James Deschner

Signal transduction by mechanical strain in chondrocytes

&NA; The beneficial effects of physiological levels of mechanical signals or exercise may be explained by their ability to suppress the signal transduction pathways of proinflammatory/catabolic mediators, while stimulating anabolic pathways. Whether these anabolic signals are a consequence of the inhibition of nuclear factor kappa B or are mediated via distinct anabolic pathways is yet to be elucidated. Purpose of review Exercise and passive motion exert reparative effects on inflamed joints, whereas excessive mechanical forces initiate cartilage destruction as observed in osteoarthritis. However, the intracellular mechanisms that convert mechanical signals into biochemical events responsible for cartilage destruction and repair remain paradoxical. This review summarizes how signals generated by mechanical stress may initiate repair or destruction of cartilage. Recent findings Mechanical strain of low magnitude inhibits inflammation by suppressing IL‐1&bgr; and TNF‐&agr;‐induced transcription of multiple proinflammatory mediators involved in cartilage degradation. This also results in the upregulation of proteoglycan and collagen synthesis that is drastically inhibited in inflamed joints. On the contrary, mechanical strain of high magnitude is proinflammatory and initiates cartilage destruction while inhibiting matrix synthesis. Investigations reveal that mechanical signals exploit nuclear factor‐kappa B as a common pathway for transcriptional inhibition/activation of proinflammatory genes to control catabolic processes in chondrocytes. Mechanical strain of low magnitude prevents nuclear translocation of nuclear factor kappa B, resulting in the suppression of proinflammatory gene expression, whereas mechanical strain of high magnitude induces transactivation of nuclear factor kappa B, and thus proinflammatory gene induction.

Calculus removal and the prevention of its formation

Periodontitis is strongly associated with the presence of dental calculus on root surfaces. Although the rough calculus surface may not in itself induce inflammation in the adjacent periodontal tissues, dental calculus serves as an ideal substrate for subgingival microbial colonization. Therefore, causerelated anti-infective therapy aims to eliminate the microbial biofilm and calcified deposits from the diseased root surfaces by means of root surface debridement. Over the past 50 years, a large number of clinical and laboratory studies have been performed to determine the efficacy of calculus removal from diseased root surfaces by various methods. These studies aimed to determine whether complete removal of subgingival calculus by root surface debridement is possible. They also evaluated the importance of operator experience in the effectiveness of calculus removal. Possible differences in efficacy between hand tools and power-driven instruments or lasers have been investigated. The impact of tooth and site characteristics, such as probing depths, tooth type, tooth surfaces and furcation areas, has also been evaluated. In addition, side-effects such as unintentional root substance removal and patient discomfort have been assessed. This review focuses on the composition and formation of calculus, its significance for the disease process, the methods available for calculus removal, and prevention of its formation.

IL-23-producing CD68+ macrophage-like cells predominate within an IL-17-polarized infiltrate in chronic periodontitis lesions

AIM To analyse antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages (Mo) or B cells depending on the regional site of chronic periodontitis (CP), and to investigate their relation to Th17 cells. MATERIAL AND METHODS Biopsies from oral mucosa as well as the coronal and bottom regions of CP were analysed by immunhistochemistry, immunofluorescence, flow cytometry and real-time PCR. RESULTS A predominance of CD68(+) Mo-like cells and CD20(+) B cells and strong Th17 infiltration was observed in the bottom region of CP lesions, while CD1a(+) DCs were only detected in the coronal regions, where Th17 infiltration was low. Furthermore, CD68(+) Mo-like cells displayed CD163 expression as a typical Mo-marker, but expressed in parallel typical DCs markers, such as CD11c or CD209 and TLR4. Interestingly, Th17-inducing cytokine IL-23p19 was produced by CD68(+) Mo-like cells, but not CD20(+) B cells. Moreover, the stimulation of in vitro generated CD68(+) Mo-like cells by Porphyromonas gingivalis-derived (Pg) lipopolysaccharide resulted in the upregulation of their IL-23p19 mRNA expression, which was inhibited by the blockage of TLR4. CONCLUSIONS In view of these data, a picture emerges that IL-17-producing cells in CP could be in part directed by CD68(+) Mo-like cells, which produce IL-23p19 upon TLR4 activation by Pg.

Interactions of Adiponectin and Lipopolysaccharide from Porphyromonas gingivalis on Human Oral Epithelial Cells

Background Periodontitis is an inflammatory disease caused by pathogenic microorganisms, such as Porphyromonas gingivalis, and characterized by the destruction of the periodontium. Obese individuals have an increased risk for periodontitis and show decreased serum levels of adiponectin. This in-vitro study was established to examine whether adiponectin modulates critical effects of lipopolysaccharide (LPS) from P. gingivalis on oral epithelial cells (OECs). Methodology/Principal Findings The presence of adiponectin and its receptors in human gingival tissue samples and OECs was analyzed by immunohistochemistry and PCR. Furthermore, OECs were treated with LPS and/or adiponectin for up to 72 h, and the gene expression and protein synthesis of pro- and anti-inflammatory mediators, matrix metalloproteinases (MMPs) and growth factors were analyzed by real-time PCR and ELISA. Additionally, cell proliferation, differentiation and in-vitro wound healing were studied. The nuclear translocation of NFκB was investigated by immunofluorescence. Gingival tissue sections showed a strong synthesis of adiponectin and its receptors in the epithelial layer. In cell cultures, LPS induced a significant up-regulation of interleukin (IL) 1β, IL6, IL8, MMP1 and MMP3. Adiponectin abrogated significantly the stimulatory effects of LPS on these molecules. Similarly, adiponectin inhibited significantly the LPS-induced decrease in cell viability and increase in cell proliferation and differentiation. Adiponectin led to a time-dependent induction of the anti-inflammatory mediators IL10 and heme oxygenase 1, and blocked the LPS-stimulated NFκB nuclear translocation. Conclusions/Significance Adiponectin may counteract critical actions of P. gingivalis on oral epithelial cells. Low levels of adiponectin, as observed in obese individuals, may increase the risk for periodontal inflammation and destruction.

Mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation during inflammation via integrin-linked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes

IntroductionThe importance of mechanical signals in normal and inflamed cartilage is well established. Chondrocytes respond to changes in the levels of proinflammatory cytokines and mechanical signals during inflammation. Cytokines like interleukin (IL)-1β suppress homeostatic mechanisms and inhibit cartilage repair and cell proliferation. However, matrix synthesis and chondrocyte (AC) proliferation are upregulated by the physiological levels of mechanical forces. In this study, we investigated intracellular mechanisms underlying reparative actions of mechanical signals during inflammation.MethodsACs isolated from articular cartilage were exposed to low/physiologic levels of dynamic strain in the presence of IL-1β. The cell extracts were probed for differential activation/inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade. The regulation of gene transcription was examined by real-time polymerase chain reaction.ResultsMechanoactivation, but not IL-1β treatment, of ACs initiated integrin-linked kinase activation. Mechanical signals induced activation and subsequent C-Raf-mediated activation of MAP kinases (MEK1/2). However, IL-1β activated B-Raf kinase activity. Dynamic strain did not induce B-Raf activation but instead inhibited IL-1β-induced B-Raf activation. Both mechanical signals and IL-1β induced ERK1/2 phosphorylation but discrete gene expression. ERK1/2 activation by mechanical forces induced SRY-related protein-9 (SOX-9), vascular endothelial cell growth factor (VEGF), and c-Myc mRNA expression and AC proliferation. However, IL-1β did not induce SOX-9, VEGF, and c-Myc gene expression and inhibited AC cell proliferation. More importantly, SOX-9, VEGF, and Myc gene transcription and AC proliferation induced by mechanical signals were sustained in the presence of IL-1β.ConclusionsThe findings suggest that mechanical signals may sustain their effects in proinflammatory environments by regulating key molecules in the MAP kinase signaling cascade. Furthermore, the findings point to the potential of mechanosignaling in cartilage repair during inflammation.

Human β-defensins differently affect proliferation, differentiation, and mineralization of osteoblast-like MG63 cells.

Purpose of this study was to investigate whether human β‐defensins (hBDs) affect maturation and proliferation of osteoblast‐like MG63 cells in vitro. Osteoblast‐like MG63 cells were stimulated with hBD‐1, ‐2, and ‐3 under control conditions and with hBD‐2 during experimental inflammation (induced by interleukin‐1β, tumor necrosis factor‐α, toll‐like receptor‐2 and ‐4 agonists). Expression of different osteogenic markers and hBDs were analyzed by real‐time PCR, immunohistochemistry, and enzyme‐linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were monitored. All tested hBDs were expressed on mRNA and protein level in MG63 cells. Only stimulation with hBD‐2 elevated the proliferation rate. hBD‐2 and hBD‐3 positively affected the differentiation of osteoblast‐like cells provided by increased transcript levels of osteogenic markers, up‐regulated ALP enzyme activity and enhanced mineralized nodule formation. All pro‐inflammatory stimuli enhanced interleukin‐6 and hBD‐2 expression and down‐regulated markers of osteoblastic differentiation. In accordance, inflammation increased transcript level of Notch‐1 (an inhibitor of osteoblastic differentiation). hBD‐2 was not able to revert effects of inflammation on differentiation. In bone cells human β‐defensins exhibit further functions than antimicrobial peptide activity. These include stimulation of proliferation and differentiation. Differentiation arrest due to inflammation could not be overcome by hBD‐2 alone. J. Cell. Physiol. 227: 994–1003, 2012.

Biomechanical Signals Suppress Proinflammatory Responses in Cartilage: Early Events in Experimental Antigen-Induced Arthritis

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.

Effects of enamel matrix derivative on periodontal wound healing in an inflammatory environment in vitro

AIM This in vitro study was established to investigate whether the regenerative capacity of periodontal ligament (PDL) cells in the presence of enamel matrix derivative (EMD) is modulated by inflammation. MATERIALS AND METHODS PDL cells were grown in the presence or absence of EMD under normal and inflammatory conditions for up to 14 days. In order to mimic an inflammatory environment, cells were incubated with interleukin (IL)-1β. Cells were also exposed to transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-1 under both conditions. For analysis of wound healing, an in vitro wound fill assay was used. The synthesis of growth factors, markers of proliferation, and osteogenic differentiation, as well as collagen was studied by real-time polymerase chain reaction, enzyme-linked immunoassay, and immunoblotting. Mineralization was assessed by alizarine red S and von Kossa staining. RESULTS EMD stimulated significantly the in vitro wound fill rate, cell proliferation and adhesion, synthesis of growth factors, and collagen, as well as mineralization. In the presence of IL-1β, these EMD effects were significantly reduced. IL-1β also inhibited significantly the wound fill rate induced by TGF-β1 and IGF-1. CONCLUSIONS Critical PDL cell functions that are associated with periodontal regeneration are reduced in an inflammatory environment.

Regulatory role of periodontal ligament fibroblasts for innate immune cell function and differentiation

Innate immunity is crucial for an effective host defense against pathogenic microorganisms in periodontal tissues. As periodontal ligament (PDL) cells synthesize immunomodulatory cytokines, the aim of this in vitro study was to investigate whether these cells can interact with innate immune cells. Resting and inflammatory primed (IL-1β, TNF-α, HMGB1) human PDL cells were co-cultured with human monocyte-derived dendritic cells or macrophages. Migration, phenotypic maturation and modulation of phagocytosis of Porphyromonas gingivalis by immune cells were investigated upon co-culture with PDL cells and/or their released soluble factors. PDL cells interacted with immune cells under both non-inflammatory and inflammatory conditions. Immune cell migration was significantly enhanced by co-culture with PDL cells, which also affected their phenotypic maturation both through cell-cell contact and through released soluble mediators. The dendritic cell maturation markers CD83 and CD86 were upregulated as much as both ‘alternatively activated’ M2 macrophage maturation markers CD23 and CD163. In contrast, the ‘classically activated’ M1 macrophage maturation marker CD64 was downregulated. Finally, PDL cells significantly enhanced the phagocytosis of Porphyromonas gingivalis by immune cells. Our experiments revealed that PDL cells are not only structural elements of the periodontium, but actively influence immune responses by interaction with innate immune cells.

Human periodontal ligament cells facilitate leukocyte recruitment and are influenced in their immunomodulatory function by Th17 cytokine release.

The objective of this in vitro study was to examine the immunomodulatory impact of human periodontal ligament (PDL) cells on the nature and magnitude of the leukocyte infiltrate in periodontal inflammation, particularly with regard to Th17 cells. PDL cells were challenged with pro-inflammatory cytokines (IL-1ß, IL-17A, and IFN-γ) and analyzed for the expression of cytokines involved in periodontal immunoinflammatory processes (IL-6, MIP-3 alpha, IL-23A, TGFß1, IDO, and CD274). In order to further investigate a direct involvement of PDL cells in leukocyte function, co-culture experiments were conducted. The expression of the immunomodulatory cytokines studied was significantly increased under pro-inflammatory conditions in PDL cells. Although PDL cells did not stimulate leukocyte proliferation or Th17 differentiation, these cells induced the recruitment of leukocytes. The results of our study suggest that PDL cells might be involved in chronic inflammatory mechanisms in periodontal tissues and thus in the transition to an adaptive immune response in periodontitis.