Svenja Memmert

LPS from P. gingivalis and Hypoxia Increases Oxidative Stress in Periodontal Ligament Fibroblasts and Contributes to Periodontitis

Oxidative stress is characterized by an accumulation of reactive oxygen species (ROS) and plays a key role in the progression of inflammatory diseases. We hypothesize that hypoxic and inflammatory events induce oxidative stress in the periodontal ligament (PDL) by activating NOX4. Human primary PDL fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (LPS-PG), a periodontal pathogen bacterium under normoxic and hypoxic conditions. By quantitative PCR, immunoblot, immunostaining, and a specific ROS assay we determined the amount of NOX4, ROS, and several redox systems. Healthy and inflamed periodontal tissues were collected to evaluate NOX4 and redox systems by immunohistochemistry. We found significantly increased NOX4 levels after hypoxic or inflammatory stimulation in PDL cells (P < 0.001) which was even more pronounced after combination of the stimuli. This was accompanied by a significant upregulation of ROS and catalase (P < 0.001). However, prolonged incubation with both stimuli induced a reduction of catalase indicating a collapse of the protective machinery favoring ROS increase and the progression of inflammatory oral diseases. Analysis of inflamed tissues confirmed our hypothesis. In conclusion, we demonstrated that the interplay of NOX4 and redox systems is crucial for ROS formation which plays a pivotal role during oral diseases.

Hypoxia and P. gingivalis Synergistically Induce HIF-1 and NF-κB Activation in PDL Cells and Periodontal Diseases

Periodontitis is characterized by deep periodontal pockets favoring the proliferation of anaerobic bacteria like Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen frequently observed in patients suffering from periodontal inflammation. Therefore, the aim of the present study was to investigate the signaling pathways activated by lipopolysaccharide (LPS) of P. gingivalis (LPS-PG) and hypoxia in periodontal ligament (PDL) cells. The relevant transcription factors nuclear factor-kappa B (NF-κB) and hypoxia inducible factor-1 (HIF-1) were determined. In addition, we analyzed the expression of interleukin- (IL-) 1β, matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) in PDL cells on mRNA and protein level. This was accomplished by immunohistochemistry of healthy and inflamed periodontal tissues. We detected time-dependent additive effects of LPS-PG and hypoxia on NF-κB and HIF-1α activation in PDL cells followed by an upregulation of IL-1β, MMP-1, and VEGF expression. Immunohistochemistry performed on tissue samples of gingivitis and periodontitis displayed an increase of NF-κB, HIF-1, and VEGF immunoreactivity in accordance with disease progression validating the importance of the in vitro results. To conclude, the present study underlines the significance of NF-κB and HIF-1α and their target genes VEGF, IL-1β, and MMP-1 in P. gingivalis and hypoxia induced periodontal inflammatory processes.

Role of Cathepsin S in Periodontal Inflammation and Infection

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1β and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1β and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

Effectiveness of early orthopaedic treatment with headgear: a systematic review and meta-analysis

Background Although the headgear appliance has been used extensively to correct anteroposterior discrepancies, its treatment effects have not yet been adequately assessed in an evidence-based manner. Objective Aim of this systematic review was to assess the therapeutic and adverse effects of early headgear treatment from controlled clinical trials on human patients in an evidence-based manner. Search methods An unrestricted electronic search of six databases from inception to December 2015. Selection criteria Randomized and prospective non-randomized controlled trials assessing the effects of headgear treatment on human patients. Data collection and analysis After duplicate study selection, data extraction, and risk of bias assessment according to the Cochrane guidelines, random-effects meta-analyses of mean differences (MDs) and relative risks (RRs), including their 95% confidence intervals (CIs) were performed, followed by subgroup and sensitivity analyses. Results A total of 18 unique studies with a total of 930 (56% male/44% female) patients were included. Headgear treatment was associated with a posterior translation of the anterior maxilla border in the short term, as seen by the mean annualized change in the SNA angle (MD = -1.63°/year; 95% CI = -2.20 to -1.06°/year; high quality evidence) compared to untreated patients. This effect was independent of the rotation of the palatal plane and the inclination of the upper incisors, while a proportional relationship with the initial discrepancy in SNA was seen. The clinical significance of this improvement diminished in the long term, although only limited evidence existed. Additionally, early headgear treatment might decrease the risk of dental trauma during the following years (RR = 0.34; 95% CI = 0.14 to 0.80; moderate quality evidence). Low quality evidence on the effect of headgear on the rotation of the palatal plane, the nasolabial angle, the occlusal outcome, and signs of temporomandibular disorders precluded robust assessments, due to risk of bias, inconsistency, imprecision, and small-study effects. Conclusions Based on existing trials, headgear is a viable treatment option to modify sagittal growth of the maxilla in the short term in Class II patients with maxillary prognathism. Registration PROSPERO (CRD42015029837). Funding None.

Impact of obesity and aging on crestal alveolar bone height in mice

Obesity and aging are associated with periodontitis, which represents a chronic inflammatory disease of the tooth-supporting tissues, i.e. the periodontium. However, if both risk factors also have a negative impact on crestal alveolar bone in a clinically healthy periodontium, has yet to be elucidated and was analyzed in this in-vivo study. Eight C57BL/6 mice were fed a normal diet during the entire study. Half of these mice were sacrificed at week 19 (group 1: younger lean mice), whereas the other half of the animals were sacrificed at week 25 (group 2: older lean mice). In addition, four mice were fed a high-fat diet until their sacrifice at week 19 (group 3: younger obese mice). Mandibles and maxillae were scanned by micro-computed tomography and, subsequently, the distance between the cementoenamel junction and alveolar bone crest (CEJ-ABC) at all molars was determined. Levels of interleukin-6, cyclooxygenase-2, visfatin and adiponectin in gingival samples were quantified by real-time PCR. For statistical analyses, the Mann-Whitney-U test was applied (p<0.05). As compared to lean mice, obese animals presented a significantly increased CEJ-ABC distance, i.e. reduced alveolar bone crest height, at week 19. The alveolar bone loss was mainly found at the first molars of the mandibles. In animals fed a normal diet, the alveolar bone crest height in the mandibles and maxillae was significantly lower in the older mice as compared to the younger animals. Furthermore, gingival cyclooxygenase-2 and visfatin expressions were higher in the obese versus lean mice and in the older versus younger mice. This in-vivo investigation shows that obesity and older age can result in reduced alveolar bone crest height and suggests that they represent risk factors even in a clinically healthy periodontium.

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.

Effect of interleukin-1β on ghrelin receptor in periodontal cells

ObjectivesPeriodontopathogens induce immunoinflammatory responses characterized by the release of inflammatory mediators, e.g., interleukin (IL)-1β, 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 methodsPeriodontal ligament (PDL) cells and gingival fibroblasts (HGFs) were exposed to IL-1β 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.ResultsIL-1β increased significantly the GHS-R1a expression in both cell types in a dose-dependent manner. The stimulatory effect of IL-1β involved the NF-κB and MAPK pathways. Exposure of cells to IL-1β also resulted in an increased production of GHS-R1 protein in both cell types. Furthermore, GHRL counteracted significantly the stimulatory actions of IL-1β on IL-6 and IL-8 in PDL cells.ConclusionsThis study demonstrates for the first time that IL-1β 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 relevanceThe 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

ObjectivesObesity 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 methodsTen 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).ResultsBody 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.ConclusionsAlthough 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 relevanceThe 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.