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The Ras homolog A (RhoA) subfamily of Rho guanosine triphosphatases (GTPases) regulates actin-based cellular functions in bone such as differentiation, migration, and mechanotransduction. Polymorphisms or genetic ablation of RHOA and some of its regulatory guanine exchange factors (GEFs) have been linked to poor bone health in humans and mice, but the effects of RhoA-specific GTPase-activating proteins (GAPs) on bone quality have not yet been identified. Therefore, we examined the consequences of RhoGAP Myo9b gene knockout on bone growth, phenotype, and cellular activity. Male and female mice lacking both alleles demonstrated growth retardation and decreased bone formation rates during early puberty. These mice had smaller, weaker bones by 4 weeks of age, but only female KOs had altered cellular numbers, with fewer osteoblasts and more osteoclasts. By 12 weeks of age, bone quality in KOs worsened. In contrast, 4-week-old heterozygotes demonstrated bone defects that resolved by 12 weeks of age. Throughout, Myo9b ablation affected females more than males. Osteoclast activity appeared unaffected. In primary osteogenic cells, Myo9b was distributed in stress fibers and focal adhesions, and its absence resulted in poor spreading and eventual detachment from culture dishes. Similarly, MC3T3-E1 preosteoblasts with transiently suppressed Myo9b levels spread poorly and contained decreased numbers of focal adhesions. These cells also demonstrated reduced ability to undergo IGF-1-induced spreading or chemotaxis toward IGF-1, though responses to PDGF and BMP-2 were unaffected. IGF-1 receptor (IGF1R) activation was normal in cells with diminished Myo9b levels, but the activated receptor was redistributed from stress fibers and focal adhesions into nuclei, potentially affecting receptor accessibility and gene expression. These results demonstrate that Myo9b regulates a subset of RhoA-activated processes necessary for IGF-1 responsiveness in osteogenic cells, and is critical for normal bone formation in growing mice. © 2017 American Society for Bone and Mineral Research.

Summary Objective Exercise is vital for maintaining cartilage integrity in healthy joints. Here we examined the exercise-driven transcriptional regulation of genes in healthy rat articular cartilage to dissect the metabolic pathways responsible for the potential benefits of exercise. Methods Transcriptome-wide gene expression in the articular cartilage of healthy Sprague–Dawley female rats exercised daily (low intensity treadmill walking) for 2, 5, or 15 days was compared to that of non-exercised rats, using Affymetrix GeneChip arrays. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for Gene Ontology (GO)-term enrichment and Functional Annotation analysis of differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway mapper was used to identify the metabolic pathways regulated by exercise. Results Microarray analysis revealed that exercise-induced 644 DEGs in healthy articular cartilage. The DAVID bioinformatics tool demonstrated high prevalence of functional annotation clusters with greater enrichment scores and GO-terms associated with extracellular matrix (ECM) biosynthesis/remodeling and inflammation/immune response. The KEGG database revealed that exercise regulates 147 metabolic pathways representing molecular interaction networks for Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, and Diseases. These pathways collectively supported the complex regulation of the beneficial effects of exercise on the cartilage. Conclusions Overall, the findings highlight that exercise is a robust transcriptional regulator of a wide array of metabolic pathways in healthy cartilage. The major actions of exercise involve ECM biosynthesis/cartilage strengthening and attenuation of inflammatory pathways to provide prophylaxis against onset of arthritic diseases in healthy cartilage.

Background: Knee osteoarthritis is a progressive degenerative joint disease and remains a leading cause of pain, physical impairment and decline in health-related quality of life in adults. Despite its incidence being amongst the highest in chronic diseases, effective biomarkers are not available to assist in its management. The main goal of this study is to identify mediators that serve as biomarkers and investigate if the levels of these biomarkers will be correlated to the efficacy of a rehabilitation program.Methods: This is a prospective cohort study with 65 participants. Patients with mild-to-moderate symptomatic knee osteoarthritis will be recruited. The Rehabilitation Program will consist of three session/week during eight weeks. Assessment about functional evaluation will be performed before and after treatment, using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and EuroQOL-5D (Euro quality of life - five dimension) scales, Visual Analog Scale (VAS), and physical function tests (time up and go, isometric strength testing and kinematic gait analysis). Serum levels of classical pro-inflammatory cytokines, hyaluronan and high mobility group box 1 protein (HMGB-1) will be evaluated. The primary outcome is the change in WOMAC scale from baseline to end. Statistical analyses will be used to determine correlation of physical improvement and serum biomarkers. Adverse events will be monitored throughout the study.Conclusions: This trial expect to study the correlation between the anti-inflammatory effects of rehabilitation program derived factors that may be involved in suppressing cytokine induction via suppressing HMGB-1.Trial registration: Clinicaltrials.gov - NCT02964624.

Background High mobility group box one protein (HMGB1) is a chromatin associated protein, which in response to stress or injury translocates from the nucleus to the extracellular milieu to serve as an alarmin. HMGB1 has a remarkable ability to form complexes with proinflammatory molecules. This is underscored by the emerging role of HMGB1 in rheumatic diseases such as systemic lupus erythematosus (SLE), where the presence of HMGB1 in complex with endogenous nuclear components such as dsDNA or nucleosomes was shown to play an important role in breaking immune tolerance against nuclear antigens.1 Although the function of HMGB1 is in-part determined by the complexes it forms with other molecules, structural modifications in the HMGB1 polypeptide that may regulate complex formation have not been described. Objectives In this study we investigated the presence of HMGB1 in large protein complexes (HMGB1c) in human plasma. The objectives of this study were to isolate and characterise HMGB1c as well as to determine the mechanism of its formation. Methods We examined the presence of HMGB1c in plasma from SLE patients and healthy controls using semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) folowed by Western blot analysis. Immunoblotting, coimmunoprecipitation, confocal microscopy, and mass spectrometry were used to detect and characterise novel high molecular weight HMGB1 variants in vitro as well as in cell lines and primary cells. Mechanisms of HMGB1c formation were delineated via mass spectrometry, RNA interference, and in vitro enzyme reactions. Results In this study we note the presence of high molecular weight, denaturing resistant HMGB1 protein complexes (HMGB1c) that were present in the plasma of SLE patients and to a much lesser extent, healthy subjects. Similarly, HMGB1c were induced when cells were incubated with endotoxin or alum. Here we report that HMGB1c formation is catalysed by the calcium-activated protein crosslinking enzyme transglutaminase-2 (TG2). HMGB1-TG2 interaction was demonstrated via coimmunoprecipitation as well as by confocal microscopy after co-transfection of cells with plasmids encoding fluorescent-tagged HMGB1 and TG2 constructs. Moreover, HMGB1c formation was suppressed in cells by TG2 siRNA. Crosslink site mapping and analysis by mass spectrometry revealed that HMGB1 can be crosslinked to TG2 as well as a number of additional proteins, including human autoantigens. Conclusions TG2 catalyses the formation of high molecular weight HMGB1 protein complexes. Given the immunoadjuvant properties of HMGB1 and the implication of TG2-mediated protein complex formation as a possible mechanism by which immune tolerance can be broken to self-molecules,2 these findings have significant physiological implications for the role of HMGB1 in cellular stress responses and innate immunity in lupus. References [1] Urbonaviciute V, Furnrohr BG, Meister S, Munoz L, Heyder P, De MF, et al. Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE. J Exp Med2008;205:3007–3018. [2] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med1997;3:797–801. Disclosure of Interest None declared

Localized juvenile periodontitis (LJP) is an aggressive periodontal disease of familial nature. Neutrophils from a majority of patients with this disease exhibit decreased Chemotaxis with increased adherence, oxidative burst, and degranulation in response to opsonized bacteria. It is proposed that the biological basis for these altered neutrophil functions in LJP may be due either to intrinsic cell abnormalities or to the effect of factors present in the sera of LJP patients, which can modulate neutrophil functions. LJP neutrophils exhibit a lower number of receptors for chemoattractants and GP-110 molecules which are known to facilitate Chemotaxis. Furthermore, these cells exhibit lower signal transduction in response to a biological stimulus. These observations suggest that intrinsic cellular defects may be responsible for altered neutrophil functions in LJP. However, healthy neutrophils, when treated with very low concentrations of proinflammatory cytokines, also exhibit the characteristics of altered or "defective" LJP neutrophils. Additionally, healthy neutrophils, when treated with LJP serum, also exhibit many of the characteristics associated with LJP neutrophils. Attempts to identify these factors have shown that cytokines like TNF-α and/or IL1 β in LJP sera may be at least partially responsible for modulating neutrophil functions in LJP. These cytokines are primarily produced by activated macrophages, indicating a role for these cells in the etiology of LJP. The hyper-responsiveness of these cells to an immunologic challenge can result in local increases in cytokines leading to excessive bone loss and tissue damage at the site of infection, while systemic elevations in cytokines would lead to decreased neutrophil Chemotaxis, both of which are observed in LJP. Present evidence indicates that neutrophil functions are indeed altered in the majority of LJP patients. However, the biological basis for the alteration may not be due to the neutrophils themselves but, rather, a consequence of an inherent hyperactive immune response during the host-pathogen interaction. J Periodontol 1996;67:337-344.

To investigate how surgically created acute full-thickness cartilage defects of similar size and location created on the medial versus lateral femoral condyle influence progression of spontaneous cartilage lesions in a rat model. Full-thickness cartilage defects of 1 mm were surgically created on the medial or lateral femoral condyles on the right leg of 20 rats (n = 10/group). Ten rats served as controls. Spontaneous lesion progression on the ipsilateral and contralateral surfaces was examined using a high-resolution digital camera along with H&E and Safranin-O staining. Chondral defects were scored grossly and histologically. Control femur displayed no cartilage disruption. Surgically treated knees exhibited created and spontaneous cartilage defects with no evidence of healing unless subchondral bone was penetrated. Ipsilateral spontaneous lesions on the lateral condyle were significantly more severe on average (p = 0.009) compared to medial lesions on gross examination. Histological examination found contralateral lesions on the lateral surface following surgically created medial lesions to be more severe (p = 0.057) compared to contralateral lesions. A trend toward more susceptible chondral damage to the lateral condyle was observed following acute lesion creation on either medial or lateral condyles. Mechanisms behind this pattern of spontaneous lesion development are unclear, requring further investigation.

Significance: Mechanosignaling is vital for maintaining the structural integrity of bone under physiologic conditions. These signals activate and suppress multiple signaling cascades regulating bone formation and resorption. Understanding these pathways is of prime importance to exploit their therapeutic potential in disorders associated with bone loss due to disuse, trauma, or disruption of homeostatic mechanisms. Recent Advances: In the case of cells of the bone, an impressive amount of data has been generated that provides evidence of a complex mechanism by which mechanical signals can maintain or disrupt cellular homeostasis by driving transcriptional regulation of growth factors, matrix proteins and inflammatory mediators in health and inflammation. Mechanical signals act on cells in a magnitude dependent manner to induce bone deposition or resorption. During health, physiological levels of these signals are essential for maintaining bone strength and architecture, whereas during inflammation, similar signals can curb inflammation by suppressing the nuclear factor kappa B (NF-κB) signaling cascade, while upregulating matrix synthesis via mothers against decapentaplegic homolog and/or Wnt signaling cascades. Contrarily, excessive mechanical forces can induce inflammation via activation of the NF-κB signaling cascade. Critical Issues: Given the osteogenic potential of mechanical signals, it is imperative to exploit their therapeutic efficacy for the treatment of bone disorders. Here we review select signaling pathways and mediators stimulated by mechanical signals to modulate the strength and integrity of the bone. Future Directions: Understanding the mechanisms of mechanotransduction and its effects on bone lay the groundwork for development of nonpharmacologic mechanostimulatory approaches for osteodegenerative diseases and optimal bone health. Antioxid. Redox Signal. 20, 970–985.

Background: UP is one of the biggest cultivators of tobacco. Rules of tobacco consumption are almost absent in the state. The state has almost 34% of young male smokers. Aims and Objectives: The idea of creating awareness among cigarette smokers and promote deaddiction among youth the present study was conducted in Muzaffarnagar Medical College to assess the cardiovascular risks according to the degree of exposure, i.e., pack-years of smoking. Materials and Methods: A total of 150 male subjects were taken for the study after proper consent. Five groups were formed - controls (no smoking), Group 1; 0–10 pack-years of smoking, Group 2; 11–20 pack-years of smoking, Group 3; 21–30 pack-years of smoking, and Group 4; 31–40 pack-years of smoking. Pack-years formula = (number of cigarettes smoked per day/20) × number of years smoked was used. Only cigarette smokers were used for the study after using exclusion criteria to rule out any confounding factor. Serum lipid profile and high-sensitivity C-reactive protein (hsCRP) were estimated. Results: As the degree of exposure is increasing, i.e. pack-years of smoking increases, the level of dyslipidemias is also becoming severe being highly significant P < 0.01** in 31–40 pack-years of smoking. hsCRP is significantly increasing P < 0.05* in Group 2 and showing highly significant P < 0.01** increase in Groups 3 and 4. Conclusion: Cardiovascular risks are increasing significantly as pack-years of smoking is increasing as indicated by dyslipidemias and hsCRP increase, means 21–30 and 31–40 pack-years are at higher risk as compared to 0–10 and 11–20 pack-years.

Massage therapy has a long history and has been widely believed effective in restoring tissue function, relieving pain and stress, and promoting overall well-being. However, the application of massage-like actions and the efficacy of massage are largely based on anecdotal experiences that are difficult to define and measure. This leads to a somewhat limited evidence-based interface of massage therapy with modern medicine. In this study, we introduce a mechatronic device that delivers highly reproducible massage-like mechanical loads to the hind limbs of small animals (rats and rabbits), where various massage-like actions are quantified by the loading parameters (magnitude, frequency and duration) of the compressive and transverse forces on the subject tissues. The effect of massage is measured by the difference in passive viscoelastic properties of the subject tissues before and after mechanical loading, both obtained by the same device. Results show that this device is useful in identifying the loading parameters that are most conducive to a change in tissue mechanical properties, and can determine the range of loading parameters that result in sustained changes in tissue mechanical properties and function. This device presents the first step in our effort for quantifying the application of massage-like actions used clinically and measurement of their efficacy that can readily be combined with various quantitative measures (e.g., active mechanical properties and physiological assays) for determining the therapeutic and mechanistic effects of massage therapies.

Osteochondral tissue-engineered grafts are proposed to hold greater potential to repair/regenerate damaged cartilage through enhanced biochemical and mechanical interactions with underlying subchondral bone as compared to simple engineered cartilage. Additionally, biomechanical stimulation of articular chondrocytes (ACs) or osteoblasts (OBs) was shown to induce greater morphogenesis of the engineered tissues composed of these cells. In this report, to define the advantages of biomechanical stimulation to osteochondral grafts for tissue engineering, we examined whether (1) ACs and OBs in three-dimensional (3D) osteochondral constructs support functional development of each other at the molecular level, and (2) biomechanical stimulation of osteochondral constructs further promotes the regenerative potential of such grafts. Various configurations of cell/scaffold assemblies, including chondral, osseous, and osteochondral constructs, were engineered with mechano-responsive electrospun poly(ɛ-caprolactone) scaffolds. These constructs were subjected to either static or dynamic (10% cyclic compressive strain at 1 Hz for 3 h/day) culture conditions for 2 weeks. The expression of bone morphogenetic proteins (BMPs) was examined to assess the regenerative potential of each treatment on the cells. Biomechanical stimulation augmented a marked upregulation of Bmp2, Bmp6, and Bmp7 as well as downregulation of BMP antagonist, Bmp3, in a time-specific manner in the ACs and OBs of 3D osteochondral constructs. More importantly, the presence of biomechanically stimulated OBs was especially crucial for the induction of Bmp6 in ACs, a BMP required for chondrocytic growth and differentiation. Biomechanical stimulation led to enhanced tissue morphogenesis possibly through this BMP regulation, evident by the improved effective compressive modulus of the osteochondral constructs (710 kPa of dynamic culture vs. 280 kPa of static culture). Similar BMP regulation was observed in the femoral cartilages of the rats subjected to gentle exercise, demonstrating the physiological relevance of in vitro biomechanical stimulation of osteochondral constructs. Overall, our findings show that biomechanical stimulation may be critical for cross signaling between ACs and OBs to support chondrocytic growth in 3D osteochondral tissues.

The Ras homolog A (RhoA) subfamily of Rho guanosine triphosphatases (GTPases) regulates actin-based cellular functions in bone such as differentiation, migration, and mechanotransduction. Polymorphisms or genetic ablation of RHOA and some of its regulatory guanine exchange factors (GEFs) have been linked to poor bone health in humans and mice, but the effects of RhoA-specific GTPase-activating proteins (GAPs) on bone quality have not yet been identified. Therefore, we examined the consequences of RhoGAP Myo9b gene knockout on bone growth, phenotype, and cellular activity. Male and female mice lacking both alleles demonstrated growth retardation and decreased bone formation rates during early puberty. These mice had smaller, weaker bones by 4 weeks of age, but only female KOs had altered cellular numbers, with fewer osteoblasts and more osteoclasts. By 12 weeks of age, bone quality in KOs worsened. In contrast, 4-week-old heterozygotes demonstrated bone defects that resolved by 12 weeks of age. Throughout, Myo9b ablation affected females more than males. Osteoclast activity appeared unaffected. In primary osteogenic cells, Myo9b was distributed in stress fibers and focal adhesions, and its absence resulted in poor spreading and eventual detachment from culture dishes. Similarly, MC3T3-E1 preosteoblasts with transiently suppressed Myo9b levels spread poorly and contained decreased numbers of focal adhesions. These cells also demonstrated reduced ability to undergo IGF-1-induced spreading or chemotaxis toward IGF-1, though responses to PDGF and BMP-2 were unaffected. IGF-1 receptor (IGF1R) activation was normal in cells with diminished Myo9b levels, but the activated receptor was redistributed from stress fibers and focal adhesions into nuclei, potentially affecting receptor accessibility and gene expression. These results demonstrate that Myo9b regulates a subset of RhoA-activated processes necessary for IGF-1 responsiveness in osteogenic cells, and is critical for normal bone formation in growing mice. © 2017 American Society for Bone and Mineral Research.

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b+F4/80+Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Exercise is the most widely recommended and used conservative therapeutic approach to improve joint function in arthritis (1–3). Recent guidelines published by Osteoarthritis Research Society International suggest that exercise is in general beneficial for patients with osteoarthritis (OA) (3). Such recommendations are supported by large cohort studies demonstrating that adults engaging in minimal to no physical activity show higher incidence of radiographically diagnosed osteoarthritis (4). Additionally, older adults engaged in moderate physical activity show reduced risk of arthritis-related disability (5). However, in some patients physical activity is either associated with a greater risk or has no effect on knee joints, making it difficult to discern which patients will or will not benefit from physical therapies (6–9). Chondrocytes, mechanoresponsive cells within the cartilage, perceive and respond to mechanical stimuli by altering their biosynthetic ability, morphology and cartilage extracellular matrix (10, 11). These cells interpret mechanical signals in a magnitude dependent manner (12, 13). Excessive loading of joints is injurious and activates proinflammatory signaling cascades, similar to those implicated in the etiology of OA (14–17). Contrarily, physiological loading is shown to be antiinflammatory and induce IL-10 production in the synovium, and upregulate synthesis of glycosaminoglycans in the cartilage of patients at increased risk of OA (18–21). In fact, exercise is vital for cartilage homeostasis and its lack atrophies cartilage (22), implicating distinct roles of physiotherapies in both preventing damage and improving joint function. The aim of this study was to determine the molecular mechanisms underlying the effectiveness of exercise in the form of gentle treadmill walking (GTW) on well-defined stages of cartilage damage, using a rat knee model of MIA (23). Others and we have shown earlier that low or physiological levels of compressive/tensile forces are antiinflammatory on chondrocytes in vitro. These forces suppress several biomarkers of inflammation, such as IL-1β, TNF-α, matrix metalloproteinases (MMPs) and aggrecanases (12, 13, 24, 25). Here, we systematically examined the efficacy of moderate exercise on the progression of MIA macroscopically, microscopically, and by microtomographic (μCT) imaging. A transcriptome-wide microarray analysis was also conducted to track the changes in gene expression subsequent to GTW therapy to gain key insights into the basis of success or failure of such therapies (1, 3, 4, 26).

As the potential range of stem cell applications in tissue engineering continues to grow, the appropriate scaffolding choice is necessary to create tightly defined artificial microenvironments for each target organ. These microenvironments determine stem cell fate via control over differentiation. In this study we examined the specific effects of scaffold stiffness on embryonic mesenchymal progenitor cell behavior. Mechanically distinct scaffolds having identical microstructures and surface chemistries were produced utilizing core-shell electrospinning. The modulus of core-shell poly(ether sulfone)-poly(ε-caprolactone) (PES-PCL) fibers (30.6 MPa) was more than four times that of pure PCL (7.1 MPa). The results for chondrogenic and osteogenic differentiation of progenitor cells on each scaffold indicate that the lower modulus PCL fibers provided more appropriate microenvironments for chondrogenesis, evident by a marked up-regulation of chondrocytic Sox9, collagen type 2, and aggrecan gene expression and chondrocyte-specific extracellular matrix glycosaminoglycan production. In contrast, the stiffer core-shell PES-PCL fibers supported enhanced osteogenesis by promoting osteogenic Runx2, alkaline phosphatase, and osteocalcin gene expression, as well as alkaline phosphatase activity. The findings demonstrate that the microstructural stiffness/modules of a scaffold and the pliability of individual fibers may play a critical role in controlling stem cell differentiation. Regulation of cytoskeletal organization may occur via a "dynamic scaffold" leading to the subsequent intracellular signaling events that control differentiation-specific gene expression.

Osteoblasts are mechanosensitive cells, which respond to biomechanical stimuli to regulate the bone structure through anabolic and catabolic gene regulation. To examine the effects of mechanical forces on the osteogenic responses through the SMAD signaling in osteoblasts, the cells were cultured in well-characterized mechanoresponsive 3-D scaffolds and exposed to 10% dynamic compressive strain (Cmp) at 1 Hz. Subsequently, SMAD phosphorylation and osteogenic gene induction was examined. Osteoblasts cultured in 3-D scaffolds exhibited increased constitutive SMAD 1/5/8 phosphorylation, as compared to monolayers cultures. This SMAD 1/5/8 phosphorylation was further upregulated after 10, 30 and 60 min in response to Cmp, exhibiting a peak activation at 30 min. No significant changes in SMAD2 phosphorylation were observed, suggesting signals generated by Cmp may not activate the Transforming Growth Factor-β signaling cascade. Subsequently, biomechanical stimulation-induced SMAD 1/5/8 phosphorylation upregulated the expression of osteogenic genes such as Osteoprotegrin, Msx2 and Runx2. Dorsomorphin, a selective inhibitor of the bone morphogenetic protein (BMP) receptor type 1 (BMPR1), blocked Cmp-induced SMAD 1/5/8 phosphorylation, as well as Osteoprotegrin, Msx2 and Runx2 gene expression. Collectively, the present findings demonstrate that biomechanical stimulation of osteoblasts activates SMAD 1/5/8 in the BMP signaling pathway through BMPR1 and may enhance osteogenesis by upregulating SMAD-dependent osteogenic genes.

Signals generated by the dynamic mechanical strain critically regulate endothelial cell proliferation and angiogenesis; however, the molecular basis remains unclear. We investigated the mechanisms by which human dermal microvascular endothelial cells (HDMECs) perceive mechanical signals and relay them intracellularly to regulate gene expression and endothelial cell proliferation. HDMECs were exposed to low/physiologic levels of dynamic strain and probed for the differential activation/inhibition of kinases in the mechanosignaling cascade associated with endothelial cell gene activation. Because angiogenesis is important at inflammatory sites, we also assessed the mechanisms of mechanosignaling in the presence of an proinflammatory cytokine IL-1β. In this article, we demonstrate that the mechanosignaling cascade is initiated by vascular endothelial growth receptor-2 (VEGFR2) activation. Mechanoactivation of VEGFR2 results in its nuclear translocation and elevation of PI3K-dependent Ser473-Akt phosphorylation. Subsequently, activated Akt inactivates the kinase activity of the serine/threonine kinase, glycogen synthase kinase-3β (GSK3β), via its Ser9 phosphorylation. Thus, inactive GSK3β fails to phosphorylate cyclin D1 and prevents its proteosomal degradation and, consequently, promotes endothelial cell survival and proliferation. In the presence of IL-1β, cyclin D1 is phosphorylated and degraded, leading to inhibition of cell proliferation. However, mechanical signals repress cyclin D1 phosphorylation and upregulate cell proliferation, despite the presence of IL-1β. The data indicate that the VEGFR2/Akt/GSK3β signaling cascade plays a critical role in sensing and phospho-relaying mechanical stimuli in endothelial cells. Furthermore, mechanical forces control highly interconnected networks of proinflammatory and Akt signaling cascades to upregulate endothelial cell proliferation.

Cartilage is a mechanosensitive tissue, which means that it can perceive and respond to biomechanical signals. Despite the known importance of biomechanical signals in the etiopathogenesis of arthritic diseases and their effectiveness in joint restoration, little is understood about their actions at the cellular level. Recent molecular approaches have revealed that specific biomechanical stimuli and cell interactions generate intracellular signals that are powerful inducers or suppressors of proinflammatory and reparative genes in chondrocytes. Biomechanical signals are perceived by cartilage in magnitude-, frequency-, and time-dependent manners. Static and dynamic biomechanical forces of high magnitudes induce proinflammatory genes and inhibit matrix synthesis. Contrarily, dynamic biomechanical signals of low/physiologic magnitudes are potent antiinflammatory signals that inhibit interleukin-1beta (IL-1beta)-induced proinflammatory gene transcription and abrogate IL-1beta/tumor necrosis factor-alpha-induced inhibition of matrix synthesis. Recent studies have identified nuclear factor-kB (NF-kB) transcription factors as key regulators of biomechanical signal-mediated proinflammatory and antiinflammatory actions. These signals intercept multiple steps in the NF-kappaB signaling cascade to regulate cytokine gene expression. Taken together, these findings provide insight into how biomechanical signals regulate inflammatory and reparative gene transcription, underscoring their potential in enhancing the ability of chondrocytes to curb inflammation in diseased joints.

Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or injured joints, but also for promoting a homeostatic equilibrium in healthy joints. Human joints provide the pivot points and physiological hinges essential for ambulation and movement to the body, and it is this mobility that in return promotes the health of the joints. But how mobilization regulates the joint microenvironment at the molecular level has remained enigmatic for many years. Recent advances in joint biomechanics and molecular approaches have facilitated an enriched understanding of how joints operate. Consequently, the mechanisms active during joint inflammation that lead to arthritic conditions, both in vivo in animal models, and in vitro at cell and tissue levels, have become increasingly detailed and defined. These efforts have produced mounting evidences supporting the premise that biomechanical signals play a fundamental role in both the etiopathogenesis of arthritic diseases and in the physiological restoration of joints. This report aims to summarize current peer-reviewed literature and available experimental data to explain how the signals generated by mechanical forces/joint mobilization generate beneficial effects on inflamed articular cartilage, and to propose the basis for using appropriate physical therapies for the optimal benefit to the patient suffering from joint associated injuries.

Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.

Despite the fact that vocal folds are subjected to extensive mechanical forces, the role of mechanical strain in vocal fold wound healing has been overlooked. Recent studies on other tissues have demonstrated that low physiological levels of mechanical forces are beneficial to injured tissues, reduce inflammation, and induce synthesis of matrix-associated proteins essential for enhanced wound healing. In this study, we speculated that mechanical strain of low magnitudes also attenuates the production of inflammatory mediators and alters the extracellular matrix synthesis to augment wound healing in cultured vocal fold fibroblasts. To test this hypothesis, fibroblasts from rabbit vocal folds were isolated and exposed to various magnitudes of cyclic tensile strain (CTS) in the presence or absence of interleukin-1beta (IL-1beta). Results suggest that IL-1beta activates proinflammatory gene transcription in vocal fold fibroblasts. Furthermore, CTS abrogates the IL-1beta-induced proinflammatory gene induction in a magnitude-dependent manner. In addition, CTS blocks IL-1beta-mediated inhibition of collagen type I synthesis, and thereby upregulates collagen synthesis in the presence of IL-1beta. These findings are the first to reveal the potential utility of low levels of mechanical signals in vocal fold wound healing, and support the emerging on vivo data suggesting beneficial effects of vocal exercise on acute phonotrauma.

Exercise/joint mobilization is therapeutic for inflammatory joint diseases like rheumatoid and osteoarthritis, but the mechanisms underlying its actions remain poorly understood. We report that biomechanical signals at low/physiological magnitudes are potent inhibitors of inflammation induced by diverse proinflammatory activators like IL-1β, TNF-α, and lipopolysaccharides, in fibrochondrocytes. These signals exert their anti-inflammatory effects by inhibiting phosphorylation of TAK1, a critical point where signals generated by IL-1β, TNF-α, and LPS converge to initiate NF-κB signaling cascade and proinflammatory gene induction. Additionally, biomechanical signals inhibit multiple steps in the IL-1β-induced proinflammatory cascade downstream of IκB kinase activation to regulate IκBα and IκBβ degradation and synthesis, and promote IκBα shuttling to export nuclear NF-κB and terminate its transcriptional activity. The findings demonstrate that biomechanical forces are but another important signal that uses NF-κB pathway to regulate inflammation by switching the molecular activation of discrete molecules involved in proinflammatory gene transcription.

Electrospun tissue engineering scaffolds pro- vide mechanical support to seeded cells that populate the structure while depositing specific extracellular matrix com- ponents. The potent sterilizing agent 1,1,1,3,3,3-hexafluoro- 2-propanol (HFIP) is often used in electrospinning investi- gations involving biologically-derived polymers. Surpris- ingly, there has been no study of solvent retention versus composition even though materials selection should influ- ence organic solvent content. We developed a method quantifying HFIP retention following electrospinning of gel- atin, polycaprolactone (PCL), and PCL-gelatin blends using electro-spray mass spectroscopy. The acetone content of ace- tone-spun PCL was also established. Pure gelatin fiber con- tained as much as 1600 ppm of HFIP. In contrast, little ace- tone or HFIP was detected in 100% PCL. Gelatin clearly has a greater affinity for HFIP than PCL and materials selection has a strong influence on the amount of retained solvent. Vacuum 1 heat treatment at 37 and 458C reduced (HFIP) to 10 and 5.6 ppm, respectively, levels having no demon- strated effects on mammalian cell viability. 2007 Wiley Periodicals, Inc. J Appl Polym Sci 107: 1547-1554, 2008

We sought to examine whether cyclic tensile strain (CTS) regulates the gene expression of tumor necrosis factor (TNF)-alpha, its receptors TNFR1 and TNFR2, and inducible nitric oxide synthase (iNOS) under inflammatory conditions, and whether these effects of CTS are sustained. Rat temporomandibular joint disc cells (TDC) were exposed to CTS in the presence or absence of interleukin (IL)-1beta for 4 and 24 h. Cells were also stimulated with IL-1beta for 24 h while being subjected to CTS only for the initial 1, 2, 4, 8, and 12 h or the entire 24 h incubation time. Furthermore, cells were incubated with IL-1beta for 24, 36, or 48 h while being exposed to CTS only for the initial 8 h. Gene expression of TNF-alpha, its receptors, and iNOS was analyzed by RT–PCR, whereas protein synthesis was determined by ELISA for TNF-alpha, immunofluorescence for TNFRs, and Griess reaction for nitric oxide. CTS inhibited the IL-1beta-stimulated synthesis of TNF-alpha, TNFR2, and iNOS. TNFR1 was constitutively expressed but not regulated by IL-1beta or CTS. Application of CTS for only 1 or 2 h during a 24 h incubation with IL-1beta was sufficient to inhibit IL-1beta-induced upregulation of TNF-alpha, TNFR2, and iNOS. However, for maximal inhibition of these genes a longer exposure of CTS was required. These findings are the first to show that biomechanical signals regulate the expression of TNFR2 but not TNFR1 under inflammatory conditions. Furthermore, the antiinflammatory effects of biomechanical signals on TDC are maintained for prolonged periods of time but are transient.

Although biomechanical signals generated during joint mobilization are vital in maintaining integrity of inflamed cartilage, the molecular mechanisms of their actions are little understood. In an experimental model of arthritis, we demonstrate that biomechanical signals are potent anti-inflammatory signals that repress transcriptional activation of proinflammatory genes and augment expression of anti-inflammatory cytokine IL-10 to profoundly attenuate localized joint inflammation.

SummaryObjectiveWe sought to determine the molecular basis for the anticatabolic effects of mechanical signals on fibrocartilage cells by studying the expression of a variety of matrix metalloproteinases (MMPs). Furthermore, we examined whether the effects of biomechanical strain on MMP gene expression are sustained.MethodsFibrochondrocytes from temporomandibular joint (TMJ) discs were exposed to dynamic tensile strain for various time intervals in the presence of interleukin (IL)-1β. The regulation of the messenger RNA (mRNA) expression and synthesis of MMPs and tissue inhibitors of MMPs (TIMPs) were examined by end-point and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Western blot analysis.ResultsFibrochondrocytes expressed mRNA for MMP-2, -3, -7, -8, -9, -11, -13, -14, -16, -17, and -19 as well as TIMP-1, -2, and -3, IL-1β induced a significant (P

The synthesis of vinyl ester resins V1, V2, and V3 was carried out using bisphenol-A based epoxy resin and methacrylic acid in the presence of triethyl-, tripropyl-, and tributyl-amines, respectively. The reaction follows first-order kinetics. The interaction between acid and amine was investigated by IR spectroscopy which shows absorptions corresponding to the formation of activated acid–catalyst complex. The specific rate constants, calculated by regression analysis, were found to obey an Arrhenius expression. The kinetic and thermodynamic parameters: activation energy, frequency factor, entropy, enthalpy, and free energy revealed that the reaction was spontaneous and irreversible with a highly ordered activated complex. The activation energy of the esterification of epoxy resin in the presence of tertiary amines increases in order V1 < V2 < V3. The experimental results were explained by proposing a reaction mechanism and deriving the rate equation. © 2005 Wiley Periodicals, Inc. Adv Polym Techn 24:1–13, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/adv.20026

Objective The mechanisms by which chondrocytes convert biomechanical signals into intracellular biochemical events are not well understood. In this study, we sought to determine the intracellular mechanisms of the magnitude-dependent actions of mechanical signals. Methods Chondrocytes isolated from rabbit articular cartilage were grown on flexible membranes. Cells were subjected to cyclic tensile strain (CTS) of various magnitudes in the presence or absence of interleukin-1β (IL-1β), which was used as a proinflammatory signal for designated time intervals. The regulation of NF-κB was measured by reverse transcriptase–polymerase chain reaction, electrophoretic mobility shift assay, and immunofluorescence. Results CTS of low magnitudes (4–8% equibiaxial strain) was a potent inhibitor of IL-1β–dependent NF-κB nuclear translocation. Cytoplasmic retention of NF-κB and reduction of its synthesis led to sustained suppression of proinflammatory gene induction. In contrast, proinflammatory signals generated by CTS of high magnitudes (15–18% equibiaxial strain) mimicked the actions of IL-1β and induced rapid nuclear translocation of NF-κB subunits p65 and p50. Conclusion Magnitude-dependent signals of mechanical strain utilize the NF-κB transcription factors as common elements to abrogate or aggravate proinflammatory responses. Furthermore, the intracellular events induced by mechanical overload are similar to those that are initiated by proinflammatory cytokines in arthritis.

Intracellular signals generated by mechanical strain profoundly affect the metabolic function of osteoblast-like periodontal ligament (PDL) cells, which reside between the tooth and alveolar bone. In response to applied mechanical forces, PDL cells synthesize bone-resorptive cytokines to induce bone resorption at sites exposed to compressive forces and deposit bone at sites exposed to tensile forces in an environment primed for catabolic processes. The intracellular mechanisms that regulate this bone remodeling remain unclear. Here, in an in vitro model system, we show that tensile strain is a critical determinant of PDL-cell metabolic functions. Equibiaxial tensile strain (TENS), when applied at low magnitudes, acts as a potent antagonist of interleukin (IL)-1beta actions and suppresses transcriptional regulation of multiple proinflammatory genes. This is evidenced by the fact that TENS at low magnitude: (i) inhibits recombinant human (rh)IL-1beta-dependent induction of cyclooxygenase-2 (COX-2) mRNA expression and production of prostaglandin estradiol (PGE2); (ii) inhibits rhIL-1beta-dependent induction matrix metalloproteinase-1 (MMP-1) and MMP-3 synthesis by suppressing their mRNA expression; (iii) abrogates rhIL-1beta-induced suppression of tissue inhibitor of metalloprotease-II (TIMP-II) expression; and (iv) reverses IL-1beta-dependent suppression of osteocalcin and alkaline phosphatase synthesis. Nevertheless, these actions of TENS were observed only in the presence of IL-1beta, as TENS alone failed to affect any of the aforementioned responses. The present findings are the first to show that intracellular signals generated by low-magnitude mechanical strain interfere with one or more critical step(s) in the signal transduction cascade of rhIL-1beta upstream of mRNA expression, while concurrently promoting the expression of osteogenic proteins in PDL cells.

Background: Obesity is a newer chronic non-communicable disease, one of the today’s most neglected public health problems according to WHO. While the complication of obesity such as diabetes, cardiovascular disease, and osteoarthritis are well established, but less emphasis is traditionally placed on the effects of obesity on the respiratory system. Aims and Objective: To study the effect of gender and correlation of body mass index (BMI) with pulmonary function tests (PFTs). Materials and Methods: This study was done on 115 first year Medical and Paramedical Students of Muzaffarnagar Medical College, Muzaffarnagar. Following the selection, the participants were divided into two groups based on BMI 25. Anthropomeric parameters and spirometeric parameters were measured using a computerized spirometer. Statistical analysis was done by applying Student’s t-test. Linear association was established using Pearson’s correlation. Results: On comparing the female participants with male participants, PFTs (forced vital capacity [FVC], forced expired volume in 1 second [FEV1], FEV1/FVC, peak expiratory flow rate) were found to be significantly lower in female than male. On observing, the correlation of pulmonary function parameter with BMI correlated positively with BMI < 25. PFTs were negatively correlated with increasing BMI in both male and female participants but the correlation is not statistically significant. Conclusion: Our finding suggests that there is a significant impairment of lung functions in the overweight individuals.

To investigate how surgically created acute full-thickness cartilage defects of similar size and location created on the medial versus lateral femoral condyle influence progression of spontaneous cartilage lesions in a rat model. Full-thickness cartilage defects of 1 mm were surgically created on the medial or lateral femoral condyles on the right leg of 20 rats (n = 10/group). Ten rats served as controls. Spontaneous lesion progression on the ipsilateral and contralateral surfaces was examined using a high-resolution digital camera along with HE and Safranin-O staining. Chondral defects were scored grossly and histologically. Control femur displayed no cartilage disruption. Surgically treated knees exhibited created and spontaneous cartilage defects with no evidence of healing unless subchondral bone was penetrated. Ipsilateral spontaneous lesions on the lateral condyle were significantly more severe on average (p = 0.009) compared to medial lesions on gross examination. Histological examination found contralateral lesions on the lateral surface following surgically created medial lesions to be more severe (p = 0.057) compared to contralateral lesions. A trend toward more susceptible chondral damage to the lateral condyle was observed following acute lesion creation on either medial or lateral condyles. Mechanisms behind this pattern of spontaneous lesion development are unclear, requring further investigation.

Background Detection of Mycobacterium leprae in slit skin smear (SSS) is a gold standard technique for the leprosy diagnosis. Over recent years, molecular diagnosis by using PCR has been increasingly used as an alternative for its diagnosis due to its higher sensitivity. This study was carried out for comparative evaluation of PCR and SSS microscopy in a cohort of new leprosy cases diagnosed in B. P. Koirala Institute of health Sciences, Dharan, Nepal. Methodology/Principal Findings In this prospective crossectional study, 50 new clinically diagnosed cases of leprosy were included. DNA was extracted from SSS and PCR was carried out to amplify 129 bp sequence of M. leprae repetitive element. Sensitivity of SSS and PCR was 18% and 72% respectively. Improvement of 54% case detection by PCR clearly showed its advantage over SSS. Furthermore, PCR could confirm the leprosy diagnosis in 66% of AFB negative cases indicating its superiority over SSS. In the paucibacillary (PB) patients, whose BI was zero; sensitivity of PCR was 44%, whereas it was 78% in the multibacillary patients. Conclusions/Significance Our study showed PCR to be more sensitive than SSS microscopy in diagnosing leprosy. Moreover, it explored the characteristic feature of PCR which detected higher level of early stage(PB) cases tested negative by SSS. Being an expensive technique, PCR may not be feasible in all the cases, however, it would be useful in diagnosis of early cases of leprosy as opposed to SSS., Author Summary Although leprosy has been eliminated at the national level, but region wise eastern Nepal has still the higher number of leprosy cases. Early diagnosis and then timely treatment is very crucial to prevent disabilities due to leprosy. Slit-skin smear (SSS), the routine diagnostic method was compared with the molecular method polymerase chain reaction (PCR) for the detection of new clinically diagnosed cases at BPKIHS. PCR was found to be much more superior than SSS in its diagnostic efficacy detecting more than half number of cases missed by SSS. Moreover our finding of PCR in detection of early stages was nearly 50% where SSS are mostly negative on such spectrum. Therefore, this costly but sensitive diagnostic tool PCR was found to be a better option in detection of early cases of leprosy in comparison to SSS.

Objective To discern the effects of continuous passive motion on inflamed temporomandibular joints (TMJ). Methods The effects of continuous passive motion on TMJ were simulated by exposing primary cultures of rabbit TMJ fibrochondrocyte monolayers to cyclic tensile strain (CTS) in the presence of recombinant human interleukin-1β (rHuIL-1β) in vitro. The messenger RNA (mRNA) induction of rHuIL-1β response elements was examined by semiquantitative reverse transcriptase–polymerase chain reaction. The synthesis of nitric oxide was examined by Griess reaction, and the synthesis of prostaglandin E2 (PGE2) was examined by radioimmunoassay. The synthesis of proteins was examined by Western blot analysis of the cell extracts, and synthesis of proteoglycans via incorporation of 35S-sodium sulfate in the culture medium. Results Exposure of TMJ fibrochondrocytes to rHuIL-1β resulted in the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), which were paralleled by NO and PGE2 production. Additionally, IL-1β induced significant levels of collagenase (matrix metalloproteinase 1 [MMP-1]) within 4 hours, and this was sustained over a period of 48 hours. Concomitant application of CTS abrogated the catabolic effects of IL-1β on TMJ chondrocytes by inhibiting iNOS, COX-2, and MMP-1 mRNA production and NO, PGE2, and MMP-1 synthesis. CTS also counteracted cartilage degradation by augmenting expression of mRNA for tissue inhibitor of metalloproteinases 2 that is inhibited by rHuIL-1β. In parallel, CTS also counteracted rHuIL-1β–induced suppression of proteoglycan synthesis. Nevertheless, the presence of an inflammatory signal was a prerequisite for the observed CTS actions, because fibrochondrocytes, when exposed to CTS alone, did not exhibit any of the effects described above. Conclusion CTS acts as an effective antagonist of rHuIL-1β by potentially diminishing its catabolic actions on TMJ fibrochondrocytes. Furthermore, CTS actions appear to involve disruption/regulation of signal transduction cascade of rHuIL-1β upstream of mRNA transcription.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are characterized by consistent inflammation of multiple joints and marked destruction of cartilage and bone (1–3). Accumulated evidence suggests that tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) are the pivotal mediators of the disease processes (1,4–6). Both of these cytokines are found in the synovial fluid of arthritic joints. Furthermore, the arthritogenic potential of TNFα has been demonstrated by the presence of severe joint inflammation and cartilage destruction in mice overexpressing TNFα (7,8). TNF neutralization by anti-TNF antibodies has been shown to reduce inflammation and provide pain relief in patients with RA (9). TNFα is involved primarily in the onset of arthritis, and induces catabolic responses in chondrocytes by stimulating expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and proteases, including stromelysin and collagenase (10–12). TNFα also synergizes with IL-1 to enhance cartilage destruction in vivo (7). Furthermore, in addition to stimulating cartilage degradation, TNFα inhibits synthesis of aggrecan and type II collagen (CII) (13,14). Collectively, induction of catabolic enzymes and inhibition of matrix synthesis by TNFα and IL-1β drive cartilage destruction in chronic inflammatory diseases such as RA or OA (1–14). A number of antiarthritic therapies aimed at neutralizing the effects of cytokines are currently being investigated. Physical therapies such as continuous passive motion have been shown to mediate reparative/anabolic effects on diseased or inflamed synovial joints, although only limited information is available regarding the mechanisms of their intracellular actions (15–20). Their effects have been attributed mainly to increased circulation and dissemination of inflammatory mediators from the inflamed joint (17,21). We have recently shown that, in vitro, cyclic tensile strain (CTS) suppresses actions of IL-1β on chondrocytes by inhibiting expression of multiple proinflammatory genes such as iNOS, COX-2, and matrix metalloproteinase 1 (MMP-1) (22,23). Additionally, CTS actions include proteoglycan synthesis and induction of reparative proteins such as tissue inhibitors of metalloproteinase 2 (TIMP-2). Because of the pivotal role of TNFα in the pathogenesis of inflammatory joint diseases, in this study we examined whether the antiinflammatory effects of CTS are also mediated via suppression of TNFα actions. By exposing articular chondrocytes to CTS in vitro, we demonstrate that CTS is a potent antagonist of TNFα actions and exerts its effects via transcriptional regulation of TNFα response elements.

In temporomandibular joint disorders, the release of proinflammatory cytokines such as interleukin-1 (IL-1) initiates an inflammatory process disrupting cartilage homeostasis, ultimately leading to cartilage destruction. Additionally, mechanical stimuli affect articular chondrocyte metabolism. While articular chondrocytes generate nitric oxide (NO) in the presence of IL-1 proteoglycan synthesis is consecutively suppressed. The purpose of this study was to assess the effects of proinflammatory cytokines and mechanical strain in the form of cyclic tensile stretch on proteoglycan synthesis in chondrocytes, as compared to the NO competitive inhibitor L-N-monomethyl arginine (LMA), and to assess whether this effect is secondarily related to the activity of growth factors such as transforming growth factor beta (TGF-beta). Lapine articular chondrocytes were exposed to one of four different treatment regimens: no cyclic tensile stretch, IL-1, cyclic tensile stretch, or IL-1 plus cyclic tensile stretch. NO production was determined as medium nitrite accumulation. TGF-beta-bioactivity in chondrocyte conditioned medium was measured with the mink-lung epithelial cell bioassay. Proteoglycan synthesis was measured as the incorporation of 35-[S]-sodium sulfate into macromolecules separated from unincorporated label by gel filtration on PD-10 columns. In resting chondrocyte cultures, only baseline levels of NO were measured and the application of stretch for 24 h did not affect NO production. Addition of IL-1 provoked a large increase in NO synthesis which was abrogated in the presence of LMA. Application of stretch decreased the IL-1 induced NO synthesis, but did not modify the effect of LMA (being a competitive inhibitor of the inducible NO synthase) inhibiting IL-1 induced NO production. Glucosaminoglycan production was noted as proteoglycan synthesis showing almost no effect of cyclic stretch alone in comparison to the control condition, which correlates with the missing NO production in control and stretch conditions. Addition of IL-1 strongly inhibited proteoglycan synthesis, which was partly restored in the presence of LMA. However, cyclic stretch acted as a stronger restorer of proteoglycan synthesis in IL-1 treated conditions in the absence, and even more in the presence, of LMA. It was concluded that motion in the form of cyclic tensile stretch is a remarkable anti-inflammatory stimulus reversing the IL-1 induced suppression of proteoglycan synthesis in chondrocytes. These findings have therapeutic implications for the treatment of temporomandibular joint disorders, supporting early onset of postoperative and post-traumatic continuous passive motion therapy.

Inflammatory cytokines play a major role in cartilage destruction in diseases such as osteoarthritis and rheumatoid arthritis. Because physical therapies such as continuous passive motion yield beneficial effects on inflamed joints, we examined the intracellular mechanisms of mechanical strain-mediated actions in chondrocytes. By simulating the effects of continuous passive motion with cyclic tensile strain (CTS) on chondrocytes in vitro, we show that CTS is a potent antagonist of IL-1β actions and acts as both an anti-inflammatory and a reparative signal. Low magnitude CTS suppresses IL-1β-induced mRNA expression of multiple proteins involved in catabolic responses, such as inducible NO synthase, cyclo-oxygenase II, and collagenase. CTS also counteracts cartilage degradation by augmenting mRNA expression for tissue inhibitor of metalloproteases and collagen type II that are inhibited by IL-1β. Additionally, CTS augments the reparative process via hyperinduction of aggrecan mRNA expression and abrogation of IL-1β-induced suppression of proteoglycan synthesis. Nonetheless, the presence of an inflammatory signal is a prerequisite for the observed CTS actions, as exposure of chondrocytes to CTS alone has little effect on these parameters. Functional analysis suggests that CTS-mediated anti-inflammatory actions are not mediated by IL-1R down-regulation. Moreover, as an effective antagonist of IL-1β, the actions of CTS may involve disruption/regulation of signal transduction cascade of IL-1β upstream of mRNA transcription. These observations are the first to show that CTS directly acts as an anti-inflammatory signal on chondrocytes and provide a molecular basis for its actions.

During orthodontic tooth movement, mechanical forces acting on periodontal ligament (PDL) cells induce the synthesis of mediators which alter the growth, differentiation, and secretory functions of cells of the PDL. Since the cells of the PDL represent a heterogeneous population, we examined mechanically stress-induced cytokine profiles in three separate clones of human osteoblast-like PDL cells. Of the four pro-inflammatory cytokines investigated, only IL-6 and TGF-beta1 were up-regulated in response to mechanical stress. However, the expression of other pro-inflammatory cytokines such as IL-1 beta, TNF-alpha, or IL-8 was not observed. To understand the consequences of the increase in TGF-beta1 expression following mechanical stress, we examined the effect of TGF-beta1 on PDL cell phenotype and functions. TGF-beta1 was mitogenic to PDL cells at concentrations between 0.4 and 10 ng/mL. Furthermore, TGF-beta1 down-regulated the osteoblast-like phenotype of PDL cells, i.e., alkaline phosphatase activity, calcium phosphate nodule formation, expression of osteocalcin, and TGF-beta1, in a dose-dependent manner. Although initially TGF-beta1 induced expression of type I collagen mRNA, prolonged exposure to TGF-beta1 down-regulated the ability of PDL cells to express type I collagen mRNA. Our results further show that, within 4 hrs, exogenously applied TGF-beta1 down-regulated IL-6 expression in a dose-dependent manner, and this inhibition was sustained over a six-day period. In summary, the data suggest that mechanically stress-induced TGF-beta1 expression may be a physiological mechanism to induce mitogenesis in PDL cells while down-regulating its osteoblast-like features and simultaneously reducing the IL-6-induced bone resorption.

The nuclear factor NF-kappaB is a pleiotropic transcription factor whose activation results in inflammation, viral replication, and growth modulation. Due to its role in pathogenesis, NF-kappaB is considered a key target for drug development. In the present report we show that sanguinarine (a benzophenanthridine alkaloid), a known anti-inflammatory agent, is a potent inhibitor of NF-kappaB activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor rapidly activated NF-kappaB, this activation was completely suppressed by sanguinarine in a dose- and time-dependent manner. Sanguinarine did not inhibit the binding of NF-kappaB protein to the DNA but rather inhibited the pathway leading to NF-kappaB activation. The reversal of inhibitory effects of sanguinarine by reducing agents suggests a critical sulfhydryl group is involved in NF-kappaB activation. Sanguinarine blocked the tumor necrosis factor-induced phosphorylation and degradation of IkappaBalpha, an inhibitory subunit of NF-kappaB, and inhibited translocation of p65 subunit to the nucleus. As sanguinarine also inhibited NF-kappaB activation induced by interleukin-1, phorbol ester, and okadaic acid but not that activated by hydrogen peroxide or ceramide, the pathway leading to NF-kappaB activation is likely different for different inducers. Overall, our results demonstrate that sanguinarine is a potent suppressor of NF-kappaB activation and it acts at a step prior to IkappaBalpha phosphorylation.

The effectiveness of an ideal antimicrobial agent depends on its ability to kill microbes with minimal toxicity to host cells. Depending on the treatment regimen, antimicrobial agents come into contact with host cells for various intervals of time. Sanguinarium (SANG), chlorhexidine (CHX) and tetracycline (TET) are 3 antimicrobial agents frequently used in the management of periodontal infections. However, their effects on host immune cells during different treatment regimens are not known. Due to their ability to serve as the first line of host defense against microbial infections, we have compared the effects of these antimicrobial agents on human neutrophil functions and viability. The results show that SANG is not lytic to neutrophils from peripheral blood or crevicular fluid, at all concentrations tested. However, exposures of neutrophils to very low concentrations of SANG (0.001%) inhibits neutrophil chemotaxis, oxidative metabolism and degranulation within 5 min. Increasing the exposure time results in a similar inhibition of neutrophil functions, albeit at 50-100 fold lower concentrations of SANG. CHX rapidly disrupts the cell membrane of both crevicular and peripheral blood neutrophils at concentrations above 0.005% within 5 min, and inhibition of all neutrophil functions is due to its lytic properties. While TET is least toxic to neutrophils, a dose dependent inhibition of neutrophil functions is dependent on the calcium concentrations of the cellular environment, and is observed only above 0.04% or higher concentrations in the absence of calcium. The data suggest that a critical cumulative concentration of these drugs is essential for their toxicity and inhibition of neutrophil functions. Therefore, both the length of exposure and the dose of the drug both are critical while considering the effectiveness of SANG, CHX or TET in the treatment of infections. Furthermore, due to differences in their mechanisms of action, the consequences of their effects on neutrophils may have significant bearing on tissue pathology as well as on their therapeutic efficacy.

Purpose To determine whether the basic science evidence supports the use of continuous passive motion (CPM) after articular cartilage injury in the knee. Methods A systematic review was performed identifying and evaluating studies in animal models that focused on the basic science of CPM of the knee. Databases included in this review were PubMed, Biosis Previews, SPORTDiscus, PEDro, and EMBASE. All functional, gross anatomic, histologic, and histochemical outcomes were extracted and analyzed. Results Primary outcomes of CPM analyzed in rabbit animal models (19 studies) included histologic changes in articular cartilage (13 studies), biomechanical changes and nutrition of intra-articular tissue (3 studies), and anti-inflammatory biochemical changes (3 studies). Nine studies specifically examined osteochondral defects, 6 of which used autogenous periosteal grafts. Other pathologies included were antigen-induced arthritis, septic arthritis, medial collateral ligament reconstruction, hemarthrosis, and chymopapain-induced proteoglycan destruction. In comparison to immobilized knees, CPM therapy led to decreased joint stiffness and complications related to adhesions while promoting improved neochondrogenesis with formation and preservation of normal articular cartilage. CPM was also shown to create a strong anti-inflammatory environment by effectively clearing harmful, inflammatory particles from within the knee. Conclusions Current basic science evidence from rabbit studies has shown that CPM for the knee significantly improves motion and biological properties of articular cartilage. This may be translated to potentially improved outcomes in the management of articular cartilage pathology of the knee. Clinical Relevance If the rabbit model is relevant to humans, CPM may contribute to improved knee health by preventing joint stiffness, preserving normal articular tissue with better histologic and biologic properties, and improving range of motion as compared with joint immobilization and intermittent active motion.