the role of nicotinamide phosphoribosyltransferase (NAMPT or visfatin) in HIF-2α-mediated osteoarthritic cartilage destruction.MethodsThe expression of HIF-2α, NAMPT and matrix-degrading enzymes was determined at the mRNA and protein levels in human osteoarthritis (OA) cartilage, mouse experimental OA cartilage and primary cultured mouse chondrocytes. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) surgery or intra-articular injection of Ad-Epas1 or Ad-Nampt in wild-type, Epas1+/−, Epas1fl/fl;Col2a1-Cre and Col2a1-Nampt transgenic (TG) mice. Primary cultured mouse chondrocytes were treated with recombinant NAMPT protein or were infected with adenoviruses.ResultsWe found that the Nampt gene is a direct target of HIF-2α in articular chondrocytes and OA cartilage. NAMPT protein, in turn, increased mRNA levels and activities of MMP3, MMP12 and MMP13 in chondrocytes, an action that was necessary for HIF-2α-induced expression of catabolic enzymes. Gain-of-function studies (intra-articular injection of Ad-Nampt; Col2a1-Nampt TG mice) and loss-of-function studies (intra-articular injection of the NAMPT inhibitor FK866) demonstrated that NAMPT is an essential catabolic regulator of osteoarthritic cartilage destruction caused by HIF-2α or DMM surgery.ConclusionsOur findings indicate that NAMPT, whose corresponding gene is a direct target of HIF-2α, plays an essential catabolic role in OA pathogenesis and acts as a crucial mediator of osteoarthritic cartilage destruction caused by HIF-2α or DMM surgery.

Dkk‐1 expression in chondrocytes inhibits experimental osteoarthritic cartilage destruction in mice

Dkk is a family of canonical Wnt antagonists with 4 members (Dkk-1, Dkk-2, Dkk-3, and Dkk-4). We undertook this study to explore the roles of Dkk-1 and Dkk-2 in osteoarthritic (OA) cartilage destruction in mice. Expression of Dkk and other catabolic factors was determined at the messenger RNA and protein levels in human and mouse OA cartilage. Experimental OA in mice was induced by destabilization of the medial meniscus (DMM) or by intraarticular injection of Epas1 adenovirus (AdEPAS-1). The role of Dkk in OA pathogenesis was examined by intraarticular injection of AdDkk-1 or by using chondrocyte-specific Dkk1 (Col2a1-Dkk1)-transgenic mice and Dkk2 (Col2a1-Dkk2)-transgenic mice. Primary culture mouse chondrocytes were also treated with recombinant Dkk proteins. We found opposite patterns of Dkk1 and Dkk2 expression in human and mouse experimental OA cartilage: Dkk1 was up-regulated and Dkk2 was down-regulated. Overexpression of Dkk1 by intraarticular injection of AdDkk-1 significantly inhibited DMM-induced experimental OA. DMM-induced OA was also significantly inhibited in Col2a1-Dkk1-transgenic mice compared with their wild-type littermates. However, Col2a1-Dkk2-transgenic mice showed no significant difference in OA pathogenesis. Wnt-3a, which activates the canonical Wnt pathway, induced Mmp13 and Adamts4 expression in primary culture chondrocytes, an effect that was significantly inhibited by Dkk-1 pretreatment or Dkk1 overexpression. Our findings indicate that expression of Dkk1, but not Dkk2, in chondrocytes inhibits OA cartilage destruction. The protective effect of Dkk-1 appears to be associated with its capacity to inhibit Wnt-mediated expression of catabolic factors, such as Mmp13, providing evidence that Dkk-1 might serve as a therapeutic target for OA treatment.

Interleukin‐6 plays an essential role in hypoxia‐inducible factor 2α–induced experimental osteoarthritic cartilage destruction in mice

Hypoxia-inducible factor 2α (HIF-2α) (encoded by Epas1) causes osteoarthritic (OA) cartilage destruction by regulating the expression of catabolic factor genes. We undertook this study to explore the role of interleukin-6 (IL-6) in HIF-2α-mediated OA cartilage destruction in mice. The expression of HIF-2α, IL-6, and catabolic factors was determined at the messenger RNA and protein levels in primary culture mouse chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild-type, HIF-2α-knockdown (Epas1+/-), and Il6-/- mice was caused by intraarticular injection of Epas1 adenovirus or destabilization of the medial meniscus. The role of IL-6 was determined by treating with recombinant IL-6 protein or by injecting HIF-2α adenovirus (AdEpas1) intraarticularly in mice with or without IL-6-neutralizing antibody. We found that Il6 is a direct target gene of HIF-2α in articular chondrocytes. Both Epas1 and Il6 were up-regulated in human and mouse OA cartilage, whereas HIF-2α knockdown in mice led to inhibition of both Il6 expression and cartilage destruction. Treatment with IL-6 enhanced Mmp3 and Mmp13 expression; conversely, Il6 knockdown inhibited HIF-2α-induced up-regulation of Mmp3 and Mmp13. Injection of IL-6 protein into mouse knee joints triggered OA cartilage destruction, whereas IL-6 neutralization led to blocking of HIF-2α-induced cartilage destruction with concomitant modulation of Mmp3 and Mmp13 expression. Moreover, Il6 knockout resulted in inhibition of AdEpas1-induced and destabilization of the medial meniscus-induced cartilage destruction as well as inhibition of Mmp3 and Mmp13 expression. Our findings indicate that IL-6 acts as a crucial mediator of HIF-2α-induced experimental OA cartilage destruction in mice via regulation of Mmp3 and Mmp13 levels.

Cytokine-like 1 Knock-out Mice (Cytl1−/−) Show Normal Cartilage and Bone Development but Exhibit Augmented Osteoarthritic Cartilage Destruction

We have shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor that regulates chondrogenesis of mouse mesenchymal cells (Kim, J. S., Ryoo, Z. Y., and Chun, J. S. (2007) J. Biol. Chem. 282, 29359-29367). In this previous work, we found that Cytl1 expression was very low in mesenchymal cells, increased dramatically during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. Moreover, exogenous addition or ectopic expression of Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells. In the current study, we generated a Cytl1 knock-out (Cytl1(-/-)) mouse to investigate the in vivo role of Cytl1. Deletion of the Cytl1 gene did not affect chondrogenesis or cartilage development. Cytl1(-/-) mice also showed normal endochondral ossification and long bone development. Additionally, ultrastructural features of articular cartilage, such as matrix organization and chondrocyte morphology, were similar in wild-type and Cytl1(-/-) mice. However, Cytl1(-/-) mice were more sensitive to osteoarthritic (OA) cartilage destruction. Compared with wild-type littermates, Cytl1(-/-) mice showed more severe OA cartilage destruction upon destabilization of the medial meniscus of mouse knee joints. In addition, expression levels of Cytl1 were markedly decreased in OA cartilage of humans and experimental mice. Taken together, our results suggest that, rather than regulating cartilage and bone development, Cytl1 is required for the maintenance of cartilage homeostasis, and loss of Cytl1 function is associated with experimental OA cartilage destruction in mice.

Effects of diallyl sulphide in chondrocyte and cartilage in experimental osteoarthritis in rabbit

Diallyl sulphide (DAS) is known for its antioxidant, anticancer and detoxifying properties. The aim of this study was to investigate the effect of DAS on rabbit articular chondrocytes and cartilage in experimental osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). DAS inhibited matrix metalloproteinase-1 (MMP-1), MMP-3 and MMP-13 expression in interleukin-1beta (IL-1β)-induced chondrocytes. In an in vivo study, DAS ameliorated cartilage degradation as assessed by morphological and histological examination. Messenger RNA expression of MMP-1, MMP-3, MMP-13 and IL-1β was inhibited by DAS in cartilage. In addition, DAS increased the collagen II level in cartilage. The results suggest that DAS may protect cartilage in the development of OA.

Thymoquinone inhibits matrix metalloproteinase expression in rabbit chondrocytes and cartilage in experimental osteoarthritis

Thymoquinone (TQ) is the main constituent of Nigella sativa oil, which has been traditionally used against arthritis in the Middle East. In this study, we investigated the effect of TQ against matrix metalloproteinase (MMP) expression in both rabbit chondrocytes and animal mode of osteoarthritis (OA) induced by anterior cruciate ligament transection and tested whether or not nuclear factor kappa B (NF-κB) was involved in this process. TQ down-regulated MMP-1, MMP-3 and MMP-13 expression and up-regulated tissue inhibitors of metalloproteinase-1 expression as assessed by quantitative realtime polymerase chain reaction. In addition, NF-κB p65 protein level as well as its translocation induced by interleukin-1β were inhibited by TQ. Our findings suggest the potential of TQ in the treatment of OA.

Trichostatin A inhibits expression of cathepsins in experimental osteoarthritis

The aim of this study was to investigate the effects of trichostatin A (TSA) on expression of cathepsins in cartilage in experimental osteoarthritis (OA). OA was induced in 18 rabbits by bilateral anterior cruciate ligament transection (ACLT). Four weeks after surgery, rabbits received intra-articular injection with TSA dissolved in the dimethylsulphoxide (DMSO) in the right knees and DMSO in the left knees once a week for 5 weeks. Rabbits were killed 7 days after the last injection. The knee joints were assessed by morphological and histological examination. Messenger RNA expression of cathepsins K, B, L, S and cystatin C was studied by real-time PCR. TSA inhibited the expression of cathepsins K, B, L, S and cystatin C accompanied with the less degradation in cartilage. The results suggest that TSA exhibits protective effects against cartilage degradation in rabbits with OA and the effects may be associated with the inhibition of cathepsins.

Correlation between the MR T2 value at 4.7 T and relative water content in articular cartilage in experimental osteoarthritis induced by ACL transection

Both animal and human studies using magnetic resonance imaging (MRI) show that cartilage degeneration increases the T2 value. However, it is unclear whether the T2 value correlates linearly with water content in cartilage with osteoarthritis. The purpose of this study was to investigate the relationship between the T2 value and water content using an animal model of cartilage injury measured at 4.7 T. Thirty Sprague Dawley rats were randomly separated into three groups (n=10 for each group). Group 1 rats were not operated on (control). Group 2 rats received a sham operation, and group 3 rats received an anterior cruciate ligament (ACL) transection. Six rats of each group were randomly assigned to T2 measurement and later subjected to ex vivo analysis of the relative water content of the knee cartilage. The other four rats in each group were killed, and the severity of cartilage degeneration was examined histologically. The knees of the six rats in the ACL transection group were imaged sequentially 4 and 13 weeks after ACL transection, and the relative water content was measured at 13 weeks. The cartilage T2 value was significantly higher 4 and 13 weeks after ACL transection in the operated knees than in the knees of the control and sham groups. The cartilage T2 value was significantly higher at 13 weeks than at 4 weeks in the operated knees. The T2 value was strongly positively correlated with the relative water content (R=0.885, P<0.0001). The trend of changes in the T2 values is consistent with an increase in the relative water content in our cartilage degeneration model. This model has potential use for the clinical evaluation of osteoarthritis.

The protective effect of licofelone on experimental osteoarthritis is correlated with the downregulation of gene expression and protein synthesis of several major cartilage catabolic factors: MMP-13, cathepsin K and aggrecanases

This study sought to evaluate the levels of mRNA expression and protein synthesis of MMP-13, cathepsin K, aggrecanase-1 (ADAMTS-4), aggrecanase-2 (ADAMTS-5) and 5-lipoxygenase (5-LOX) in cartilage in the experimental anterior cruciate ligament (ACL) dog model of osteoarthritis (OA), and to examine the effects of treatment with licofelone, a 5-lipoxygenase (LOX)/cyclooxygenase (COX) inhibitor, on the levels of these catabolic factors. Sectioning of the ACL of the right knee was performed in three experimental groups: group 1 received no active treatment (placebo group); and groups 2 and 3 received therapeutic concentrations of licofelone (2.5 or 5.0 mg/kg/day orally, respectively) for 8 weeks, beginning the day following surgery. A fourth group consisted of untreated dogs that were used as normal controls. Specimens of cartilage were selected from lesional areas of OA femoral condyles and tibial plateaus, and were processed for real-time quantitative PCR and immunohistochemical analyses. The levels of MMP-13, cathepsin K, ADAMTS-4, ADAMTS-5 and 5-LOX were found to be significantly increased in OA cartilage. Licofelone treatment decreased the levels of both mRNA expression and protein synthesis of the factors studied. Of note was the marked reduction in the level of 5-LOX gene expression. The effects of the drug were about the same at both tested dosages. In vivo treatment with therapeutic dosages of licofelone has been found to reduce the degradation of OA cartilage in experimental OA. This, coupled with the results of the present study, indicates that the effects of licofelone are mediated by the inhibition of the major cartilage catabolic pathways involved in the destruction of cartilage matrix macromolecules. Moreover, our findings also indicate the possible auto-regulation of 5-LOX gene expression by licofelone in OA cartilage.

Effect of caffeic acid phenethyl ester on cartilage in experimental osteoarthritis.

Activation of nuclear factor kappa B (NF-kappaBeta) in synovial cells is seen in RA and OA patients. Caffeic acid phenethyl ester (CAPE) is a specific and potent inhibitor of NF-kappaBeta. We aimed to determine the in vivo effects of intra-articular injections of CAPE on cartilage in an experimental rabbit osteoarthritis (OA) model. Two groups of six New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT). Four weeks after ACLT, the test group was injected with 150 micro g/kg CAPE in 0.5% ethanol once daily for 2 weeks and the control group was injected the same amount of 0.5% ethanol intra-articularly. All rabbits were killed 2 weeks after the last injection, and cartilage tissue was evaluated morphologically. A histological score totaling 7 points was determined for each knee. The CAPE group showed significantly decreased cartilage destruction and reduced loss of matrix proteoglycans. The histological score for cartilage tissue was significantly better in the CAPE group than in the control group (3.0+/-0.25 vs 5.3+/-0.55, P=0.005). This study suggests that intra-articular injection of CAPE may protect cartilage against the development of experimentally induced OA.

Effects of intermittently applied cyclic loading on proteoglycan metabolism and swelling behaviour of articular cartilage explants

Objective: The aim of this study was to evaluate the effect of tissue load, frequency and load duration on the biosynthesis and release of proteoglycans (PGs) as well as on the swelling behaviour of cultured mature bovine articular cartilage superimposed with intermittent loads.Methods: Cyclic compressive pressure was introduced for 1, 3 or 6 days using a sinusoidal waveform of 0.5Hz-frequency with a peak stress of 0.1, 0.5 or 1.0MPa. The loads were applied for a period of 10 seconds (s) followed by a load-free period of 10, 100 or 1000s. The incorporation of [35S]-SO4into glycosaminoglycans (GAGs) during the final 18h, the content of GAGs and DNA as well as the deformation of loaded explants were determined.Results: The PG synthesis is sensitive to changes in the loading conditions applied, whereas the release of newly synthesized PG is not. A maximum PG synthesis is observed at day 3, and under load-free intervals of 100s. After 6 days of loading the release of endogenous PGs is significantly elevated, the viability of superficial chondrocytes decreased, and cartilage swelling is observed.Conclusions: Considering numerous reports of elevated PG levels synthesized as well as released from human and experimental osteoarthritic cartilage, our results implicate that degenerative processes can also be mimicked by applying well-defined mechanical conditions as described here.

Increased proteoglycan synthesis in cartilage in experimental canine osteoarthritis does not reflect a permanent change in chondrocyte phenotype.

To determine whether chondrocytes in early experimental osteoarthritic (OA) cartilage continue to show increased synthesis and turnover of proteoglycans (PGs) during explant culture. A comparison was also made between the responsiveness of experimental OA and control cartilage to interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) after 1 day and 3 days in culture. OA was induced in mature animals by sectioning of the anterior cruciate ligament followed by 3 months of normal exercise. PG synthesis in the articular cartilage was determined by measuring 35S-sulfate incorporation during explant culture over 1-3 days. Inhibition of PG synthesis was also determined with various concentrations of IL-1 beta and TNF alpha after 1 and 3 days in culture. PGs extracted from the articular cartilage over 1-3 days in culture were examined by agarose-polyacrylamide gel electrophoresis. Up to 24 hours after excision from the joint, PG synthesis was higher in experimental OA cartilage than in control cartilage. It was also less sensitive to inhibition by TNF alpha. These differences were no longer detected after 48-72 hours in culture. There were no changes in the relative proportions of aggrecan and decorin/biglycan extracted from and synthesized by control and experimental OA cartilage over the 3 days in culture. Previous results indicated that PG synthesis and turnover in articular cartilage was increased for many months after induction of experimental OA. Our present results show that the enhanced rate of PG synthesis and turnover were evident in freshly explanted tissue, but the differences were lost over 3 days in culture. A decreased responsiveness to TNF alpha was also lost. The hypermetabolic activity of experimental OA chondrocytes was thus reversible and not a permanent change in chondrocyte phenotype.