Influence of Occlusal Hypofunction on Alveolar Bone Healing in Rats.

The aim of this in vivo study was to investigate the effect of occlusal hypofunction on alveolar bone healing in the absence or presence of an enamel matrix derivative (EMD). A standardized fenestration defect over the root of the mandibular first molar in 15 Wistar rats was created. Occlusal hypofunction was induced by extraction of the antagonist. Regenerative therapy was performed by applying EMD to the fenestration defect. The following three groups were established: (a) normal occlusion without EMD treatment, (b) occlusal hypofunction without EMD treatment, and (c) occlusal hypofunction with EMD treatment. After four weeks, all animals were sacrificed, and histological (hematoxylin and eosin, tartrate-resistant acid phosphatase) as well as immunohistochemical analyses (periostin, osteopontin, osteocalcin) were performed. The occlusal hypofunction group showed delayed bone regeneration compared to the group with normal occlusion. The application of EMD could partially, but not completely, compensate for the inhibitory effects of occlusal hypofunction on bone healing, as evidenced by hematoxylin and eosin and immunohistochemistry for the aforementioned molecules. Our results suggest that normal occlusal loading, but not occlusal hypofunction, is beneficial to alveolar bone healing. Adequate occlusal loading appears to be as advantageous for alveolar bone healing as the regenerative potential of EMD.

Cellular effects of glycine and trehalose air-polishing powders on human gingival fibroblasts in vitro.

OBJECTIVES: Air-polishing has been used in the treatment of periodontitis and gingivitis for years. The introduction of low-abrasive powders has enabled the use of air-polishing devices for subgingival therapy. Within the last decade, a wide range of different low-abrasive powders for subgingival use has been established. In this study, the effects of a glycine powder and a trehalose powder on human gingival fibroblasts (HGF) were investigated. METHODS: HGF were derived from three systemically and periodontally healthy donors. After 24 h and 48 h of incubation time, mRNA levels, and after 48 h, protein levels of TNFα, IL-8, CCL2, and VEGF were determined. In addition, NF-κB p65 nuclear translocation and in vitro wound healing were assessed. Statistical analysis was performed by ANOVA and post hoc Dunnett's and Tukey's tests (p < 0.05). RESULTS: Glycine powder significantly increased the expression of proinflammatory genes and showed exploitation of the NF-κB pathway, albeit trehalose powder hardly interfered with cell function and did not trigger the NF-κB pathway. In contrast to trehalose, glycine showed a significant inhibitory effect on the in vitro wound healing rate. CONCLUSION: Subgingivally applicable powders for air-polishing devices can regulate cell viability and proliferation as well as cytokine expression. Our in vitro study suggests that the above powders may influence HGF via direct cell effects. Trehalose appears to be relatively inert compared to glycine powder. CLINICAL RELEVANCE: With the limitations of an in vitro design, our study suggests that in terms of cell response, trehalose-based air-polishing powders show a reduced effect on inflammation.

Interaction of periodontitis and orthodontic tooth movement-an in vitro and in vivo study.

OBJECTIVES: The aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2). MATERIALS AND METHODS: The effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA. RESULTS: Periodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro. CONCLUSIONS: Our study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process. CLINICAL RELEVANCE: Orthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

Effect of Bacterial Infection on Ghrelin Receptor Regulation in Periodontal Cells and Tissues.

The effect of bacterial infection on the expression of growth hormone secretagogue receptor (GHS-R) was investigated in periodontal cells and tissues, and the actions of ghrelin were evaluated. GHS-R was assessed in periodontal tissues of rats with and without periodontitis. Human gingival fibroblasts (HGFs) were exposed to Fusobacterium nucleatum in the presence and absence of ghrelin. GHS-R expression was determined by real-time PCR and immunocytochemistry. Furthermore, wound healing, cell viability, proliferation, and migration were evaluated. GHS-R expression was significantly higher at periodontitis sites as compared to healthy sites in rat tissues. F. nucleatum significantly increased the GHS-R expression and protein level in HGFs. Moreover, ghrelin significantly abrogated the stimulatory effects of F. nucleatum on CCL2 and IL-6 expressions in HGFs and did not affect cell viability and proliferation significantly. Ghrelin stimulated while F. nucleatum decreased wound closure, probably due to reduced cell migration. Our results show original evidence that bacterial infection upregulates GHS-R in rat periodontal tissues and HGFs. Moreover, our study shows that ghrelin inhibited the proinflammatory actions of F. nucleatum on HGFs without interfering with cell viability and proliferation, suggesting that ghrelin and its receptor may act as a protective molecule during bacterial infection on periodontal cells.

Effects of Obesity on Bone Healing in Rats.

Although the association between periodontitis and obesity is well explored, it is unclear whether obesity is associated with a worse therapeutic outcome after periodontal treatment. The aim of this study was to investigate the effects of obesity on bone healing with and without the application of regeneration-promoting molecules. A standardized bone fenestration-type defect was created over the root of the mandibular first molar in 15 Wistar rats. Ten animals received a high-fat, high-sucrose diet (HFSD), while the remaining five animals were fed a standard diet. During surgery, the fenestration defects from half of the HFSD-fed, i.e., obese animals, were treated with regeneration-promoting molecules (enamel matrix derivative; EMD). After four weeks, bone healing was evaluated by histomorphometry, TRAP staining and immunohistochemistry for RUNX2 and osteopontin. The analyses revealed that the spontaneous healing of the periodontal defects was compromised by obesity. Application of EMD partially compensated for the negative effect of obesity. Nevertheless, EMD-stimulated bone healing in obese animals was not better than the spontaneous healing in the obesity-free control group, indicating that obesity may also inhibit the stimulatory effects of regeneration-promoting molecules. Our results show that obesity can negatively influence bone healing and suggest that bone healing may be compromised in humans.

Impact of Leptin on Periodontal Ligament Fibroblasts during Mechanical Strain.

Orthodontic treatment to correct dental malocclusions leads to the formation of pressure zones in the periodontal ligament resulting in a sterile inflammatory reaction, which is mediated by periodontal ligament fibroblasts (PDLF). Leptin levels are elevated in obesity and chronic inflammatory responses. In view of the increasing number of orthodontic patients with these conditions, insights into effects on orthodontic treatment are of distinct clinical relevance. A possible influence of leptin on the expression profile of PDLF during simulated orthodontic mechanical strain, however, has not yet been investigated. In this study, PDLF were exposed to mechanical strain with or without different leptin concentrations. The gene and protein expression of proinflammatory and bone-remodelling factors were analysed with RT-qPCR, Western-blot and ELISA. The functional analysis of PDLF-induced osteoclastogenesis was analysed by TRAP (tartrate-resistant acid phosphatase) staining in coculture with human macrophages. Pressure-induced increase of proinflammatory factors was additionally elevated with leptin treatment. PDLF significantly increased RANKL (receptor activator of NF-kB ligand) expression after compression, while osteoprotegerin was downregulated. An additional leptin effect was demonstrated for RANKL as well as for subsequent osteoclastogenesis in coculture after TRAP staining. Our results suggest that increased leptin concentrations, as present in obese patients, may influence orthodontic tooth movement. In particular, the increased expression of proinflammatory factors and RANKL as well as increased osteoclastogenesis can be assumed to accelerate bone resorption and thus the velocity of orthodontic tooth movement in the orthodontic treatment of obese patients.

Filifactor alocis and Tumor Necrosis Factor-Alpha Stimulate Synthesis of Visfatin by Human Macrophages.

There is little known about the effect of the periodontopathogen Filifactor alocis on macrophages as key cells of the innate immune defense in the periodontium. Therefore, the aim of the present study was to investigate the effect of F. alocis and additionally of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) on visfatin and other pro-inflammatory and proteolytic molecules associated with periodontitis in human macrophages. The presence of macrophage markers CD14, CD86, CD68, and CD163 was examined in gingival biopsies from healthy individuals and periodontitis patients. Human macrophages were incubated with F. alocis and TNFα for up to 2 d. The effects of both stimulants on macrophages were determined by real-time PCR, ELISA, immunocytochemistry, and immunofluorescence. F. alocis was able to significantly stimulate the synthesis of visfatin by human macrophages using TLR2 and MAPK pathways. In addition to visfatin, F. alocis was also able to increase the synthesis of cyclooxygenase 2, TNFα, and matrix metalloproteinase 1. Like F. alocis, TNFα was also able to stimulate the production of these proinflammatory and proteolytic molecules. Our results highlight the pathogenetic role of F. alocis in periodontal diseases and also underline the involvement of visfatin in the aetiopathogenesis of periodontitis.

Regulation of Anti-Apoptotic SOD2 and BIRC3 in Periodontal Cells and Tissues.

The aim of the study was to clarify whether orthodontic forces and periodontitis interact with respect to the anti-apoptotic molecules superoxide dismutase 2 (SOD2) and baculoviral IAP repeat-containing protein 3 (BIRC3). SOD2, BIRC3, and the apoptotic markers caspases 3 (CASP3) and 9 (CASP9) were analyzed in gingiva from periodontally healthy and periodontitis subjects by real-time PCR and immunohistochemistry. SOD2 and BIRC3 were also studied in gingiva from rats with experimental periodontitis and/or orthodontic tooth movement. Additionally, SOD2 and BIRC3 levels were examined in human periodontal fibroblasts incubated with Fusobacterium nucleatum and/or subjected to mechanical forces. Gingiva from periodontitis patients showed significantly higher SOD2, BIRC3, CASP3, and CASP9 levels than periodontally healthy gingiva. SOD2 and BIRC3 expressions were also significantly increased in the gingiva from rats with experimental periodontitis, but the upregulation of both molecules was significantly diminished in the concomitant presence of orthodontic tooth movement. In vitro, SOD2 and BIRC3 levels were significantly increased by F. nucleatum, but this stimulatory effect was also significantly inhibited by mechanical forces. Our study suggests that SOD2 and BIRC3 are produced in periodontal infection as a protective mechanism against exaggerated apoptosis. In the concomitant presence of orthodontic forces, this protective anti-apoptotic mechanism may get lost.

Resistin Is Increased in Periodontal Cells and Tissues: In Vitro and In Vivo Studies.

Resistin, a proinflammatory adipokine, is elevated in many inflammatory diseases. However, little is known about its performance in periodontitis. The present study is aimed at evaluating resistin expression and synthesis in periodontal cells and tissues under inflammatory/microbial stress in addition to its effects on the periodontium. In vivo, 24 male rats were randomly divided into two groups: control and ligature-induced periodontal disease. After 6 and 12 days, animals were sacrificed to analyze gene expression of adipokines, bone loss, inflammation, and resistin synthesis. In vitro, human periodontal ligament (PDL) fibroblasts were used to evaluate the expression of resistin after inflammatory stimuli. In addition, PDL fibroblasts were exposed to resistin to evaluate its role on soft and hard tissue metabolism markers. The periodontitis group demonstrated significant bone loss, an increase in the number of inflammatory cells and vascular structures, an increase in resistin expression and synthesis, and a decrease in the expression of adiponectin, leptin, and its functional receptor. PDL fibroblasts showed a significant increase in resistin expression and synthesis in response to the inflammatory stimulus by IL-1ß. Resistin induced an increase in cytokine expression and a decrease in the regulation of some hard tissue and matrix formation genes in PDL fibroblasts. These data indicate that resistin is produced by periodontal cells and tissues, and this effect is enhanced by inflammatory stimuli. Moreover, resistin seems to interfere with soft and hard tissue metabolism during periodontitis by reducing markers related to matrix formation and bone tissue.

CXCL1, CCL2, and CCL5 modulation by microbial and biomechanical signals in periodontal cells and tissues-in vitro and in vivo studies.

OBJECTIVES: This study was established to investigate whether the chemokines CXCL1, CCL2, and CCL5 are produced in periodontal cells and tissues and, if so, whether their levels are regulated by microbial and/or mechanical signals. MATERIALS AND METHODS: The chemokine expression and protein levels in gingival biopsies from patients with and without periodontitis were analyzed by RT-PCR and immunohistochemistry. The chemokines were also analyzed in gingival biopsies from rats subjected to experimental periodontitis and/or orthodontic tooth movement. Additionally, chemokine levels were determined in periodontal fibroblasts exposed to the periodontopathogen Fusobacterium nucleatum and mechanical forces by RT-PCR and ELISA. RESULTS: Higher CXCL1, CCL2, and CCL5 levels were found in human and rat gingiva from sites of periodontitis as compared with periodontally healthy sites. In the rat experimental periodontitis model, the bacteria-induced upregulation of these chemokines was significantly counteracted by orthodontic forces. In vitro, F. nucleatum caused a significant upregulation of all chemokines at 1 day. When the cells were subjected simultaneously to F. nucleatum and mechanical forces, the upregulation of chemokines was significantly inhibited. The transcriptional findings were paralleled at protein level. CONCLUSIONS: This study provides original evidence in vitro and in vivo that the chemokines CXCL1, CCL2, and CCL5 are regulated by both microbial and mechanical signals in periodontal cells and tissues. Furthermore, our study revealed that biomechanical forces can counteract the stimulatory actions of F. nucleatum on these chemokines. CLINICAL RELEVANCE: Mechanical loading might aggravate periodontal infection by compromising the recruitment of immunoinflammatory cells.

Regulation of matrix metalloproteinase-1 by Filifactor alocis in human gingival and monocytic cells.

OBJECTIVES: Periodontitis is a highly prevalent chronic inflammatory disease caused by periodontopathogens, such as Filifactor alocis. This study sought to examine the matrix metalloproteinase (MMP)-1 synthesis by monocytic and fibroblastic cells in response to F. alocis and to unravel the underlying cellular mechanisms. MATERIAL AND METHODS: Gingival biopsies from periodontally healthy and periodontitis individuals were analyzed for the presence of F. alocis and MMP-1 by RT-PCR. Human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells were stimulated with F. alocis in the presence and absence of a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. MMP-1 expression and protein levels were studied by RT-PCR and ELISA, respectively. RESULTS: F. alocis was highly prevalent in biopsies from periodontitis patients but barely present in the healthy gingiva. Significantly higher MMP-1 expression levels were found in the inflamed gingiva as compared with healthy biopsies. F. alocis caused a significant and dose-dependent MMP-1 upregulation in both cells. The stimulatory effect of F. alocis on MMP-1 was TLR2- and MAPK-dependent and more pronounced on THP-1 cells as compared with HGF-1 cells. CONCLUSIONS: Our results demonstrate that F. alocis and MMP-1 are more prevalent at periodontitis sites. Additionally, our study provides original evidence that F. alocis can stimulate MMP-1 production by fibroblastic and monocytic cells, suggesting that F. alocis may contribute to periodontal breakdown through MMP-1. CLINICAL RELEVANCE: F. alocis and MMP-1 are linked to each other and key players in periodontitis, which may have significant implications for future diagnostic and treatment strategies.

Role and Regulation of Mechanotransductive HIF-1α Stabilisation in Periodontal Ligament Fibroblasts.

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.

Autophagy in periodontal ligament fibroblasts under biomechanical loading.

Autophagy (cellular self-consumption) is an adaptive stress response and an important aspect of adaption to mechanical loading. If mechanical forces are associated with autophagy regulation in periodontal ligament (PDL) fibroblasts is still unknown. The aim of this study was to analyze the influence of force magnitude on autophagy regulation and subsequently on cell death in human PDL fibroblasts. Autophagy-associated genes were analyzed with a specific PrimePCR assay after 24 h of stimulation with high (STSH) and low magnitudes (STSL) of static tensile strain applied to PDL fibroblasts. Based on the results, targets were selected for further real-time PCR analysis. The autophagic flux was assessed by immunoblotting for autophagy marker microtubule-associated protein 1, light chain 3, and by autophagosome staining. Cell death was determined by TUNEL assay and Cell Death Detection ELISAPLUS. Autophagy was induced pharmacologically by rapamycin and inhibited by chloroquine. For statistical analysis, the Kruskal Wallis test followed by the post-hoc Dunnett's test was used. Static tensile strain had regulatory effects on mRNA expression of multiple autophagy-associated targets. Stimulation with STSH induced mRNA expression changes in more autophagy-associated targets than STSL. The autophagic flux was induced by STSH while STSL had no significant effect on autophagosome formation. Furthermore, autophagy inhibition led to increased cell death. Low magnitudes of tensile strain seem to have cell-protective properties. Taken together, our findings provide novel insights about autophagy regulation by biomechanical loading in human PDL fibroblasts. Our results suggest a gradual response of autophagy to static tensile strain in human PDL fibroblasts.

Regulation of tyrosine hydroxylase in periodontal fibroblasts and tissues by obesity-associated stimuli.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamines and has been connected to aggravated progression of periodontal disease under chronic stress. Obesity is known to increase the risk of periodontitis and adipokines have been suggested to be a pathomechanistic link. This study examines if obesity-associated stimuli have regulatory effects on TH levels in periodontal cells and tissues. Human periodontal ligament fibroblasts were cultured in the presence of leptin or visfatin for up to 2 days. Untreated cells served as control. TH regulation was analyzed by real-time PCR, immunocytochemistry and ELISA. TH gene expression in periodontal tissues of normal-weight and obese rodents was determined. Examination of gingival biopsies from rats and patients with and without periodontal disease was performed by real-time PCR or immunohistochemistry. For statistics, ANOVA and post hoc tests were applied (p < 0.05). In vitro, TH gene expression and protein levels were increased by leptin and visfatin. In vivo, TH gene expression was upregulated in periodontal tissues of obese rodents as compared to normal-weight animals. Additionally, increased TH gene expression was found in rat gingival biopsies with experimental periodontitis. Human gingival biopsies from sites of periodontitis confirmed the animal data by demonstrating elevated TH levels at periodontally diseased sites. This study provides original evidence that obesity-associated stimuli induce a TH upregulation in periodontal cells and tissues. Since TH levels were also increased at periodontitis sites, our in vitro and animal findings suggest that this enzyme could represent a pathomechanism whereby obesity contributes to periodontitis.

Effect of interleukin-1ß on ghrelin receptor in periodontal cells.

OBJECTIVES: Periodontopathogens induce immunoinflammatory responses characterized by the release of inflammatory mediators, e.g., interleukin (IL)-1ß, IL-6, and IL-8. Ghrelin (GHRL) is an appetite hormone which mediates its effect via the functional receptor GHS-R1a. This study was to examine the effect of an inflammatory insult on GHS-R1a in human periodontal cells. MATERIALS AND METHODS: Periodontal ligament (PDL) cells and gingival fibroblasts (HGFs) were exposed to IL-1ß in the presence and absence of GHRL. Cells were also pre-incubated with specific inhibitors of NF-κB or MEK1/MEK2 signaling. Gene expression of GHS-R1a and proinflammatory mediators was assessed by real-time PCR, GHS-R1 protein level by immunocytochemistry, and NF-κB nuclear translocation by immunofluorescence. RESULTS: IL-1ß increased significantly the GHS-R1a expression in both cell types in a dose-dependent manner. The stimulatory effect of IL-1ß involved the NF-κB and MAPK pathways. Exposure of cells to IL-1ß also resulted in an increased production of GHS-R1 protein in both cell types. Furthermore, GHRL counteracted significantly the stimulatory actions of IL-1ß on IL-6 and IL-8 in PDL cells. CONCLUSIONS: This study demonstrates for the first time that IL-1ß upregulates the functional ghrelin receptor in periodontal fibroblastic cells. Moreover, these results further support the assumption that the GHRL/GHS-R system exerts anti-inflammatory effects. Therefore, the upregulation of ghrelin receptor in periodontal cells in response to an inflammatory stimulus may represent a negative feedback mechanism to attenuate the initial inflammatory process in periodontal diseases. CLINICAL RELEVANCE: The anti-inflammatory GHRL/GHS-R system may serve as a promising target for the prevention and therapy of periodontal diseases.

Characterization of a diet-induced obesity rat model for periodontal research.

OBJECTIVES: Obesity is associated with periodontitis, but the mechanisms underlying this association have yet to be unraveled. The present investigation was to evaluate a common rat model, in which obesity is induced by high-fat, high-sucrose diet (HFSD), for its applicability in periodontal research. MATERIALS AND METHODS: Ten male Wistar rats were fed a 3-month HFSD along with a matching control group. Afterwards, the body weight, adipocyte morphology, leptin and adiponectin levels in adipose tissue, gingiva, and serum as well as the serum levels of triglyceride, cholesterol, and glucose were analyzed. For statistical analyses, parametric and non-parametric tests were applied (p < 0.05). RESULTS: Body weight was significantly higher in the HFSD group after dieting as compared to control. HFSD caused a significant increase in serum triglyceride, low-density lipoprotein cholesterol, and leptin levels and a significant decrease in high-density lipoprotein cholesterol. Furthermore, adipose tissue from HFSD rats exhibited significantly larger adipocytes, displayed a significant upregulation of leptin and, surprisingly, elevated adiponectin levels, which is in contrast to chronic obesity in humans. Although leptin and adiponectin were also observed in gingival biopsies, no obvious differences between the groups were found. CONCLUSIONS: Although this rat diet-induced obesity model is characterized by changes typical of obesity, it also has limitations, which have to be considered when data, especially with regard to adipokines, are extrapolated to humans. CLINICAL RELEVANCE: The rodent diet-induced obesity model may be useful for unraveling pathomechanisms underlying the association between obesity and periodontal destruction but conclusions have to be drawn with caution.

Mechanosensor polycystin-1 potentiates differentiation of human osteoblastic cells by upregulating Runx2 expression via induction of JAK2/STAT3 signaling axis.

Polycystin-1 (PC1) has been proposed as a chief mechanosensing molecule implicated in skeletogenesis and bone remodeling. Mechanotransduction via PC1 involves proteolytic cleavage of its cytoplasmic tail (CT) and interaction with intracellular pathways and transcription factors to regulate cell function. Here we demonstrate the interaction of PC1-CT with JAK2/STAT3 signaling axis in mechanically stimulated human osteoblastic cells, leading to transcriptional induction of Runx2 gene, a master regulator of osteoblastic differentiation. Primary osteoblast-like PC1-expressing cells subjected to mechanical-stretching exhibited a PC1-dependent increase of the phosphorylated(p)/active form of JAK2. Specific interaction of PC1-CT with pJAK2 was observed after stretching while pre-treatment of cells with PC1 (anti-IgPKD1) and JAK2 inhibitors abolished JAK2 activation. Consistently, mechanostimulation triggered PC1-mediated phosphorylation and nuclear translocation of STAT3. The nuclear phosphorylated(p)/DNA-binding competent pSTAT3 levels were augmented after stretching followed by elevated DNA-binding activity. Pre-treatment with a STAT3 inhibitor either alone or in combination with anti-IgPKD1 abrogated this effect. Moreover, PC1-mediated mechanostimulation induced elevation of Runx2 mRNA levels. ChIP assays revealed direct regulation of Runx2 promoter activity by STAT3/Runx2 after mechanical-stretching that was PC1-dependent. Our findings show that mechanical load upregulates expression of Runx2 gene via potentiation of PC1-JAK2/STAT3 signaling axis, culminating to possibly control osteoblastic differentiation and ultimately bone formation.

Role of cathepsin S In periodontal wound healing-an in vitro study on human PDL cells.

BACKGROUND: Cathepsin S is a cysteine protease, which is expressed in human periodontal ligament (PDL) cells under inflammatory and infectious conditions. This in vitro study was established to investigate the effect of cathepsin S on PDL cell wound closure. METHODS: An in vitro wound healing assay was used to monitor wound closure in wounded PDL cell monolayers for 72 h in the presence and absence of cathepsin S. In addition, the effects of cathepsin S on specific markers for apoptosis and proliferation were studied at transcriptional level. Changes in the proliferation rate due to cathepsin S stimulation were analyzed by an XTT assay, and the actions of cathepsin S on cell migration were investigated via live cell tracking. Additionally, PDL cell monolayers were treated with a toll-like receptor 2 agonist in the presence and absence of a cathepsin inhibitor to examine if periodontal bacteria can alter wound closure via cathepsins. RESULTS: Cathepsin S enhanced significantly the in vitro wound healing rate by inducing proliferation and by increasing the speed of cell migration, but had no effect on apoptosis. Moreover, the toll-like receptor 2 agonist enhanced significantly the wound closure and this stimulatory effect was dependent on cathepsins. CONCLUSIONS: Our findings provide original evidence that cathepsin S stimulates PDL cell proliferation and migration and, thereby, wound closure, suggesting that this cysteine protease might play a critical role in periodontal remodeling and healing. In addition, cathepsins might be exploited by periodontal bacteria to regulate critical PDL cell functions.

Regulation of Ghrelin Receptor by Periodontal Bacteria In Vitro and In Vivo.

Ghrelin plays a major role in obesity-related diseases which have been shown to be associated with periodontitis. This study sought to analyze the expression of the functional receptor for ghrelin (GHS-R1a) in periodontal cells and tissues under microbial conditions in vitro and in vivo. The GHS-R1a expression in human periodontal cells challenged with the periodontopathogen Fusobacterium nucleatum, in gingival biopsies from periodontally healthy and diseased individuals, and from rats with and without ligature-induced periodontitis was analyzed by real-time PCR, immunocytochemistry, and immunofluorescence. F. nucleatum induced an initial upregulation and subsequent downregulation of GHS-R1a in periodontal cells. In rat experimental periodontitis, the GHS-R1a expression at periodontitis sites was increased during the early stage of periodontitis, but significantly reduced afterwards, when compared with healthy sites. In human gingival biopsies, periodontally diseased sites showed a significantly lower GHS-R1a expression than the healthy sites. The expression of the functional ghrelin receptor in periodontal cells and tissues is modulated by periodontal bacteria. Due to the downregulation of the functional ghrelin receptor by long-term exposure to periodontal bacteria, the anti-inflammatory actions of ghrelin may be diminished in chronic periodontal infections, which could lead to an enhanced periodontal inflammation and tissue destruction.