Single Nucleotide Polymorphisms in RUNX2 and BMP2 contributes to different vertical facial profile.

The vertical facial profile is a crucial factor for facial harmony with significant implications for both aesthetic satisfaction and orthodontic treatment planning. However, the role of single nucleotide polymorphisms (SNPs) in the development of vertical facial proportions is still poorly understood. This study aimed to investigate the potential impact of some SNPs in genes associated with craniofacial bone development on the establishment of different vertical facial profiles. Vertical facial profiles were assessed by two senior orthodontists through pre-treatment digital lateral cephalograms. The vertical facial profile type was determined by recommended measurement according to the American Board of Orthodontics. Healthy orthodontic patients were divided into the following groups: "Normodivergent" (control group), "Hyperdivergent" and "Hypodivergent". Patients with a history of orthodontic or facial surgical intervention were excluded. Genomic DNA extracted from saliva samples was used for the genotyping of 7 SNPs in RUNX2, BMP2, BMP4 and SMAD6 genes using real-time polymerase chain reactions (PCR). The genotype distribution between groups was evaluated by uni- and multivariate analysis adjusted by age (alpha = 5%). A total of 272 patients were included, 158 (58.1%) were "Normodivergent", 68 (25.0%) were "Hyperdivergent", and 46 (16.9%) were "Hypodivergent". The SNPs rs1200425 (RUNX2) and rs1005464 (BMP2) were associated with a hyperdivergent vertical profile in uni- and multivariate analysis (p-value < 0.05). Synergistic effect was observed when evaluating both SNPs rs1200425- rs1005464 simultaneously (Prevalence Ratio = 4.0; 95% Confidence Interval = 1.2-13.4; p-value = 0.022). In conclusion, this study supports a link between genetic factors and the establishment of vertical facial profiles. SNPs in RUNX2 and BMP2 genes were identified as potential contributors to hyperdivergent facial profiles.

Longitudinal analysis of microcirculatory parameters in gingival tissues after tooth extraction in patients with different risk profiles for wound healing disorders - a pilot study.

OBJECTIVES: We aimed to establish a risk profile for intraoral wound healing disorders based on measurements of microcirculation in gingival tissues. MATERIALS AND METHODS: Oxygen saturation (SO2) and blood flow in gingival tissues were measured with tissue spectrometry and laser doppler spectroscopy in 37 patients before/after tooth extractions. Patients were assigned to four groups: anamnestically and periodontally healthy patients (n = 7), anamnestically healthy but suffering from periodontitis (n = 10), anamnestically healthy but smoking and suffering from periodontitis (n = 10) and suffering from diabetes and periodontitis (n = 10). Measurements were performed at three different time points: Baseline measurement (T0), one day post extractionem (p.e.) (T1) and seven days p.e. (T2). RESULTS: Baseline SO2 values were higher in control patients (p = .038). This effect was most evident in comparison to smokers suffering from periodontitis (p = .042), followed by diabetics suffering from periodontitis (p = .09). An opposite trend was seen for blood flow. Patients suffering from periodontitis demonstrated higher blood flow values (p = .012). Five patients, which belonged to the group of smokers suffering from periodontitis, showed clinically a delayed wound healing. CONCLUSION: Differences in SO2 and blood flow of gingival tissue could be detected in different groups of patients with existing periodontitis compared to control patients. CLINICAL RELEVANCE: Lower baseline SO2 values could be a warning signal for possible wound healing disorders after oral surgery.

Association between dental age and malocclusions: a systematic review.

BACKGROUND: The evidence in the literature suggests that some skeletal or dental malocclusions are involved with dental development, resulting in advanced or delayed dental age (DA). The purpose of this systematic review was to investigate the association between DA and different types of malocclusions. METHODS: The search was carried out on PubMed, Scopus, Web of Science, Virtual Health Library, and in the gray literature. Observational studies that evaluated the association between DA and sagittal, vertical, or transversal malocclusions were included. The quality assessment was performed using the Newcastle-Ottawa Scale (NOS). The data from primary studies were narratively synthesized. The certainty of evidence was evaluated using the GRADE approach. The study was conducted from August 2023 to October 2023. RESULTS: One Thousand Nine Hundred Ninety-One records were identified in the initial search. Twenty (n = 20) studies were included. Most of the studies (n=15) presented a moderate quality according to NOS. Twelve studies evaluated the association between DA and sagittal discrepancies; eight studies evaluated vertical discrepancies, and only one study analyzed a transversal discrepancy. Demirjian's method for DA assessment was the most used among the studies. The primary studies observed that patients of both sexes presenting a vertical growth pattern and males with skeletal Class III malocclusion tend to have advanced DA. The study that investigated transversal malocclusion found that unilateral posterior cross-bite is associated with delayed DA. The certainty of evidence was very low for all outcomes evaluated. CONCLUSION: DA may be associated with the type of malocclusion. It is suggested that DA can be used as an initial diagnostic tool in orthodontics. Future well-designed studies should be performed in order to investigate the association between DA and different types of malocclusions in more detail. TRIAL REGISTRATION: This study was registered in the PROSPERO database (CRD42023454207).

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.

Impact of glycine and erythritol/chlorhexidine air-polishing powders on human gingival fibroblasts: An in vitro study.

BACKGROUND: Supra- and subgingival air-polishing has been used in periodontitis and gingivitis therapy for years. Low-abrasive types of powders have facilitated the application in subgingival areas. In this study, the cellular effects of a glycine powder and an erythritol/chlorhexidine (CHX) powder on human gingival fibroblasts (HGF) were investigated. METHODS: HGF were obtained from sound gingiva of three healthy donors. After 12 h and 24 h of incubation time, cell viability testing and, after 24 h and 48 h, a cell proliferation assay was conducted. Additionally, the individual components erythritol and CHX were investigated for cell viability. In vitro wound healing was monitored for 48 h and scanning electron microscopy (SEM) analysis was performed after 24 h. Statistical analysis was accomplished by ANOVA and post hoc Dunnett's and Tukey's tests (p < 0.05) were performed. RESULTS: Erythritol/CHX powder and in a lower extent, glycine powder decreased cell viability and cell proliferation. The negative effect of erythritol/CHX was mainly based on the CHX component. In vitro wound healing was negatively influenced in both types of powders compared to control. Cell size was altered in both test groups, whereas cell morphology was affected only in the erythritol/CHX group. CONCLUSIONS: The investigated powders for subgingival air-polishing can influence cell viability, morphology, and proliferation, as well as wound closure in vitro. These actions on fibroblasts are discernible, with the cytotoxic effect of erythritol/CHX powder being very clear and mainly due to the CHX component. Our results suggest that subgingivally applied powders can exert direct effects on gingival fibroblasts.

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.

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.

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.

Autophagy Induces Expression of IL-6 in Human Periodontal Ligament Fibroblasts Under Mechanical Load and Overload and Effects Osteoclastogenesis in vitro.

Objective: Autophagy is an important cellular adaptation mechanism to mechanical stress. In animal experiments, inhibition of autophagy during orthodontic tooth movement triggered increased expression of inflammation-related genes and decreased bone density. The aim of this study was to investigate how autophagy affects cytokine levels of interleukin 6 (IL-6) in human periodontal ligament (hPDL) fibroblasts under mechanical pressure and the resulting influence on osteoblast communication. Methods: hPDL fibroblasts were subjected to physiologic mechanical load, constant overload, or rapamycin treatment for 16 to 24 h ± autophagy inhibitor 3-MA. Autophagosomes were quantified by flow cytometry. Gene expression of il-6 as well as IL-6 levels in the supernatant were determined with rtPCR and ELISA. To investigate the influence of mechanically-induced autophagy on cell-cell communication, an osteoblast-culture was subjected to supernatant from stimulated hPDL fibroblasts ± soluble IL-6 receptor (sIL-6R). After 24 h, osteoprotegerin (opg) and receptor activator of nuclear factor κB ligand (rankl) gene expressions were detected with rtPCR. Gene expression of a disintegrin and metalloproteinases (adam) 10 and 17 in stimulated hPDL fibroblasts was examined via rtPCR. Results: Autophagy was induced by biomechanical stress in hPDL fibroblasts in a dose-dependent manner. Mechanical load and overload increased IL-6 expression at gene and protein level. Autophagy inhibition further enhanced the effects of mechanical stimulation on IL-6 expression. Mechanical stimulation of hPDL fibroblasts downregulated adam10 and adam17 expressions. Inhibition of autophagy had stimulus-intensity depending effects: autophagy inhibition alone or additional application of physiological stress enhanced adam10 and adam17 expressions, whereas mechanical overload had adverse effects. Osteoblasts showed significantly reduced opg expression in the presence of supernatant derived of hPDL fibroblasts treated with autophagy inhibitor and sIL-6R. Conclusion: IL-6 levels were increased in response to pressure in hPDL fibroblasts, which was further enhanced by autophagy inhibition. This caused a decrease in opg expression in osteoblasts. This may serve as an explanatory model for accelerated tooth movement observed under autophagy inhibition, but may also represent a risk factor for uncontrolled bone loss.

Influences of cold atmospheric plasma on apoptosis related molecules in osteoblast-like cells in vitro.

BACKGROUND: Cold atmospheric plasma (CAP) has recently been identified as a novel therapeutic strategy for supporting processes of wound healing. Since CAP is additionally known to kill malignant cells, our study intends to determine the influence of CAP on crucial molecules involved in the molecular mechanism of apoptosis in osteoblast-like cells. METHODS: Human osteoblast-like cells were CAP-treated for 30 and 60 s. CAP effects on critical factors related to apoptosis were studied at transcriptional and protein level using real time-PCR, immunofluorescence staining and western blot. Phalloidin / DAPI staining was used for analyzing the cell morphology. In addition, apoptotic outcomes of CAP were displayed using flow cytometry analysis. For studying intracellular signaling pathways, MAP kinase MEK 1/2 and PI3K were blocked. Finally, the effects of CAP on caspase-3 activity were examined using a caspase-3 assay. RESULTS: CAP treatment resulted in a significant downregulation of p53 and apoptotic protease activating factor (APAF)-1, caspase (CASP)9, CASP3, BCL2 Antagonist/Killer (BAK)1, and B-Cell Lymphoma (BCL)2 mRNA expression at 1 d. An inhibitory effect of CAP on apoptotic genes was also shown under inflammatory and apoptotic conditions. Nuclear translocation of p53 was determined in CAP treated cells at the early and late stage, after 15 min, 30 min, and 1 h. p53 and APAF-1 protein levels were reduced at 1 d, visualized by immunofluorescence and western blot, respectively. Moreover, a morphological cytoskeleton modification was observed after CAP treatment at 1 d. Further, both CAP-treated and untreated (control) cells remained equally vital as detected by flow cytometry analysis. Interestingly, CAP-associated downregulation of CASP9 and CASP3 mRNA gene expression was also visible after blocking MAP kinase and PI3K. Finally, CAP led to a decrease in CASP3 activity in osteoblast-like cells under normal and apoptotic conditions. CONCLUSIONS: Our in vitro-study demonstrated, that CAP decreases apoptosis related molecules in osteoblast-like cells, underlining a beneficial effect on hard-tissue cells.

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.

CXCL5, CXCL8, and CXCL10 regulation by bacteria and mechanical forces in periodontium.

OBJECTIVE: The aim of the present study was to evaluate the expressions of CXCL5, CXCL8, and CXCL10 in periodontal cells and tissues in response to microbial signals and/or biomechanical forces. METHODS: Human gingival biopsies from inflamed and healthy sites were used to examine the chemokine expressions and protein levels by real-time PCR and immunohistochemistry. The chemokines were also investigated in gingival biopsies from rats submitted to experimental periodontitis and/or tooth movement. Furthermore, chemokine levels were determined in human periodontal fibroblasts stimulated by the periodontopathogen Fusobacterium nucleatum and/or constant tensile forces (CTS) by real-time PCR and ELISA. Additionally, gene expressions were evaluated in periodontal fibroblasts exposed to F. nucleatum and/or CTS in the presence and absence of a MAPK inhibitor by real-time PCR. RESULTS: Increased CXCL5, CXCL8, and CXCL10 levels were observed in human and rat gingiva from sites of inflammation as compared with periodontal health. The rat experimental periodontitis caused a significant (p<0.05) increase in alveolar bone resorption, which was further enhanced when combined with tooth movement. In vitro, F. nucleatum caused a significant upregulation of CXCL5, CXCL8, and CXCL10 at 1 day. Once the cells were exposed simultaneously to F. nucleatum and CTS, the chemokines regulation was significantly enhanced. The transcriptional findings were also observed at protein level. Pre-incubation with the MEK1/2 inhibitor significantly (p<0.05) inhibited the stimulatory actions of F. nucleatum either alone or in combination with CTS on the expression levels of CXCL5, CXCL8, and CXCL10 at 1d. CONCLUSIONS: Our data provide original evidence that biomechanical strain further increases the stimulatory actions of periodontal bacteria on the expressions of these chemokines. Therefore, biomechanical loading in combination with periodontal infection may lead to stronger recruitment of immunoinflammatory cells to the periodontium, which might result in an aggravation of periodontal inflammation and destruction.

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.

Regulation of Autophagic Signaling by Mechanical Loading and Inflammation in Human PDL Fibroblasts.

Autophagy (cellular self-consumption) is a crucial adaptation mechanism during cellular stress conditions. This study aimed to examine how this important process is regulated in human periodontal ligament (PDL) fibroblasts by mechanical and inflammatory stress conditions and whether the mammalian target of rapamycin (mTOR) signaling pathway is involved. Autophagy was quantified by flow cytometry. Qualitative protein phosphorylation profiling of the mTOR pathway was carried out. Effects of mTOR regulation were assessed by quantification of important synthesis product collagen 1, cell proliferation and cell death with real-time PCR and flow cytometry. Autophagy as a response to mechanical or inflammatory treatment in PDL fibroblasts was dose and time dependent. In general, autophagy was induced by stress stimulation. Phosphorylation analysis of mTOR showed regulatory influences of mechanical and inflammatory stimulation on crucial target proteins. Regulation of mTOR was also detectable via changes in protein synthesis and cell proliferation. Physiological pressure had cell-protective effects (p = 0.025), whereas overload increased cell death (p = 0.003), which was also promoted in long-term inflammatory treatment (p < 0.001). Our data provide novel insights about autophagy regulation by mechanical and inflammatory stress conditions in human PDL fibroblasts. Our results suggest some involvement of the mTOR pathway in autophagy and cell fate regulation under the named conditions.

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.

Molecular biology of periodontal ligament fibroblasts and orthodontic tooth movement : Evidence and possible role of the circadian rhythm.

PURPOSE: The circadian clock plays an important role in many physiological states and pathologies. The significance of its core genes in bone formation and tooth development has already been demonstrated. However, regulation of these genes and their influence on periodontal and bone remodeling in periodontal ligament (PDL) fibroblasts remains to be elucidated. Our hypothesis was that the circadian clock influences markers for periodontal and bone remodeling and therefore orthodontic tooth movement itself. MATERIALS AND METHODS: Human PDL fibroblasts were cultured and synchronized in circadian rhythms with the help of a dexamethasone shock. Cells were harvested at 4 h intervals. Reverse transcription and quantitative RT PCR (real time polymerase chain reaction) were performed to assess the mRNA levels of the clock genes ARNTL, CLOCK1, PER1, and PER2. Subsequently, mRNA expression of important marker genes for periodontal and bone remodeling, OPG, RANKL, OCN, OPN, RUNX2, COL1A1, IL1ß, KI67, and POSTN, were examined at time points of ARNTL amplitude expression. RESULTS: Gene expression of core clock genes varied over 48 h in accordance with the circadian rhythm. Functional markers, except KI67, showed significant differences at time points of maximum fluctuation especially of ARNTL. CONCLUSIONS: PDL fibroblasts express circadian clock genes. Our results suggest that genes associated with bone and periodontal remodeling are influenced by the circadian rhythm. Further research will have to refine the understanding of this influence for orthodontic treatment.

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 ghrelin receptor by microbial and inflammatory signals in human osteoblasts.

Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1ß and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1ß and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1ß-induced GHS-R1a upregulation. IL-1ß and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1ß and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1ß and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1ß and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.

Regulation of somatostatin receptor 2 by proinflammatory, microbial and obesity-related signals in periodontal cells and tissues.

BACKGROUND: Periodontitis is a chronic disease characterized by a progressive and irreversible destruction of the tooth-supporting tissues, including gingiva and periodontal ligament (PDL). Microorganisms, such as Fusobacterium nucleatum, evoke an inflammatory host response, which leads to increased levels of inflammatory mediators, such as interleukin (IL)-1ß. Periodontitis has been linked to obesity, and adipokines have been suggested to represent a pathomechanistic link. The hormone somatostatin (SST) exerts antiproliferative, antiangiogenetic, proapoptotic, anti-nociceptive and other effects through binding to its receptors, such as SSTR2. Therefore, the objective of the present study was to examine the regulation of SSTR2 in periodontal cells and tissues under inflammatory, microbial and obesity-related conditions. METHODS: In-vitro, human PDL fibroblasts were exposed to IL-1ß, F. nucleatum, leptin or visfatin. The SSTR2 regulation was assessed by real-time PCR and immunocytochemistry. In-vivo, the SSTR2 expression was analyzed in gingival biopsies of periodontally diseased and healthy subjects by real-time PCR and immunohistochemistry. Additionally, the SSTR2 expression was determined in gingival biopsies of rats with ligature-induced periodontitis, rats with diet-induced obesity, and periodontally and systemically healthy control animals. For statistical analyses, the Mann-Whitney-U test and ANOVA with post-hoc tests were applied (p < 0.05). RESULTS: Exposure of PDL cells to IL-1ß and F. nucleatum caused a significant SSTR2 upregulation by 2.6-fold and 6.4-fold, respectively. Additionally, leptin and visfatin increased significantly the SSTR2 gene expression by 3.0-fold and 2.8-fold, respectively. These stimulatory effects were also observed at protein level. SSTR2 expressions in human gingival biopsies from sites of periodontitis were significantly higher than those in healthy biopsies. Similarly, SSTR2 expression levels were significantly enhanced at periodontally-diseased sites in rat experimental periodontitis. Finally, the SSTR2 expression was significantly upregulated in gingival biopsies of obese rats as compared to normal weight control animals. CONCLUSIONS: Our study provides original insights into the SSTR2 regulation in cells and tissues of the periodontium. We demonstrate for the first time that proinflammatory, microbial and obesity-associated molecules result in an SSTR2 upregulation. Since SST has been shown to be antiproliferative, antiangiogenetic, and proapoptotic, our study suggests that SSTR2 might play a critical role in the aetiopathogenesis of periodontitis.