Do model polymer therapeutics sufficiently diffuse through articular cartilage to be a viable therapeutic route?

The ability of a polymer therapeutic to access the appropriate subcellular location is crucial to its efficacy and is defined to a large part by the many and complex cellular biological and biochemical barriers such that a construct must traverse. It is shown here that model dextrin conjugates are able to pass through a cartilaginous extracellular matrix into chondrocytes, with little perturbation of the matrix structure, indicating that targeting of potential therapeutics through a cartilaginous extracellular matrix should be proven possible. Rapid chondrocytic targeting of drugs which require intra cellularisation for their activity and uniform extracellular concentrations of drugs with an extracellular target, is thus enabled though polymer conjugation.

Histopathology of chondronecrosis development in knee articular cartilage in a rat model of Kashin-Beck disease using T-2 toxin and selenium deficiency conditions.

The objective of this study is to observe pathogenic lesions of joint cartilages in rats fed with T-2 toxin under a selenium deficiency nutrition status in order to determine possible etiological factors causing Kashin-Beck disease (KBD). Sprague-Dawley rats were fed selenium-deficient or control diets for 4 weeks prior to their being exposed to T-2 toxin. Six dietary groups were formed and studied 4 weeks later, i.e., controls, selenium-deficient, low T-2 toxin, high T-2 toxin, selenium-deficient diet plus low T-2 toxin, and selenium-deficient diet plus high T-2 toxin. Selenium deficiencies were confirmed by the determination of glutathione peroxidase activity and selenium levels in serum. The morphology and pathology (chondronecrosis) of knee joint cartilage of experimental rats were observed using light microscopy and the expression of proteoglycans was determined by histochemical staining. Chondronecrosis in deep zone of articular cartilage of knee joints was seen in both the low and high T-2 toxin plus selenium-deficient diet groups, these chondronecrotic lesions being very similar to chondronecrosis observed in human KBD. However, the chondronecrosis observed in the rat epiphyseal growth plates of animals treated with T-2 toxin alone or T-2 toxin plus selenium-deficient diets were not similar to that found in human KBD. Our results indicate that the rat can be used as a suitable animal model for studying etiological factors contributing to the pathogenesis (chondronecrosis) observed in human KBD. However, those changes seen in epiphyseal growth plate differ from those seen in human KBD probably because of the absence of growth plate closure in the rat.

The effects of mycotoxins and selenium deficiency on tissue-engineered cartilage.

OBJECTIVE: To investigate the effects of 3 mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin, in the presence and absence of selenium (Se) on the metabolism of tissue-engineered cartilage to mimic conditions found in Kashin-Beck disease (KBD) environments. MATERIALS AND METHODS: Chondrocytes were seeded onto bone matrix gelatin (BMG) to construct engineered cartilage. The 3 toxins were added to the culture media for 3 weeks followed by immunhistochemical analyses of collagens type II and X, aggrecan, matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3), MMP inhibitors 1 and 3 (TIMP-1 and TIMP-3) and α(2) macroglobulin (α2M). RESULTS: Type II collagen was decreased while type X collagen was increased in response to DON, NIV and T-2 toxin. Aggrecan was reduced by all 3 mycotoxins. Compared with the control, the 3 toxins decreased the expression of α2M, TIMP-1 and TIMP-3, and increased the expression of MMP-1 and MMP-3. Se could partially inhibit the effects of DON, NIV and T-2 toxins. CONCLUSION: Under the low Se condition, the 3 mycotoxins produced procatabolic changes in cartilage resulting in the loss of aggrecan and type II collagen and promoted a hypertrophic phenotype of chondrocytes characterized by increasing type-X-collagen expression, enhancing the expression of MMPs, while weakening the TIMPs. Se could partially block the effects mentioned above. These results support the hypothesis that the combination of mycotoxin stress and Se deficiency would be the causative factors for KBD.

Novel phosphoramidate prodrugs of N-acetyl-(D)-glucosamine with antidegenerative activity on bovine and human cartilage explants.

(D)-Glucosamine and other nutritional supplements have emerged as safe alternative therapies for osteoarthritis (OA), a chronic and degenerative articular joint disease. In our preceding paper, a series of novel O-6 phosphate N-acetyl (d)-glucosamine prodrugs aimed at improving the oral bioavailability of N-acetyl-(d)-glucosamine as its putative bioactive phosphate form were shown to have greater chondroprotective activity in vitro when compared to the parent agent. In order to extend the SAR studies, this work focuses on the O-3 and O-4 phosphate prodrugs of N-acetyl-(d)-glucosamine bearing a 4-methoxy phenyl group and different amino acid esters on the phosphate moiety. Among the compounds, the (l)-proline amino acid-containing prodrugs proved to be the most active of the series, more effective than the prior O-6 compounds, and well processed in chondrocytes in vitro. Data on human cartilage support the notion that these novel O-3 and O-4 regioisomers may represent novel promising leads for drug discovery for osteoarthritis.

Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin-Beck disease.

OBJECTIVE: To investigate the effects of mycotoxin moniliformin (MON) on the metabolism of aggrecan and type II collagen in human chondrocytes in vitro and the relationship between MON and Kashin-Beck disease (KBD). METHODS: Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without MON toxin. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of aggrecan and type II collagen in the cartilage was determined using immunocytochemical staining. RESULTS: MON toxin inhibited chondrocyte viability in dose-dependent and time-dependent manners. MON reduced aggrecan and type II collagen syntheses in the tissue-engineered cartilage. MON also increased the expression of matrix metalloproteinase-1 (MMP-1), MMP-13, BC4 epitopes, and CD44 in cartilages. However, the expression of 3B3(-) epitopes in cartilages was inhibited by MON. Selenium partially alleviated the damage of aggrecan induced by MON toxin. CONCLUSION: MON toxin promoted the catabolism of aggrecan and type II collagen in human chondrocytes.

Contrasting effects of n-3 and n-6 fatty acids on cyclooxygenase-2 in model systems for arthritis.

Cyclooxygenase-2 (COX-2) is intimately involved in symptoms of arthritis while dietary n-3 polyunsaturated fatty acids (PUFA) are thought to be beneficial. In these experiments, using both bovine and human in vitro systems that mimic features of arthritis, we show that the n-3 PUFA eicosapentaenoic acid (EPA) is able to reduce mRNA and protein levels of COX-2. Activity, as assessed through prostaglandin E(2) formation, was also reduced in a dose-dependent manner. These effects of EPA contrasted noticeably with the n-6 PUFA, arachidonic acid. The data provide direct evidence for a molecular mechanism by which dietary n-3 PUFA, such as EPA, can reduce inflammation and, hence, associated symptoms in arthritis.

Modification of palm oil for anti-inflammatory nutraceutical properties.

Palm oil is one of the most important edible oils in the world. Its composition (rich in palmitate and oleate) make it suitable for general food uses but its utility could be increased if its fatty acid quality could be varied. In this study, we have modified a palm olein fraction by transesterification with the n-3 polyunsaturated fatty acids, alpha-linolenate or eicosapentaenoic acid (EPA). Evaluation of the potential nutritional efficacy of the oils was made using chondrocyte culture systems which can be used to mimic many of the degenerative and inflammatory pathways involved in arthritis. On stimulation of such cultures with interleukin-1alpha, they showed increased expression of cyclooxygenase-2, the inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-1beta and the proteinase ADAMTS-4. This increased expression was not affected by challenge of the cultures with palm olein alone but showed concentration-dependent reduction by the modified oil in a manner similar to EPA. These results show clearly that it is possible to modify palm oil conveniently to produce a nutraceutical with effective anti-inflammatory properties.

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect.

OBJECTIVE: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD), the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. METHODS: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA), soluble CD44 (sCD44), IL-1beta and TNF-alpha levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was determined by flow cytometry (FCM). CD44, hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. RESULTS: T-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1beta and TNF-alpha levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. CONCLUSION: T-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.

Biological basis for the benefit of nutraceutical supplementation in arthritis.

Arthritis is a common disease in which the end-point results in joint replacement surgery. This article reviews the use of nutraceuticals as alternative treatments for pathological manifestations of arthritic disease. The efficacy of fish oils (e.g. cod liver oil) in the diet has been demonstrated in several clinical trials, animal feeding experiments and in vitro models that mimic cartilage destruction in arthritic disease. In addition, there is some evidence for beneficial effects of other nutraceuticals, such as green tea, herbal extracts, chondroitin sulphate and glucosamine. However, in most cases, there is little scientific evidence at the cellular and molecular levels to explain their mechanisms of action.

Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing.

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.

Effects of n-3 fatty acids on cartilage metabolism.

Although the clinical benefits of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) has been recognised for a number of years, the molecular mechanisms by which particular PUFA affect metabolism of cells within the synovial joint tissues are not understood. This study set out to investigate how n-3 PUFA and other classes of fatty acids affect both degradative and inflammatory aspects of metabolism of articular cartilage chondrocytes using an in vitro model of cartilage degradation. Using well-established culture models, cartilage explants from normal bovine and human osteoarthritic cartilage were supplemented with either n-3 or n-6 PUFA, and cultures were subsequently treated with interleukin 1 to initiate catabolic processes that mimic cartilage degradation in arthritis. Results show that supplementation specifically with n-3 PUFA, but not n-6 PUFA, causes a decrease in both degradative and inflammatory aspects of chondrocyte metabolism, whilst having no effect on the normal tissue homeostasis. Collectively, our data provide evidence supporting dietary supplementation of n-3 PUFA, which in turn may have a beneficial effect of slowing and reducing inflammation in the pathogenesis of degenerative joint diseases in man.

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids.

OBJECTIVE: To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage. METHODS: The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). RESULTS: Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis. CONCLUSION: These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.