Poor cell adhesion to orthopaedic and dental implants may result in implant failure. Cellular adhesion to biomaterial surfaces primarily is mediated by integrins, which act as signal transduction and adhesion proteins. Because integrin function depends on divalent cations, we investigated the effect of magnesium ions modified bioceramic substrata (Al(2)O(3)-Mg(2+)) on human bone-derived cell (HBDC) adhesion, integrin expression, and activation of intracellular signalling molecules. Immunohistochemistry, flow cytometry, cell adhesion, cell adhesion blocking, and Western blotting assays were used. Our findings demonstrated that adhesion of HBDC to Al(2)O(3)-Mg(2+) was increased compared to on the Mg(2+)-free Al(2)O(3). Furthermore, HBDC adhesion decreased significantly when the fibronectin receptor alpha5beta1- and beta1-integrins were blocked by functional blocking antibodies. HBDC grown on the Mg(2+)-modified bioceramic expressed significantly enhanced levels of beta1-, alpha5beta1-, and alpha3beta1-integrins receptors compared to those grown on the native unmodified Al(2)O(3). Tyrosine phosphorylation of intracellular integrin-dependent signalling proteins as well as the expression of key signalling protein Shc isoforms (p46, p52, p66), focal adhesion kinase, and extracellular matrix protein collagen type I were significantly enhanced when HBDC were grown on Al(2)O(3)-Mg(2+) compared to the native Al(2)O(3). We conclude that cell adhesion to biomaterial surfaces is probably mediated by alpha5beta1- and beta1-integrin. Cation-promoted cell adhesion depends on 5beta1- and beta1-integrins associated signal transduction pathways involving the key signalling protein Shc and results also in enhanced gene expression of extracellular matrix proteins. Therefore, Mg(2+) supplementation of bioceramic substrata may be a promising way to improve integration of implants in orthopaedic and dental surgery.

Osmotically inactive skin Na+ storage is characterized by Na+ accumulation without water accumulation in the skin. Negatively charged glycosaminoglycans (GAGs) may be important in skin Na+ storage. We investigated changes in skin GAG content and key enzymes of GAG chain polymerization during osmotically inactive skin Na+ storage. Female Sprague-Dawley rats were fed a 0.1% or 8% NaCl diet for 8 wk. Skin GAG content was measured by Western blot analysis. mRNA content of key dermatan sulfate polymerization enzymes was measured by real-time PCR. The Na+ concentration in skin was determined by dry ashing. Skin Na+ concentration during osmotically inactive Na+ storage was 180–190 mmol/l. Increasing skin Na+ coincided with increasing GAG content in cartilage and skin. Dietary NaCl loading coincided with increased chondroitin synthase mRNA content in the skin, whereas xylosyl transferase, biglycan, and decorin content were unchanged. We conclude that osmotically inactive skin Na+ storage is an active process characterized by an increased GAG content in the reservoir tissue. Inhibition or disinhibition of GAG chain polymerization may regulate osmotically inactive Na+ storage.

IntroductionCurrently available treatments for osteoarthritis (OA) are restricted to nonsteroidal anti-inflammatory drugs, which exhibit numerous side effects and are only temporarily effective. Thus novel, safe and more efficacious anti-inflammatory agents are needed for OA. Naturally occurring polyphenolic compounds, such as curcumin and resveratrol, are potent agents for modulating inflammation. Both compounds mediate their effects by targeting the NF-κB signalling pathway.MethodsWe have recently demonstrated that in chondrocytes resveratrol modulates the NF-κB pathway by inhibiting the proteasome, while curcumin modulates the activation of NF-κB by inhibiting upstream kinases (Akt). However, the combinational effects of these compounds in chondrocytes has not been studied and/or compared with their individual effects. The aim of this study was to investigate the potential synergistic effects of curcumin and resveratrol on IL-1β-stimulated human chondrocytes in vitro using immunoblotting and electron microscopy.ResultsTreatment with curcumin and resveratrol suppressed NF-κB-regulated gene products involved in inflammation (cyclooxygenase-2, matrix metalloproteinase (MMP)-3, MMP-9, vascular endothelial growth factor), inhibited apoptosis (Bcl-2, Bcl-xL, and TNF-α receptor-associated factor 1) and prevented activation of caspase-3. IL-1β-induced NF-κB activation was suppressed directly by cocktails of curcumin and resveratrol through inhibition of Iκκ and proteasome activation, inhibition of IκBα phosphorylation and degradation, and inhibition of nuclear translocation of NF-κB. The modulatory effects of curcumin and resveratrol on IL-1β-induced expression of cartilage specific matrix and proinflammatory enzymes were mediated in part by the cartilage-specific transcription factor Sox-9.ConclusionsWe propose that combining these natural compounds may be a useful strategy in OA therapy as compared with separate treatment with each individual compound.

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This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML-1. Cultured on a three-dimensional clinostat, ML-1 cells formed three-dimensional MCTSs (MCTS diameter: 0.3 +/- 0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up-regulated the thyroid-stimulating hormone-receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML-1 cells grown on the clinostat showed elevated amounts of the apoptosis-associated Fas protein, of p53, and of bax but showed reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4', 6-diamidino-2-phenylindole staining, DNA laddering, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADP-ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

Resveratrol, a polyphenol derived from red grapes, berries, and peanuts, has been shown to mediate death of a wide variety of cells. The mechanisms by which resveratrol mediates cell death include necrosis, apoptosis, autophagy, and others. While most studies suggest that resveratrol kills tumor cells selectively, evidence is emerging that certain normal cells such as endothelial cells, lymphocytes, and chondrocytes are vulnerable to resveratrol. Cell killing by this stilbene may be mediated through any of numerous mechanisms that involve activation of mitochondria and of death caspases; upregulation of cyclin-dependent kinase inhibitors, tumor suppressor gene products, or death-inducing cytokines and cytokine receptors; or downregulation of cell survival proteins (survivin, cFLIP, cIAPs, X-linked inhibitor of apoptosis protein (XIAP), bcl-2, bcl-XL) or inhibition of cell survival kinases (e.g., mitogen-activiated protein kinases (MAPKs), AKT/phosphoinositide 3-kinase (PI3K), PKC, EGFR kinase) and survival transcription factors (nuclear factor-kappaB (NF-kappaB), activating protein 1 (AP-1), HIF-1alpha, signal transducer and activator of transcription (STAT3)). Induction of any of these pathways by resveratrol leads to cell death. While cell death is a hallmark of resveratrol, this polyphenol also has been linked with suppression of inflammation, arthritis, and cardiovascular diseases and delaying of aging. These attributes of resveratrol are discussed in detail in this review.

ObjectiveInterleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes.MethodsPrimary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy.ResultsPretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling.ConclusionThe results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects.

Perception of mechanical signals and the biological responses to such stimuli are fundamental properties of load bearing articular cartilage in diarthrodial joints. Chondrocytes utilize mechanical signals to synthesize an extracellular matrix capable of withstanding high loads and shear stresses. Recent studies have shown that chondrocytes undergo changes in shape and volume in a coordinated manner with load induced deformation of the matrix. These matrix changes, together with alterations in hydrostatic pressure, ionic and osmotic composition, interstitial fluid and streaming potentials are, in turn, perceived by chondrocytes. Chondrocyte responses to these stimuli are specific and well coordinated to bring about changes in gene expression, protein synthesis, matrix composition and ultimately biomechanical competence. In this hypothesis paper we propose a chondrocyte mechanoreceptor model incorporating key extracellular matrix macromolecules, integrins, mechanosensitive ion channels, the cytoskeleton and subcellular signal transduction pathways that maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression.