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.

Antimicrobial Efficiency of Pistacia lentiscus L. Derivates against Oral Biofilm-Associated Diseases-A Narrative Review.

Pistacia lentiscus L. (PlL) has been used for centuries in traditional medicine. The richness in antimicrobial biomolecules of Pll derivates can represent an alternative to chemically formulated agents used against oral infections. This review summarizes the knowledge on the antimicrobial activity of PlL essential oil (EO), extracts, and mastic resin against microorganisms being of relevance in oral biofilm-associated diseases. Results demonstrated that the potential of PlL polyphenol extracts has led to increasing scientific interest. In fact, the extracts are a significantly more effective agent than the other PlL derivates. The positive findings regarding the inhibition of periodontal pathogens and C. albicans, together with the antioxidant activity and the reduction of the inflammatory responses, suggest the use of the extracts in the prevention and/or reversal of intraoral dysbiosis. Toothpaste, mouthwashes, and local delivery devices could be effective in the clinical management of these oral diseases.

Salivary Pellicle Modification with Grape-seed Extract: In Vitro Study on the Effect on Bacterial Adhesion and Biofilm Formation.

PURPOSE: Grape-seed extract (GSE) contains polyphenols that readily adhere to proteins and modify the acquired enamel pellicle (AEP). The first step in biofilm formation is bacterial adhesion to the AEP-covered enamel. The aim of this in vitro study was to test whether AEP modification with GSE, fluoride (F-), or their combination (GSE+F-) modulates bacterial adhesion, biofilm metabolism and composition, or cariogenic demineralisation of the enamel. MATERIALS AND METHODS: The study comprised 3 parts: 1) single-strain Streptococcus gordonii species, 2) a five-species biofilm model, or 3) biofilm (re-)formation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Human whole-mouth stimulated saliva was used to form an AEP on human enamel specimens. The AEP was incubated in water (control), or modified with GSE, F-, or GSE+F-. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and cariogenic demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 4 h or 22 h. Differences between groups were analysed with one-way ANOVA and post-hoc Bonferroni tests. RESULTS: GSE and GSE+F- statistically significantly decreased single-strain S. gordonii adhesion, but had no relevant influence when the five-species biofilm model was used. In the biofilm (re-)formation model, GSE reduced bacterial adhesion compared to GSE+F-, while F- caused less cariogenic demineralisation than was found in the control group. CONCLUSION: AEP modified with GSE retards S. gordonii adhesion, but it does not influence the formation, metabolism and composition of a cariogenic multi-species biofilm.

Leaves and Fruits Preparations of Pistacia lentiscus L.: A Review on the Ethnopharmacological Uses and Implications in Inflammation and Infection.

There is an increasing interest in revisiting plants for drug discovery, proving scientifically their role as remedies. The aim of this review was to give an overview of the ethnopharmacological uses of Pistacia lentiscus L. (PlL) leaves and fruits, expanding the search for the scientific discovery of their chemistry, anti-inflammatory, antioxidative and antimicrobial activities. PlL is a wild-growing shrub rich in terpenoids and polyphenols, the oil and extracts of which have been widely used against inflammation and infections, and as wound healing agents. The more recurrent components in PlL essential oil (EO) are represented by α-pinene, terpinene, caryophyllene, limonene and myrcene, with high variability in concentration depending on the Mediterranean country. The anti-inflammatory activity of the oil mainly occurs due to the inhibition of pro-inflammatory cytokines and the arachidonic acid cascade. Interestingly, the capacity against COX-2 and LOX indicates PlL EO as a dual inhibitory compound. The high content of polyphenols enriching the extracts provide explanations for the known biological properties of the plant. The protective effect against reactive oxygen species is of wide interest. In particular, their anthocyanins content greatly clarifies their antioxidative capacity. Further, the antimicrobial activity of PlL oil and extracts includes the inhibition of Staphylococcus aureus, Escherichia coli, periodontal bacteria and Candida spp. In conclusion, the relevant scientific properties indicate PlL as a nutraceutical and also as a therapeutic agent against a wide range of diseases based on inflammation and infections.

Enamel matrix derivative and subgingival reinstrumentation: a pilot study.

OBJECTIVES: To verify the effect of adjunctive enamel matrix derivative (EMD) in subgingival reinstrumentation during supportive periodontal therapy. METHOD AND MATERIALS: Using a split-mouth design, residual periodontal pockets with probing depth (PD) of 5 to 8 mm in 13 patients were treated by subgingival reinstrumentation with (test teeth) and without (control teeth) EMD. At baseline and after 6 and 12 months the clinical variables PD, clinical attachment level (CAL), and bleeding on probing (BOP) were recorded. At the same appointments gingival crevicular fluid (GCF) was collected to analyze for interleukin (IL)-1ß, matrix metalloproteinase (MMP)-8, IL-10, and transforming growth factor (TGF)-ß. RESULTS: Statistically significant improvements in PD, CAL, and BOP occurred in both groups. The reduction of PD was significantly higher in the test group than in the control group after 12 months (P = .005). The change of IL-1ß within 12 months was significantly different between both groups (P = .019). No other significant differences were detected between both groups. CONCLUSION: The study suggests that subgingival reinstrumentation with adjunctive EMD could additionally reduce probing pocket depth and the need for periodontal surgery. (Quintessence Int 2021;52:506-513; doi: 10.3290/j.qi.b1044079).

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.

Peri-Implant Diseases: Characteristics of the Microbiota and of the Host Response in Humans - A Narrative Review.

The present narrative review provides a summary of the temporal and spatial reactions of the oral microbiome to the placement of a dental implant into the oral cavity, depicting the most important interactions between the oral microbiota and the host response involved in the development of peri-implant infections in humans (i.e., peri-implant mucositis and peri-implantitis). Starting with the formation of a pellicle to acute and rampant peri-implant inflammation, a number of steps, including biofilm formation, aggressive bacterial invasion, and host defense mechanisms, are involved. Better understanding of the factors related to the host response and changes in the composition of microbiota has led to the development of novel treatment modalities. Finally, a short outlook into the future is provided.

Biofilms.

In reality, most microorganisms are not free floating. They exist in biofilms, a community of many of them from the same species or from other genera and attached to surfaces.Microorganisms undergo a transition from free-floating, planktonic microorganisms to a sessile, surface-attached one. Contact with a surface induces changes in gene expression, and a strong attachment of microcolonies occurs only after a few hours. The maturation of a biofilm is associated with matrix formation. The matrix is of importance as it provides stability and protects against environmental insults, it consists of polysaccharides, water, lipids, proteins, and extracellular DNA. Biofilms can be found everywhere - in the environment, in water systems - and they play an important role in medicine and dentistry. In medicine, infections of chronic wounds, of the respiratory tract in cystic fibrosis infections, or when linked with incorporated biomaterial are mostly biofilm associated. In the oral cavity, the most prevalent oral diseases, dental caries, and periodontitis are multi-species biofilm-associated diseases. Although not acting alone, key pathogens drive the development of the microbial shift. Microorganisms metabolize sugar and create an acidic environment where aciduric bacteria (including mutans streptococci) become dominant, which leads to the demineralization of enamel and dentine. Porphyromonas gingivaliscauses biofilm dysbiosis in the development of periodontal disease. Biofilm-associated infections are extremely difficult to treat. The matrix serves as a barrier to antimicrobial agents and there are subpopulations of dormant bacteria resistant to antimicrobials requiring metabolically active cells. Approaches to treat biofilm-associated infections include the modification of the biofilm composition, inhibitors of quorum-sensing molecules, or interfering with matrix constituents.

Clinical outcomes following periodontal surgery and root surface decontamination by erythritol-based air polishing. A randomized, controlled, clinical pilot study.

AIM: To evaluate the outcomes following surgical periodontal treatment and root surface decontamination by means of air polishing using an erythritol powder or conventional mechanical root debridement. MATERIAL AND METHODS: Thirty systemically healthy patients (44.38 ± 8.2 years old, 11 smokers, 19 women) diagnosed with periodontitis stages III-IV were included. Each patient, with one single-rooted tooth, with one probing pocket depth (PD) ≥ 6 mm associated with horizontal bone loss, was treated by means of simplified papilla preservation flap (SPPF) and randomized to either test treatment (careful removal of the calculus with the tip of a blade, air polishing of the root surfaces with erythritol) or to the control group (scaling and root planing with hand curettes, ultrasonic instruments). PD, clinical attachment (CAL), bone sounding (BS), and radiographic bone level (BL) were evaluated at baseline and 12 months postsurgically. RESULTS: Twenty-seven patients completed the 12-month follow-up (test: n = 14, control: n = 13). In both groups, statistically significant improvements were obtained (p < 0.05, mean CAL gain/PD reduction: test, 2.50 ± 1.60 mm/3.00 ± 0.96 mm; control, 2.85 ± 1.21 mm/3.38 ± 1.12 mm). No statistically significant differences were observed between the groups for any of the investigated parameters (p < 0.05). CONCLUSION: Within their limits, the present results indicate that the use of air polishing with an erythritol powder during periodontal surgery may represent a valuable minimally invasive adjunct following calculus removal by means of hand and ultrasonic instruments or a valuable alternative to these, for root surfaces without calculus. CLINICAL RELEVANCE: The use of air polishing with an erythritol powder during periodontal surgery appears to represent a valuable minimally invasive adjunct following calculus removal by means of hand and ultrasonic instruments or a valuable alternative to these, for root surfaces without calculus.

Adjunctive air-polishing with erythritol in nonsurgical periodontal therapy: a randomized clinical trial.

BACKGROUND: This study was aimed to investigate if the adjunctive use of erythritol air-polishing powder applied with the nozzle-system during subgingival instrumentation (SI) has an effect on the outcome of non-surgical periodontal treatment in patients with moderate to severe periodontitis. METHODS: Fourty-two individuals with periodontitis received nonsurgical periodontal therapy by SI without (controls, n = 21) and with adjunctive air-polishing using nozzle + erythritol powder (test, n = 21). They were analyzed for the clinical variables BOP (primary outcome at six months), probing depth (PD), attachment level, four selected microorganisms and two biomarkers at baseline, before SI as well as three and six months after SI. Statistical analysis included nonparametric tests for intra- and intergroup comparisons. RESULTS: In both groups, the clinical variables PD, attachment level and BOP significantly improved three and six months after SI. The number of sites with PD ≥ 5 mm was significantly lower in the test group than in the control group after six months. At six months versus baseline, there were significant reductions of Tannerella forsythia and Treponema denticola counts as well as lower levels of MMP-8 in the test group. CONCLUSIONS: Subgingival instrumentation with adjunctive erythritol air-polishing powder does not reduce BOP. But it may add beneficial effects like reducing the probing depth measured as number of residual periodontal pocket with PD ≥ 5 mm when compared with subgingival instrumentation only. CLINICAL RELEVANCE: The adjunctive use of erythritol air-polishing powder applied with the nozzle-system during SI may improve the clinical outcome of SI and may reduce the need for periodontal surgery. Trial registration The study was retrospectively registered in the German register of clinical trials, DRKS00015239 on 6th August 2018, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL .

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.

The Pharmaceutical Ability of Pistacia lentiscus L. Leaves Essential Oil Against Periodontal Bacteria and Candida sp. and Its Anti-Inflammatory Potential.

BACKGROUND: Given the increasing request for natural pharmacological molecules, this study assessed the antimicrobial capacity of Pistacia lentiscus L. essential oil (PLL-EO) obtained from the leaves of wild plants growing in North Sardinia (Italy) toward a wide range of periodontal bacteria and Candida, including laboratory and clinical isolates sp., together with its anti-inflammatory activity and safety. METHODS: PLL-EO was screened by gas chromatography/mass spectrometry. The minimal inhibitory concentration (MIC) was determined. The anti-inflammatory activity was measured by cyclooxygenase (COX-1/2) and lipoxygenase (LOX) inhibition, while the antioxidant capacity was determined electro-chemically and by the MTT assay. The WST-1 assay was used to ascertain cytotoxicity toward four lines of oral cells. RESULTS: According to the concentrations of terpens, PLL-EO is a pharmacologically-active phytocomplex. MICs against periodontal bacteria ranged between 3.13 and 12.5 µg/ml, while against Candida sp. they were between 6.25 and 12.5 µg/mL. Oxidation by COX-1/2 and LOX was inhibited by 80% and 20% µg/mL of the oil, respectively. Antioxidant activity seemed negligible, and no cytotoxicity arose. CONCLUSIONS: PLL-EO exhibits a broad-spectrum activity against periodontal bacteria and Candida, with an interesting dual inhibitory capacity toward COX-2 and LOX inflammatory enzymes, and without side effects against oral cells.

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 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.

Effect of enamel matrix derivative on wound healing following gingival recession coverage using the modified coronally advanced tunnel and subepithelial connective tissue graft: a randomised, controlled, clinical study.

OBJECTIVES: The potential effect of enamel matrix derivative (EMD) on wound healing following recession coverage surgery is still controversially discussed in the literature. The aim of this randomised, controlled, single blinded clinical study was, therefore, to investigate clinically and immunologically the potential effects of EMD on early wound healing and clinical results following treatment of single and multiple gingival recessions by the modified coronally advanced tunnel technique (MCAT) and subepithelial connective tissue graft (sCTG). MATERIALS AND METHODS: A total of 40 systemically healthy patients with Miller class I, II or III single or multiple gingival recessions were treated with MCAT + sCTG with or without EMD. Patients were consecutively enrolled and randomly assigned to test or control treatment. Inflammatory markers (interleukin (IL)-1ß, IL-8, IL-10 and matrix metalloprotease (MMP)-8) were measured at baseline, 2 days and 1 week postoperatively. The following clinical parameters were assessed at baseline and at 6 months postoperatively: Recession Depth (RD), Recession Width (RW), Width of Keratinized Tissue (KT) and Probing Depth (PD). Patient-reported outcomes were analysed by means of a visual analogue scale. RESULTS: No statistically significant differences were detected between the 2 groups in terms of inflammatory markers and patient-reported outcomes during early wound healing. In the test group, RD was reduced from 4.0 ± 1.2 mm at baseline to 0.9 ± 1.3 mm at 6 months (p < 0.001), while the corresponding values in the control group were 4.5 ± 2.0 mm at baseline and 1.0 ± 1.0 mm at 6 months, respectively. At 6 months, mean root coverage measured 78 ± 26% in the test group and 77 ± 18% in the control group, respectively. CONCLUSION: Within their limits, the present data have failed to show an influence of EMD on the clinical and immunological parameters related to wound healing following recession coverage surgery using MCAT and sCTG. CLINICAL RELEVANCE: Early wound healing following recession coverage by means of MCAT and sCTG does not seem to be influenced by the additional application of EMD.

Bacterial Contamination of ePTFE Membranes Following Regenerative Surgery of Intrabony Defects Treated with Platelet-rich Plasma and Natural Bone Mineral.

PURPOSE: The rationale of using platelet-rich plasma (PRP) in reconstructive periodontal surgery is to amplify or accelerate the wound healing through the growth factors contained in platelets. On the other hand, bacterial colonisation of membranes may negatively affect the healing process. The aim of this study was to evaluate bacterial contamination of non-bio-resorbable membranes (ePTFE) used for regenerative periodontal therapy of intrabony defects and the clinical attachment level (CAL) gain with or without PRP. MATERIALS AND METHODS: Seventeen patients were treated with a natural bone mineral (NBM) and guided tissue regeneration (GTR) with an ePTFE membrane (NBM + GTR group; ie, control group), while in another 17 patients PRP was additionally applied (NBM + PRP + GTR group; ie, test group). Furthermore, the retrieved membranes were analysed for the presence of periodontopathogens and data were related to the gain of clinical attachment. In addition, the in vitro sensitivity of selected microbes to PRP was checked by using agar diffusion test. RESULTS: Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were not detected in the PRP group whereas in the controls A. actinomycetemcomitans was detected in five patients (p = 0.022) and P. gingivalis in two cases (difference not statistically significant, p = 0.242). Detection of A. actinomycetemcomitans was not associated with less CAL gain. If the samples were positively tested for Prevotella intermedia/nigrescens and/or P. gingivalis, the CAL gains were lower compared with the negative samples (p = 0.002). PRP did not show any inhibitory effect on bacterial growth in vitro. CONCLUSION: Within their limits, the present results appear to suggest that the presence of P. intermedia/nigrescens and/or P. gingivalis at the regenerated site may negatively influence the clinical outcomes. However, the potential influence of PRP on bacterial colonisation and the impact on the clinical outcome is still unclear and remains to be elucidated.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

In Vitro Effect of Er:YAG Laser on Different Single and Mixed Microorganisms Being Associated with Endodontic Infections.

Objective: The purpose of this in vitro study was to evaluate the antimicrobial effect of activated irrigation with different modes of erbium-doped yttrium aluminum garnet (Er:YAG) laser application on microorganisms related to secondary endodontic infection. Background: Er:YAG laser has been recommended as an adjuvant tool for root canal disinfection during endodontic treatment. Materials and methods: Laser-activated irrigation (LAI) with 300 or 600 µm tips were tested with or without intermittent irrigation with 0.9% sodium chloride (NaCl) solution against different microorganisms (five single strains and dual species (Streptococcus gordonii combined with Actinomyces oris or Fusobacterium nucleatum) in root canals after 3 days of incubation. In a 21-day infection model, LAI was used together with intermittent rinsing with sodium hypochlorite (NaOCl) against the dual-species mixtures; here the incidence of microbial regrowth after up to 7 days was monitored. Results: In the 3-day root infection model, LAI protocols did not show any significant reduction of the microbial load when compared with manual irrigation with saline solution. In the 21-day infection, S. gordonii combined with A. oris were not detectable anymore after applying the LAI protocol with a 600 µm tip (30 mJ/10 pps) up to 7 days after treatment. Conclusions: Application of LAI with a 600 µm tip by using an Er:YAG laser might be advantageous in treatment of endodontic infections.

Gingipains impair attachment of epithelial cell to dental titanium abutment surfaces.

The study investigated in vitro the effect of Porphyromonas gingivalis and its cysteine proteases (gingipains) on epithelial cell adhesion to titanium-zirconium alloy surfaces. Titanium-zirconium discs with a standard machined (M) or chemically modified hydrophilic surface (modM) were coated with lamin-5 and incubated with telomerase-inactivated gingival keratinocytes (TIGK). Three P. gingivalis strains or gingipains were either added simultaneously with TIGK or after TIGK cells were already attached to the disks. Adhered TIGK cells were counted at 24 h. All P. gingivalis strains clearly inhibited adhesion of TIGK cells to M and modM surfaces. Compared with bacteria/gingipain-free TIGK cell cultures, the number of attached TIGK cells was reduced by about 80% and 60% when P. gingivalis was added simultaneously or after TIGK cells were already attached to the disks (each p < 0.01), respectively. Counts of attached cells were similarly reduced when only gingipains were used. Adhesion molecules of TIGK cells, in particular E-cadherin, were cleaved by P. gingivalis. In conclusion, P. gingivalis and gingipains interfere with the adhesion of epithelial cells to titanium-zirconium alloy surfaces by cleaving adhesion molecules, while a chemically modified hydrophilic titanium-zirconium alloy surface did not yield any protection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2549-2556, 2019.

Regulation of ghrelin receptor by microbial and inflammatory signals in human osteoblasts

Abstract: 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)-1β 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-1β 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-1β-induced GHS-R1a upregulation. IL-1β 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-1β and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1β 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-1β 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.