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.

Non-Invasive Physical Plasma Reduces the Inflammatory Response in Microbially Prestimulated Human Gingival Fibroblasts.

Non-invasive physical plasma (NIPP), an electrically conductive gas, is playing an increasingly important role in medicine due to its antimicrobial and regenerative properties. However, NIPP is not yet well established in dentistry, although it has promising potential, especially for periodontological applications. The aim of the present study was to investigate the effect of NIPP on a commercially available human gingival fibroblast (HGF) cell line and primary HGFs in the presence of periodontitis-associated bacteria. First, primary HGFs from eight patients were characterised by immunofluorescence, and cell numbers were examined by an automatic cell counter over 5 days. Then, HGFs that were preincubated with Fusobacterium nucleatum (F.n.) were treated with NIPP. Afterwards, the IL-6 and IL-8 levels in the cell supernatants were determined by ELISA. In HGFs, F.n. caused a significant increase in IL-6 and IL-8, and this F.n.-induced upregulation of both cytokines was counteracted by NIPP, suggesting a beneficial effect of physical plasma on periodontal cells in a microbial environment. The application of NIPP in periodontal therapy could therefore represent a novel and promising strategy and deserves further investigation.

Influence of cold atmospheric plasma on dental implant materials - an in vitro analysis.

BACKGROUND AND OBJECTIVES: Alterations in the microenvironment of implant surfaces could influence the cellular crosstalk and adhesion patterns of dental implant materials. Cold plasma has been described to have an influence on cells, tissues, and biomaterials. Hence, the mechanisms of osseointegration may be altered by non-thermal plasma treatment depending on different chemical compositions and surface coatings of the biomaterial. The aim of the present study is to investigate the influence of cold atmospheric plasma (CAP) treatment on implant surfaces and its biological and physicochemical side effects. MATERIALS AND METHODS: Dental implant discs from titanium and zirconia with different surface modifications were treated with CAP at various durations. Cell behavior and adhesion patterns of human gingival fibroblast (HGF-1) and osteoblast-like cells (MG-63) were examined using scanning electron microscopy and fluorescence microscopy. Surface chemical characterization was analyzed using energy-dispersive X-ray spectroscopy (EDS). Quantitative analysis of cell adhesion, proliferation, and extracellular matrix formation was conducted including real-time PCR. RESULTS: CAP did not affect the elemental composition of different dental implant materials. Additionally, markers for cell proliferation, extracellular matrix formation, and cell adhesion were differently regulated depending on the application time of CAP treatment in MG-63 cells and gingival fibroblasts. CONCLUSIONS: CAP application is beneficial for dental implant materials to allow for faster proliferation and adhesion of cells from the surrounding tissue on both titanium and zirconia implant surfaces with different surface properties. CLINICAL RELEVANCE: The healing capacity provided through CAP treatment could enhance osseointegration of dental implants and has the potential to serve as an effective treatment option in periimplantitis therapy.

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.

Bevacizumab and sunitinib mediate osteogenic and pro-inflammatory molecular changes in primary human alveolar osteoblasts in vitro.

Antiangiogenic medications target the de novo blood vessel formation in tumorigenesis. However, these novel drugs have been linked to the onset of medication-related osteonecrosis of the jaw (MRONJ). The aim of this in vitro study was to examine the effects of the vascular endothelial growth factor A (VEGFA) antibody bevacizumab (BEV) and the receptor tyrosine kinase inhibitor (RTKI) sunitinib (SUN) on primary human osteoblasts derived from the alveolar bone. Primary human alveolar osteoblasts (HAOBs) were treated with BEV or SUN for 48 h. Cellular metabolic activity was examined by XTT assay. Differentially regulated genes were identified by screening of 22 selected osteogenic and angiogenic markers by quantitative real-time reverse transcriptase polymerase chain reaction (qRT2-PCR). Protein levels of alkaline phosphatase (ALP), collagen type 1, α1 (COL1A1) and secreted protein acidic and cysteine rich (SPARC) were examined by enzyme-linked immunoassay (ELISA). Treatment with BEV and SUN did not exhibit direct cytotoxic effects in HAOBs as confirmed by XTT assay. Of the 22 genes examined by qRT2-PCR, four genes were significantly regulated after BEV treatment and eight genes in the SUN group as compared to the control group. Gene expression levels of ALPL, COL1A1 and SPARC were significantly downregulated by both drugs. Further analysis by ELISA indicated the downregulation of protein levels of ALP, COL1A1 and SPARC in the BEV and SUN groups. The effects of BEV and SUN in HAOBs may be mediated by alterations to osteogenic and catabolic markers. Therapeutic or preventive strategies in MRONJ may address drug-induced depression of osteoblast differentiation.

Effect of Cold Atmospheric Plasma (CAP) on Osteogenic Differentiation Potential of Human Osteoblasts.

Bone regeneration after oral and maxillofacial surgery is a long-term process, which involves various mechanisms. Recently, cold atmospheric plasma (CAP) has become known to accelerate wound healing and have an antimicrobial effect. Since the use of CAP in dentistry is not yet established, the aim of the present study was to investigate the effect of CAP on human calvaria osteoblasts (HCO). HCO were treated with CAP for different durations of time and distances to the cells. Cell proliferation was determined by MTT assay and cell toxicity by LDH assay. Additionally, RT-qPCR was used to investigate effects on osteogenic markers, such as alkaline phosphatase (ALP), bone morphogenic protein (BMP)2, collagen (COL)1A1, osteonectin (SPARC), osteoprotegerin (OPG), osterix (OSX), receptor activator of NF-κB (RANK), RANK Ligand (RANKL), and Runt-related transcription factor (RUNX)2. There were small differences in cell proliferation and LDH release regarding treatment duration and distance to the cells. However, an increase in the expression of RANK and RANKL was observed at longer treatment times. Additionally, CAP caused a significant increase in mRNA expression of genes relevant to osteogenesis. In conclusion, CAP has a stimulating effect on osteoblasts and may thus represent a potential therapeutic approach in the regeneration of hard tissue defects.

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.

Cold Atmospheric Plasma Promotes Regeneration-Associated Cell Functions of Murine Cementoblasts In Vitro.

The aim of the study was to examine the efficacy of cold atmospheric plasma (CAP) on the mineralization and cell proliferation of murine dental cementoblasts. Cells were treated with CAP and enamel matrix derivates (EMD). Gene expression of alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (BGLAP), periostin (POSTN), osteopontin (OPN), osterix (OSX), collagen type I alpha 1 chain (COL1A1), dentin matrix acidic phosphoprotein (DMP)1, RUNX family transcription factor (RUNX)2, and marker of proliferation Ki-67 (KI67) was quantified by real-time PCR. Protein expression was analyzed by immunocytochemistry and ELISA. ALP activity was determined by ALP assay. Von Kossa and alizarin red staining were used to display mineralization. Cell viability was analyzed by XTT assay, and morphological characterization was performed by DAPI/phalloidin staining. Cell migration was quantified with an established scratch assay. CAP and EMD upregulated both mRNA and protein synthesis of ALP, POSTN, and OPN. Additionally, DMP1 and COL1A1 were upregulated at both gene and protein levels. In addition to upregulated RUNX2 mRNA levels, treated cells mineralized more intensively. Moreover, CAP treatment resulted in an upregulation of KI67, higher cell viability, and improved cell migration. Our study shows that CAP appears to have stimulatory effects on regeneration-associated cell functions in cementoblasts.

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.

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.

Regulation of Cyclooxygenase 2 by Filifactor alocis in Fibroblastic and Monocytic Cells.

Periodontitis is a prevalent chronic inflammatory disease triggered by a synergistic and dysbiotic microbiota present in the oral biofilm. This in vitro study is aimed at evaluating the regulation of cyclooxygenase (COX)2 expression and production by the periodontopathogen Filifactor alocis in human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells and also at investigating the underlying cellular pathway mechanisms. HGF-1 and THP-1 cells were exposed either to F. alocis or to the proinflammatory cytokine tumor necrosis factor alpha (TNFα) for 1 and 2 d to examine the COX2 expression by qPCR. COX2 protein levels were evaluated by ELISA in F. alocis-stimulated cells. Both types of cells were also stimulated with a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. F. alocis significantly (p < 0.05) increased COX2 at both transcriptional and protein levels in both HGF-1 and THP-1 cells. Moreover, the stimulatory effect of F. alocis on COX2 was more pronounced in HGF-1 cells in comparison to THP-1 cells. F. alocis upregulated the COX2 expression in a dose-dependent manner in both type cells at 1 d. TNFα also significantly (p < 0.05) increased the COX2 expression in both cells. After preincubation of HGF-1 and THP-1 cells either with a neutralizing anti-TLR2 antibody or with specific MAPK inhibitors, the F. alocis-upregulated COX2 expression was significantly (p < 0.05) suppressed at 1 d. Our in vitro study provides original evidence that F. alocis stimulates COX2 production in fibroblastic and monocytic cells through TLR2 and MAPK mechanisms, suggesting a role of this periodontopathogen in the etiopathogenesis of periodontitis.

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.

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.

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.