Combination of enamel matrix derivative and hyaluronic acid inhibits lipopolysaccharide-induced inflammatory response on human epithelial and bone cells.

OBJECTIVES: The aim of this study was to evaluate the in vitro effect of enamel matrix derivative (EMD) and hyaluronic acid (HA) and their synergistic combination on lipopolysaccharides (LPS)-induced inflammation in human keratinocytes and osteoblasts. MATERIAL AND METHODS: Cells were challenged with LPS (1 µg/ml) and cultured in the following treatment groups with EMD (30 mg/ml) and HA (30 mg/ml): LPS, EMD, HA, EMD + HA, EMD + LPS, HA + LPS, and EMD + HA + LPS. Cell viability, inflammatory cytokine expression, and cell migration were determined using colorimetric assay, quantitative real-time polymerase chain reaction (qPCR), and scratch wound healing assay, respectively. RESULTS: Cell viability was decreased when exposed to LPS compared to the controls. Overall, LPS treatment expressed upregulation on inflammatory cytokine tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6). EMD and HA reduced up to 3.0-fold the cytokine expression caused by LPS (p < 0.05). EMD and HA statistically induced higher migration in osteoblasts and keratinocytes, respectively. Migration was impaired by LPS, whereas it significantly increased after addition of EMD and HA. CONCLUSIONS: EMD and HA are advantageous biomaterials that individually generate strong directional migratory keratinocyte and osteoblast response. Their combination also enhances cell viability, and anti-inflammatory and migratory abilities to promote healing specially under LPS inflammatory stimulus. Future in vivo and animal research is necessary to further characterize the effect of EMD and HA on periodontal regeneration. CLINICAL RELEVANCE: The use of EMD in conjunction with HA resulted in a reduction of inflammation and improvement of tissue healing at wound sites. Both biomaterials combined may potentially improve the effectiveness of bone regeneration in periodontal bone defects, pointing to the potential clinical relevance of both materials in regenerative periodontal surgery.

Evaluation of air polishing with a sterile powder and mechanical debridement during regenerative surgical periimplantitis treatment: a study in dogs.

OBJECTIVES: To evaluate the effectiveness of mechanical debridement and/or air polishing on the healing of ligature-induced buccal periimplantitis dehiscence defects in dogs. MATERIAL AND METHODS: Forty-eight implants were placed in the mandibles of twelve beagle dogs, and periimplantitis was induced for 2 months using ligatures. The resulting buccal dehiscence-type defects were surgically cleaned and augmented (xenogenic filler and resorbable membrane) according to one of the following treatments: (1) Cleaning with carbon curette (debridement - D) and guided bone regeneration (GBR/G): DG, (2) air polishing cleaning (A) and GBR: AG, (3) a combination of D/A/G: DAG, and (4) D/A without GBR: DA. After 2 months, histomorphometric and inflammatory evaluations were conducted. RESULTS: The median bone gain after therapy ranged between 1.2 mm (DG) and 2.7 mm (AG). Relative bone gain was between 39% (DG) and 59% (AG). The lowest inflammation scores were obtained in DA without GBR (5.84), whereas significantly higher values between 8.2 and 9.4 were found in the groups with augmentation. At lingual sites without defects, scores ranged from 4.1 to 5.9. According to ISO, differences above 2.9 were considered representative for irritative properties. CONCLUSIONS: All treatments resulted in partial regeneration of the defects. No treatment group showed a significantly (p < 0.05) better outcome. However, pretreatment with air polishing showed a tendency for less inflammation. Noteworthy, inflammation assessment showed an overall irritative potential after GBR in the evaluated early healing phase. CLINICAL RELEVANCE: Periimplantitis treatment still represents a big issue in daily practice and requires additional preclinical research in order to improve treatment concepts.

Origin of MMP-8 and Lactoferrin levels from gingival crevicular fluid, salivary glands and whole saliva.

BACKGROUND: Pathologically elevated levels of matrix metalloproteinase-8 (MMP-8) and Lactoferrin in oral fluids have been associated with the presence of gingivitis/periodontitis. This study aimed to assess the origin of MMP-8 and Lactoferrin in periodontitis patients and to identify the degree to which conventional clinical parameters correlate with their presence. METHODS: A total of ten periodontitis and ten healthy patients were included in this study. Whole saliva (stimulated and unstimulated), parotid/sublingual glandular fluid and gingival crevicular fluid from pockets and sulci were tested for MMP-8 and Lactoferrin and protein concentrations were quantified using an ELISA assay. Clinical parameters were checked for potential associations with MMP-8 and Lactoferrin levels. RESULTS: Periodontal patients presented higher concentrations of MMP-8 and Lactoferrin in pockets than other sources (P = 0.03). Lactoferrin measurement was higher in the parotid compared to sublingual glandular fluid in periodontitis patients (P = 0.03). Increased probing pocket depth was positively correlated with high MMP-8 and Lactoferrin levels. CONCLUSIONS: Periodontal pockets appear to be the major source of active matrix metalloproteinase and Lactoferrin, which also may also enter the oral cavity through the salivary glands. Since clinically healthy sites in periodontitis patients also had elevated biomarker levels, gingival crevicular fluid biomarker testing may be more predictive of future tissue breakdown than conventional clinical parameters.

Dose-dependent green tea effect on decrease of inflammation in human oral gingival epithelial keratinocytes: in vitro study.

OBJECTIVES: This in vitro study aimed to analyze the anti-inflammatory and wound healing potential of green tea extract (GTE) in human gingival epithelial keratinocytes (HGEK) treated with lipopolysaccharides (LPS). MATERIALS AND METHODS: A cell viability assay was conducted using MTT to determine nontoxic levels of GTE on immortalized HGEK. Cells were concomitantly treated with LPS (1 µg/ml) and GTE (1 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml) to assess inflammation. Gene expression levels of inflammatory markers IL-ß1, IL-6, and TNFα were measured by RT-PCR and their protein production was assessed by ELISA. The scratch wound healing assay was used to investigate the effects of different concentrations of GTE on cell migration. We also explored the effect of GTE on the induction of the Nrf2/HO-1 pathway in the cells with or without LPS. RESULTS: GTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml significantly enhanced cell viability (p < 0.05). And IL-ß1, IL-6, and TNFα gene expression presented up to 10-fold decrease compared with LPS-treated cells, which was also similarly found on the protein levels. At the same concentrations, cell migration increased. CONCLUSIONS: The mechanism results showed that GTE produced the anti-inflammatory response by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and increasing the level of anti-oxidant protein heme oxygenase-1 (HO-1). CLINICAL RELEVANCE: GTE may be potentially used as oral rinse anti-inflammatory drug for treatment and prevention of oral inflammatory diseases, which is shown here by the ability to reduce the inflammation and increase in cell migration in a dose-dependent manner.

Bacterial supernatants elevate glucose-dependent insulin secretion in rat pancreatic INS-1 line and islet ß-cells via PI3K/AKT signaling.

Diabetes and periodontitis are considered associated chronic diseases, and hyperinsulinemia in prediabetes has been shown to be present in normoglycemic animals with periodontitis. As periodontal bacterial species are significant sources of endotoxemia and may directly stimulate insulin secretion, we hypothesized that increased bacterial virulence may exert an adverse effect on rat pancreatic ß-cell function via PI3K/AKT signaling. INS-1 cells and isolated pancreatic islets were cultured separately with the following supernatants: Streptococcus anginosus, Streptococcus mutans, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis (P.g), and Treponema denticola (T.d). Supernatants were purified from single bacterial cultures and prepared at different dilutions (100 pg/ml, 50 ng/ml, 200 ng/ml, and 500 ng/ml) to challenge INS-1 and islets. Gene expression (IL-1ß, TNFα, IL-6, TLR2, TLR4, Ins1, and Ins2) and insulin secretion were measured. The results showed upregulation of gene expression up to 5.5-fold, not only as a result of the different dilutions used, but also due to bacterial virulence (p < 0.05). P.g and T.d supernatants demonstrated an increase in insulin secretion to fivefold at hypo- and hyperglycemia, yet stimulation from hypo- to hyperglycemia stays in the same ratio. Activation of TLR4/PI3K/AKT signaling by supernatants in INS-1 cells resulted in increased IL-1ß, TNFα, IL-6 gene expression levels, and AKT phosphorylation, which were abolished by TLR4 and PI3K/AKT signaling inhibitor. We demonstrated that bacterial supernatants derived from gram-negative species increasingly stimulate insulin secretion in ß-cells and TLR4 may promote inflammation by activating the PI3K/AKT signaling pathway to induce pro-inflammatory molecules. Bacterial species, depending on their virulence, appear to play a role in the relationship between periodontitis and prediabetes by promoting insulin resistance and ß-cell compensatory response.

[Autologous platelet concentrates in regenerative dentistry - A narrative literature review. Part II: clinical application of PRF in periodontology and implantology].

The clinical impact of platelet-rich fibrin (PRF) and plasma rich in growth factors (PRGF®) respectively has been studied extensively in the field of regenerative dentistry during the last two decades. Literature supports evidence for additional benefits in regenerative periodontal therapy, alveolar ridge preservation, management of extraction sockets, implantology including guided bone regeneration as well as defect management in oral surgery. Regarding gingival wound healing and soft tissue regeneration, there is sufficient evidence for their positive effects which have been confirmed in several systematic reviews. The effects seem less clear in conjunction with osseous regenerative treatments, where the inter-study heterogenity in terms of different PRF-protocols, indications and application forms might hinder a systematic comparison. Nevertheless there is evidence that PRF might have beneficial effects on hard-tissue or its regeneration respectively.For being able to facilitate conclusions in systematic reviews, precise reporting of the used PRF-protocols is mandatory for future (clinical) research in the field of autologous platelet concentrates.

[Autologous platelet concentrates in regenerative dentistry - A narrative literature review. Part I: Theoretical and legal aspects of the PR(G)F application].

The use of autologous platelet concentrates (APC) such as platelet-rich fibrin (PRF) and/or plasma rich in growth factors (PRGF®) is considered an established treatment modality in re-generative dentistry. The possibility of delivering growth factors over aclinically relevant time of several days seems particularly interesting in the context of wound healing.The growing body of evidence in the field of APC requires a continuous and actual knowledge of the literature for being able to make evidence-based treatment recommendations with a realistic assessment of possible advantages of this technology.PR(G)F can be applied in solid or liquid form, pure or in combination with other biomaterials. Both appear to be reasonable, depending on the clinical indication and/or desired treatment outcomes. Because of the many different factors that can affect the PR(G)F products final characteristics, a basic understanding of these parameters is desirable for choosing the most suitable product and/or optimizing its clinical application. This review aims to provide an over-view of relevant theoretical, practical, legal and biologic aspects of APCs.

Injectable Platelet-Rich Fibrin and Advanced Platelet-Rich Fibrin Demonstrate Enhanced Anti-Biofilm Effect Compared to Enamel Matrix Derivatives on Decontaminated Titanium Surfaces.

BACKGROUND: The search for effective antimicrobial agents to mitigate peri-implant infections remains a crucial aspect of implant dentistry. This study aimed to evaluate and compare the antimicrobial efficacy of i-PRF, A-PRF+, and enamel matrix derivative (EMD) on decontaminated rough and smooth titanium (Ti) discs. MATERIALS AND METHODS: Rough and smooth Ti discs were coated with multispecies biofilm and thoroughly debrided using a chitosan-bristled brush. Subsequently, i-PRF, A-PRF+, and EMD were applied. Untreated discs served as control. Residual adherent bacteria present on the treated Ti discs were visualized by SEM and quantified using culture technique, and colony-forming units (CFUs) were measured after 48 h and 7 days. RESULTS: i-PRF demonstrated better antimicrobial effectiveness on both smooth and rough implant surfaces as compared to A-PRF+ and EMD (p < 0.001). In all the experimental groups, smooth Ti discs displayed a greater reduction in microbes compared to rough Ti discs when treated with the biologics. The major reduction in CFU values was determined after seven days. CONCLUSIONS: i-PRF as a regenerative material may also be suitable for decontaminating implant surfaces, which could influence tissue healing and regenerative outcomes positively.

Fibroblast growth factor 9 (FGF9)-pituitary homeobox 2 (PITX2) pathway mediates transforming growth factor ß (TGFß) signaling to regulate cell proliferation in palatal mesenchyme during mouse palatogenesis.

Cleft palate represents one of the most common congenital birth defects. Transforming growth factor ß (TGFß) signaling plays crucial functions in regulating craniofacial development, and loss of TGFß receptor type II in cranial neural crest cells leads to craniofacial malformations, including cleft palate in mice (Tgfbr2(fl/fl);Wnt1-Cre mice). Here we have identified candidate target genes of TGFß signaling during palatal formation. These target genes were selected based on combining results from gene expression profiles of embryonic day 14.5 palates from Tgfbr2(fl/fl);Wnt1-Cre mice and previously identified cleft palate phenotypes in genetically engineered mouse models. We found that fibroblast growth factor 9 (Fgf9) and transcription factor pituitary homeobox 2 (Pitx2) expressions are significantly down-regulated in the palate of Tgfbr2(fl/fl);Wnt1-Cre mice, and Fgf9 and Pitx2 loss of function mutations result in cleft palate in mice. Pitx2 expression is down-regulated by siRNA knockdown of Fgf9, suggesting that Fgf9 is upstream of Pitx2. We detected decreased expression of both cyclins D1 and D3 in the palates of Tgfbr2(fl/fl);Wnt1-Cre mice, consistent with the defect in cell proliferation. Significantly, exogenous FGF9 restores expression of cyclins D1 and D3 in a Pitx2-dependent manner and rescues the cell proliferation defect in the palatal mesenchyme of Tgfbr2(fl/fl);Wnt1-Cre mice. Our study indicates that a TGFß-FGF9-PITX2 signaling cascade regulates cranial neural crest cell proliferation during palate formation.

Inflammatory stimulation of osteoblasts and keratinocytes from a SAPHO patient for implant risk evaluation.

The present report exemplifies a translational method, which could assist the clinical preevaluation of patients at risk before surgical interventions. In this study, a presurgical implant decision in a case of SAPHO (synovitis, acne, palmoplantar pustulosis, hyperostosis, osteitis) is described. Since the etiology of this syndrome is likely to involve genetic, infectious and immunological components, lipopolysaccharides (LPS) may conceptually trigger cytokine production leading to a specific chronic inflammation and immunological host response. This may hamper proper healing or accentuate the destruction of periodontal host tissues. In our approach, we examined the ex vivo cell viability and immune responses of primary osteoblasts and keratinocytes under sterile inflammation induced by P. gingivalis LPS. Keratinocytes and osteoblasts were obtained from biopsies of the keratinized gingiva and alveolar bone tissues of a SAPHO human subject. Enzymatically dissociated cells were thus cultured and incubated to LPS at different concentrations (50ng/ml, 200ng/ml, 500ng/ml and 1µg/ml) for 24 h in order to test inflammatory cytokine response (quantitative real time PCR) and toxicity (cell viability). Healthy primary keratinocytes and osteoblasts were used as control cells. The highest concentration of LPS (1µg/ml) significantly reduced cell viability (p < 0.05). Meanwhile, all tested LPS concentrations similarly enhanced the mRNA expressions of selected inflammatory cytokines (TNFα, IL-6, IL-8, IL-1ß and IL-1𝛼) up to ≈3.5-fold, when compared to the healthy cell controls (p < 0.05). This study demonstrated a valuable inflammatory risk evaluation before implant placement, which was successfully performed based on the presented laboratory diagnostic/prognostic approach.Sapho.

Notch Signaling Pathway in Tooth Shape Variations throughout Evolution.

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth crown shape modifications convergent to those observed along Muridae evolution. RNA sequencing analysis revealed that these alterations are due to the modulation of more than 2000 genes and that Notch signaling is a hub for significant morphogenetic networks, such as Wnts and Fibroblast Growth Factors. The modeling of these tooth crown changes in mutant mice, via a three-dimensional metamorphosis approach, allowed prediction of how Jagged1-associated mutations in humans could affect the morphology of their teeth. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution.

Cytotoxic and Inflammatory Effects of Electronic and Traditional Cigarettes on Oral Gingival Cells Using a Novel Automated Smoking Instrument: An In Vitro Study.

Information about the potential oral health effects of vaping from electronic cigarettes (e-cigs) is still sparse and inconsistent. The purpose of this study was to compare the safety and cytotoxicity of e-cig liquid aerosols versus traditional cigarette (t-cig) smoke on human epithelial oral cells. T-cig smoke and e-cig aerosols were generated by a newly developed automated smoking instrument in order to simulate realistic user puffing behaviors. Air−liquid interface transwell cell cultures were exposed to standardized puff topography (puff duration: 2 s, puff volume: 35 mL, puff frequency: 1 puff every 60 s) of reference t-cigs or commercially available e-cigs at different air dilutions. Cell viability, morphology, and death rate were evaluated with MTT and TUNEL assays. The inflammatory cytokine gene expression of inflammatory genes was assessed by quantitative RT-PCR. E-cigs and t-cigs indicated similar adverse effects by enhancing cytotoxicity and cell death in a dose-dependent manner. E-cig aerosol and t-cig smoke treatment expressed upregulation of inflammatory cytokines up to 3.0-fold (p < 0.05). These results indicate that e-cig smoking may contribute to oral tissue−cell damage and tissue inflammation. Our approach allows the production of e-cig aerosol and t-cig smoke in order to identify harmful effects in oral tissues in vitro.

Effect of Titanium and Zirconium Oxide Microparticles on Pro-Inflammatory Response in Human Macrophages under Induced Sterile Inflammation: An In Vitro Study.

The wear-debris particles released by shearing forces during dental implant insertion may contribute to inflammatory reactions or osteolysis associated with peri-implantitis by stimulating inflammasome-activation. The study aim was to examine cytotoxic and pro-inflammatory effects of titanium (TiO2) and zirconia (ZrO2) particles in macrophages regarding their nature/particle concentration over time under sterile lipopolysaccharide (LPS) inflammation. Macrophages were exposed to TiO2 and ZrO2 particles (≤5 µm) in cell culture. Dental glass was used as inert control and LPS (1 µg/mL) was used to promote sterile inflammation. Cytotoxicity was determined using MTT assays and cytokine expression of TNF-α, IL-1ß and IL-6 was evaluated by qRT-PCR. Data were analyzed using Student's t-test and ANOVA (p ≤ 0.05). Cytotoxicity was significantly increased when exposed to higher concentrations of glass, TiO2 and ZrO2 (≥107 particles/mL) compared to controls (p ≤ 0.05). Macrophages challenged with TiO2 particles expressed up to ≈3.5-fold higher upregulation than ZrO2 from 12 to 48 h. However, when exposed to LPS, TiO2 and ZrO2 particle-induced pro-inflammatory gene expression was further enhanced (p ≤ 0.05). Our data suggest that ZrO2 particles produce less toxicity/inflammatory cytokine production than TiO2. The present study shows that the biological reactivity of TiO2 and ZrO2 depends on the type and concentration of particles in a time-dependent manner.

Oral Diagnostic Methods for the Detection of Periodontal Disease.

Periodontitis is a common immune-inflammatory oral disease. Early detection plays an important role in its prevention and progression. Saliva is a reliable medium that mirrors periodontal health and is easily obtainable for identifying periodontal biomarkers in point-of-care diagnostics. The aim of this study is to evaluate the effectiveness of diagnostic salivary tests to determine periodontal status. Whole saliva (stimulated/unstimulated) from twenty healthy and twenty stage III grade B generalized periodontitis patients was tested for lactoferrin, alkaline phosphatase, calcium, density, osmolarity, pH, phosphate, buffer capacity, salivary flow rate and dynamic viscosity. A semi-quantitative urinary strip test was used to evaluate markers of inflammation in saliva (erythrocytes, leukocytes, urobilinogen, nitrite, glucose, bilirubin, and ketones), clinical periodontal parameters and pathogenic bacteria. Concentrations of lactoferrin, hemoglobin, and leukocytes were found to be significantly higher in the stimulated and unstimulated saliva in periodontitis patients compared to healthy patients, whereas alkaline phosphatase levels were higher in unstimulated saliva of periodontitis patients (p < 0.05). Periodontal biomarker analysis using test strips may be considered rapid and easy tool for distinguishing between periodontitis and healthy patients. The increase in lactoferrin, hemoglobin, and leucocytes-determined by strip tests-may provide a non-invasive method of periodontal diagnosis.

Transcriptional activity analysis of promoter region of human PAX9 gene under dexamethasone, retinoic acid, and ergocalciferol treatment in MCF-7 and MDPC23.

PAX9 gene is a member of the family homeobox of transcription factors and performs important function in development and organogenesis. Mutations in PAX9 coding sequences have been implicated in autosomal dominant oligodontia affecting predominantly permanent molars and second premolars. Previous studies have shown that PAX9 is required for secondary palate development and teratogens have been identified as inducers of a tooth and craniofacial malformations. This work focused on the analysis on the 5'-flanking region of the PAX9 gene studying the influence of retinoic acid, dexamethasone, and vitamin D on the expression of PAX9 by expression constructs that carry the reporter gene luciferase. As results, retinoic acid and dexamethasone showed progressive decrease of PAX9 expression. PAX9-pGL3B1 and PAX9-pGL3B2 promoter was inhibited under the treatment of dexamethasone and ergocalciferol. Retinoic acid and dexamethasone did not alter PAX9-pGL3B3 behavior indicating that sequences present between -1106 and +92 were important for the transcriptional activity of PAX9 promoter. In this study, we characterized the transcriptional activity of specific regions of the PAX9 promoter gene and we demonstrated that retinoic acid and ergocalciferol can modulate the transcriptional activity of PAX9 gene.

Effect of high glucose levels and lipopolysaccharides-induced inflammation on osteoblast mineralization over sandblasted/acid-etched titanium surface.

BACKGROUND AND PURPOSE: Poorly controlled diabetes mellitus has been related to higher risk of implant treatment complications due to increased susceptibility to infection and delayed wound healing. Lipopolysaccharides (LPS) stimulate cytokine production leading to chronic inflammation and immunological host response that accentuates the destruction of periodontal tissues. This study aimed to evaluate the effect of different glycemic conditions on secretion and mineralization of bone matrix under sterile inflammation induced by LPS on osteoblasts seeded over sandblasted/acid-etched (SLA) titanium surface. MATERIALS AND METHODS: Osteoblast cell viability was performed to determine the influence of different glucose concentrations (5.5, 8, 12, and 24 mM), which were chosen to reflect normal, postprandial, and high glucose values, similar to those typically seen in Diabetes mellitus under clinical conditions. Cells were seeded on titanium SLA discs (Straumann AG, Waldenburg, Switzerland) and exposed to glucose concentrations and LPS (1µg/mL) in order to test inflammatory response (qPCR) and mineralization (Alizarin Red staining). RESULTS: Osteoblast viability was severely decreased when exposed to higher glucose levels (≥12 mM) and LPS (P < .05) compared to control. When the osteoblasts were exposed to LPS and glucose at ≥8 mM, the gene transcripts of inflammatory cytokines were ≈2.5-fold upregulated, while ≤8 mM glucose elicited no significant change compared to control without glucose treatment (P > .05). Osteoblasts exposed to LPS produced sparse extracellular matrix mineralization, especially combined with higher glucose values (≥12 mM), together with decreased calcium deposition compared to control (P < .05). CONCLUSIONS: High glucose levels combined with LPS inflammatory stimulation elicited an adverse effect on the volume and quality of mineralized hard tissue formation on SLA titanium surfaces in vitro. Hence, both normal glucose levels and infection control including low levels of circulating LPS during initial osseointegration period may be required to increase the success rate of dental implants.

In Vitro Effect of Modified Polyetheretherketone (PEEK) Implant Abutments on Human Gingival Epithelial Keratinocytes Migration and Proliferation.

Improving soft tissue attachment to implant abutments is a crucial factor for enduring health and maintenance of soft peri-implant tissue health. In this in vitro study we aimed to compare the biocompatibility of three different abutment surfaces: titanium, zirconia and modified polyetheretherketone (PEEK). Surface topography, roughness and wettability were investigated with scanning electron microscopy, profilometer and contact angle meter, respectively. Human gingival epithelial keratinocytes were examined for viability, morphology, proliferation and migration by using tetrazolium salt colorimetric assay, scanning electron microscopy imaging, immunofluorescence bromodeoxyuridine analysis and scratch wound healing assays. Roughness measurements revealed differences between the investigated surfaces. Keratinocytes cultured on all examined surfaces indicated adhesion and attachment by means of scanning electron microscopy imaging. Cell viability assays showed no significant differences between the groups (p > 0.05). The modified PEEK surface similarly improved surface roughness in comparison to titanium and zirconia, which resulted in greater and equivalent cell proliferation and migration. The study methodology showed here may emphasize the importance of cell interactions with different abutment materials, which in part increases the changes of implant success. PEEK, titanium and zirconia surface types used in this study showed mostly similar epithelial biological responses.

Remineralization of Artificial Dentin Caries Using Dentin and Enamel Matrix Proteins.

To assess the remineralizing potential of dentin matrix proteins and enamel matrix derivatives (DMPs and EMDs) after application on artificially induced dentin lesions, given the hypothesis that these materials increase the mineral uptake, binding, and mineralization. Forty-eight caries-free human premolars were used. Teeth were cut, polished, and embedded, leaving an open window on the root surface, of which one-third was covered with a flowable composite to preserve the healthy untreated dentin. Then, samples were demineralized in Buskes solution for 33 days. A micro-CT scan prior to treatment was performed. Next, the samples were randomly allocated into four groups: (A) An untreated negative control (CON), (B) application of porcine dentin matrix proteins (DMP), (C) treatment with enamel matrix derivatives (EMD, Emdogain, Straumann), and (D) amine fluoride application (AMF, Elmex fluid, GABA). All samples were placed in artificial saliva for 21 days. A second micro-CT scan was performed, after which the change in gray scaling within a defined region of interest (0.25 mm3) was analyzed. ANCOVA was applied to discover statistical differences between the different treatments. Both, treatment with AMF; (P = 0.011 versus CON) as well as with DMP (P = 0.043 versus CON) yielded a statistically significant difference compared to the control treatment. EMD treatment was not found to differ (P > 0.05). Mainly the top layer of the defects showed clear signs of remineralization, which was also evident in CON. This study was able to visually confirm the remineralization potential of demineralized dentin especially after DMP application, which, however, did not outperform AMF. Based on this, additional studies combining proteins and fluorides are now warranted and ongoing.

Periodontal bacterial supernatants modify differentiation, migration and inflammatory cytokine expression in human periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSC) play an important role in periodontal tissue homeostasis/turnover and could be applied in cell-based periodontal regenerative therapy. Bacterial supernatants secreted from diverse periodontal bacteria induce the production of cytokines that contribute to local periodontal tissue destruction. However, little is known about the impact of whole bacterial toxins on the biological behavior of PDLSC. Therefore this study investigated whether proliferation, migration, inflammatory cytokines expression and transcriptional profile would be affected by exposure to endotoxins from bacterial species found in the subgingival plaque. PDLSC were cultured with the following bacterial supernatants: S. mutans, S. anginosus, P. intermedia, F. nucleatum, P. gingivalis and T. denticola. These supernatants were prepared in dilutions of 1:1000, 1:500, 1:300 and 1:50. Using quantitative RT-PCR, gene expression of selected inflammatory cytokines (IL-6, IL-8 and IL-1ß) and cell-surface receptors (TLR2, TLR4) showed upregulation of ≈2.0- to 3.0-fold, when exposed to P. intermedia, F. nucleatum, P. gingivalis and T. denticola. However, supernatants did not affect proliferation (MTT) and migration (wound scratch assays) of PDLSC. Next generation RNA sequencing confirmed modified lineage commitment of PDLSC by stimulating chondrogenesis, adipogenesis and inhibition of osteogenesis under P. gingivalis supernatant treatment compared to control. Taken together, this study shows stem cell immunomodulatory response to different periodontal bacteria supernatant and suggests that stem cell transcriptional capacity, migration/proliferation and osteogenesis may differ in the presence of those pathogens. These results bring into question stem cell contribution to periodontal tissue regeneration and onset of inflammation.

In Vitro Evaluation of Substantivity, Staining Potential, and Biofilm Reduction of Guava Leaf Extract Mouth Rinse in Combination with its Anti-Inflammatory Effect on Human Gingival Epithelial Keratinocytes.

This study aimed to assess the biofilm reduction, staining potential, and cytotoxicity of guava extract mouth rinse compared to chlorhexidine (CHX). Substantivity, staining, and antibiofilm potential were investigated by spectrophotometry, colony-forming units, and luminosity color meter, respectively. The cell viability assay was conducted using a colorimetric assay to determine nontoxic levels of guava (0.15%) and CHX in human gingival epithelial keratinocytes (HGEK-16). Cells were treated with lipopolysaccharides (LPS, 1µg/mL) and guava to assess inflammatory gene expression levels of interleukin-ß1, tumor necrosis factor-α, and Prostaglandin E2. A scratch wound healing assay investigated the effects of guava on cell migration. The teeth coated in guava mouth rinse displayed 19.4% higher substantivity compared to CHX (0.2%), and the anti-biofilm reduction was observed with both guava and CHX mouth rinses (P < 0.05). The overall discoloration changes were higher with CHX and distilled water compared to guava. Also, guava significantly enhanced HGEK-16 cell viability (P < 0.05), and IL-ß1, TNFα and PGE2 expression presented a 0.6-fold decrease when exposed to guava and LPS (P < 0.05). The present study showed that guava mouth rinse fulfilled the requirement for an effective and useful oral care product with desirable substantivity and anti-biofilm action. In addition, guava reduced the inflammation response in HGEK-16 and may be a potential oral rinse for oral anti-inflammatory therapies.