Background:The role of DNA methylation in musculoskeletal diseases is poorly understood. Results: Methylation of specific CpG sites in the MMP13 and IL1B promoters correlates strongly with gene activity in human cartilage. Conclusion: Specific CpG site methylation inhibits MMP13 transactivation by HIF-2␣. Significance: Our findings offer new insight on the impact of epigenetic control of a joint disease that can affect adults throughout life.

Objective Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1β, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1β and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma–Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 2 mM GlcN, (v) cultured with 1.0 μM BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 1.0 μM BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1β and MMP13 proximal promoter were quantified by pyrosequencing. Result IL1β expression was enhanced over 580-fold in articular chondrocytes treated with IL-1β and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1β expression by 3-fold. In the presence of BAY, IL-1β induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the −256 CpG site in the IL1β promoter was 65% in control cultures and decreased to 36% in the presence of IL-1β/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1β/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. Conclusion We demonstrate for the first ti...

Objective To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. Methods Expression of iNOS was quantified by quantitative reverse transcriptase–polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. Results The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at −289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at −5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. Conclusion These findings demonstrate the association between demethylation of specific NF-κB–responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.

ObjectiveTo investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter.MethodsExpression levels were determined using quantitative reverse transcription–polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation.ResultsAlthough COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter.ConclusionThis first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment.

Abstract. Laboratory hypervelocity impact experiments were conducted to verify the performance of aerogel dust collectors used for gathering meteorolds and space debris in the near-Earth environment and to derive the relationships of various parameters characterizing the projectile with morphology of tracks left by t•he penetrating projectile in the aerogel collector pad. Silica aerogel collectors of 0.03 g/cm -• density were impacted at velocities ranging from 1 to 14 km/s with projectiles of aluminum oxide, olivine, or sodalime glass, with diameters ranging from 10 to 400/xm. At impact velocities below 6 km/s the projectiles were captured without fragmentation by the aerogel collector and, in many instances, without complete ablation even at 12 km/s. The shapes and dimensions of the penetration tracks left in the aerogel collector were correlated with the impact parameters, and the results permitted derivation of a series of equations relating the track dimensions to incoming projectile size, impact energy, and other projectile parameters. A simplified model, similar to meteor-entry phenomena, was used to predict the trends in experimental penetration track lengths and the diameters of captured projectiles. IntroductionSolid particles present in the near-Earth environment derive from either natural or artificial origin and are termed "meteoroids" and "space debris", respectively. Meteoroids are considered to be supplied from comets, asteroids, and planets, and some known as interstellar dust particles or interstellar grains are from outside the solar system [Griin et al., 1993]. Since meteoroids are thought to be closely related with the evolution of the solar system, study of these materials provides us with crucial information on the source materials for the solar system. In situ sampling of meteoroids in space can avoid contamination by terrestrial sources, which is unavoidable in sampling on Earth.Space debris is the product of normal satellite operations, the deterioration of satellites, and the fragmentation or breakup of satellites [Johnson and McKnight, 1991]. It is important to investigate micrometer-to-millimeter-sized debris, the range within which the majority of debris particles lie, because this changes the characteristics of the materials of spacecraft or of the on-board parts after colliding with them. The distribution and composition of small-sized debris are not well known, as these particles are too small to be observed with ground-based telescopes or radars. In situ sampling of dust particles is useful for getting material information on the de- The authors developed a dust collector made from silica aerogel to take the Manipulator Flight Demonstration (MFD) flight opportunity aboard the Space Shuttle (STS-85),

Objectives Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling that function via the JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription). Eight SOCS proteins, SOCS1–SOCS7 and CIS-1 (cytokine-inducible SH2-domain, with similar structure to the other SOCS proteins) have been identified, of which SOCS1, 2, and 3 and CIS-1 are the best characterised. A characteristic feature of osteoarthritis (OA) is increased production by articular chondrocytes of proinflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα), which may be induced by mechanotransduction and contribute to cartilage destruction. In this study, we have compared the gene expression of SOCS1, 2, 3 and CIS-1 in healthy and OA human chondrocytes, and also analyzed the effects of IL-1β and TNFα on the levels of mRNA encoding these SOCS family members. In addition, SOCS2 protein production was assessed and the CpG methylation status of the SOCS2 promoter was analyzed to determine the role of epigenetics in its regulation. Methods Femoral heads were obtained after joint replacement surgery for late stage OA and hemiarthroplasty following a fracture of the neck of femur (#NOF). Chondrocytes from the superficial layer of OA cartilage and the deep zone of #NOF cartilage were isolated by sequential treatment with trypsin, hyaluronidase and collagenase B. Total DNA and RNA were extracted from the same chondrocytes, and the levels of SOCS1, 2, 3 and CIS-1 mRNA were determined by qRT-PCR. The percentage of methylation in the CpG sites of the SOCS2 proximal promoter was quantified by pyrosequencing. Alternatively, healthy chondrocytes were isolated from #NOF cartilage and cultured with and without a mixture of IL-1β and oncostatin M (OSM, both 2.5 ng/ml) or TNFα (10 ng/ml). The short-term cultures with single cytokine treatment were harvested 24 and 72 h after treatment, and the long-term cultures were maintained for 4–5 weeks until confluent with periodical cytokine stimulation. Total RNA was extracted and mRNA levels were determined by qRT-PCR. Results The SOCS2 and CIS-1 mRNA levels were reduced by approximately 10-fold in OA samples compared to control samples, while SOCS1 and SOCS3 showed similar expression patterns in OA and control chondrocytes. The SOCS2 and CIS-1 mRNA levels declined by 6-fold and 3-fold with long-term treatment with IL-1β and OSM in combination and TNFα, respectively. There was no significant difference in the CpG methylation status of the SOCS2 promoter between healthy and OA chondrocytes. Similarly, cytokine stimulation did not change the CpG methylation status of the SOCS2 promoter. Conclusions This study demonstrates the reduced expression of SOCS2 and CIS-1 in OA, while SOCS1 and SOCS3 were unaffected. The observation that long-term treatment with inflammatory cytokines attenuated the expression of SOCS2 and CIS-1 suggests a potential positive feedback mechanism, and a role of SOCS in the pathology of OA.

The photochemical reaction of poly(ether ether ketone) (PEEK) sheets under tensile loads has been investigated. Two types of UV irradiation tests were carried out in a vacuum environment: with and without a cooling apparatus. Chemical structures, thermal properties, and mechanical properties were measured to clarify photo deterioration. Chemical analysis based on Fourier Transform Infrared Spectrometer (FT-IR) and X-ray Photoelectron Spectroscopy (XPS) showed photochemical scission caused by UV exposure. Thermal properties, measured by Differential Scanning Calorimeter (DSC), indicated that a crosslinking reaction occurred during the radiation tests. Tensile properties of PEEK sheets after UV radiation clearly showed a tendency to embrittlement affected not only by crosslinking but also by the orientation of molecular chains resulting from the temperature rise of the specimens. Furthermore, applied tensile stress during exposure accelerated molecular scission and disturbed the crosslinking effects of the tensile properties.

In this paper, we proposed to utilize a reconstructed cardiac tissue as microactuator with easy assembly. In a glucose solution, cardiomyocytes can contract autonomously using only chemical energy. However, a single cardiomyocyte is not enough to actuate a microrobot or a mechanical system. Though the output power will increase by using multiple cardiomyocyte, it is difficult to assemble those cardiomyocyte to predefined positions one-by-one using a micromanipulator. Reconstructed cardiac tissue not only will enable researchers to assemble the cells easily and but also has a potential to improve the contractile ability. To realize a bio-actuator in this paper, we reconstructed a microcardiac tissue using an extracellular matrix, and their displacements, displacement frequency, contractile force, and lifetime of the reconstructed cardiac tissue were evaluated. Electrical and pharmacological responses of the reconstructed cardiac tissue were also evaluated. Finally, a bioactuator, a primitive micropillar actuator, was fabricated and applicability of the reconstructed cardiac tissue for bioactuators was evaluated.