Dental implant material related changes in molecular signatures in peri-implantitis - A systematic review of omics in-vivo studies.

OBJECTIVE: Titanium particles have been shown in in-vitro studies to lead to the activation of specific pathways, this work aims to systematically review in- vivo studies examining peri-implant and periodontal tissues at the transcriptome, proteome, epigenome and genome level to reveal implant material-related processes favoring peri-implantitis development investigated in animal and human trials. METHODS: Inquiring three literature databases (Medline, Embase, Cochrane) a systematic search based on a priori defined PICOs was conducted: '-omics' studies comparing molecular signatures in healthy and infected peri-implant sites and/or healthy and periodontitis-affected teeth in animals/humans. After risk of bias assessments, lists of differentially expressed genes and results of functional enrichment analyses were compiled whenever possible. RESULTS: Out of 2187 screened articles 9 publications were deemed eligible. Both healthy and inflamed peri-implant tissues showed distinct gene expression patterns compared to healthy/diseased periodontal tissues in animal (n = 4) or human studies (n = 5), with immune response, bone metabolism and oxidative stress being affected the most. Due to the lack of available re-analyzable data and inconsistency in methodology of the eligible studies, integrative analyses on differential gene expression were not applicable CONCLUSION: The differences of transcriptomic signatures in between peri-implant lesions compared to periodontal tissue might be related to titanium particles arising from dental implants and are in line with the in-vitro data recently published by our group. Nevertheless, limitations emerge from small sample sizes of included studies and insufficient publication of re-analyzable data.

Evaluation of the biological effects of amelogenin on human oral keratinocytes.

OBJECTIVES: Amelogenins are clinically used in periodontal regeneration as main components of root surface modifying agents, even without specifically preventing the premature colonization of the healing tissue defect by means of a physical barrier membrane. The objective of this study was to investigate the effects of human amelogenin on the proliferation, migration, and morphology of Immortalized Human Oral Keratinocytes (iHOKs). METHODS: Immortalized Human Oral Keratinocytes were expanded in Keratinocyte Growth Medium-2 (KGM-2). Full-length recombinant amelogenin protein was diluted in KGM-2 in five concentrations (10 ng/ml, 100 ng/ml, 1.000 ng/ml, 5.000 ng/ml and 10.000 ng/ml). iHOKs were cultured in medium supplemented with the amelogenin dilutions. Samples without amelogenin served as control. Cell metabolism and cell proliferation together with cell migration were evaluated at day 7, 14, 21. RESULTS: At day 7, iHOKs treated with 10,000 ng/ml showed a significant decrease in keratinocytes´ proliferation. The metabolic activity at this timepoint was significantly lower for concentrations ≥ 1000 ng/ml. At days 14 and 21, both the addition of 5000 ng/ml and even more 10,000 ng/ml amelogenin reduced significantly the proliferation of keratinocytes. The effects on the metabolic activity for these timepoints were visible already with 100 ng/ml. Treatment of iHOKs with amelogenin of ≥ 1000 ng/ml led to inhibitory effects on cell migration already after 24 h. CONCLUSIONS: The full-length recombinant amelogenin has a significant biological impact on iHOKs. The increasing dose dependent inhibitory effects of amelogenin shown on iHOKs might explain the disruption of the apical migration of the junctional epithelium during regenerative healing. CLINICAL SIGNIFICANCE: Amelogenin, presents time- and dose-dependent inhibitory effects on the growth of keratinocytes, which might explain the biological rationale behind its application in periodontal regeneration.

Dental implant material related changes in molecular signatures in peri-implantitis - A systematic review and integrative analysis of omics in-vitro studies.

OBJECTIVE: Since peri-implantitis differs clinically and histopathologically from periodontitis, implant wear debris is considered to play a role in the destructive processes. This work aims to systematically review if titanium particles affect oral-related cells through changes in molecular signatures (e.g., transcriptome, proteome, epigenome), thereby promoting peri-implantitis. METHODS: Leveraging three literature databases (Medline, Embase, Cochrane) a systematic search based on a priori defined PICOs was conducted: '-omics' studies examining titanium exposure in oral-related cells. After risk of bias assessments, lists of differentially expressed genes, proteins, and results of functional enrichment analyses were compiled. The significance of overlapping genes across multiple studies was assessed via Monte Carlo simulation and their ranking was verified using rank aggregation. RESULTS: Out of 2104 screened articles we found 12 eligible publications. A significant overlap of gene expression in oral-related cells exposed to titanium particles was found in four studies. Furthermore, changes in biological processes like immune/inflammatory or stress response as well as toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling pathways were linked to titanium in transcriptome and proteome studies. Epigenetic changes caused by titanium were detected but inconsistent. CONCLUSION: An influence of titanium implant wear debris on the development and progression of peri-implantitis is plausible but needs to be proven in further studies. Limitations arise from small sample sizes of included studies and insufficient publication of re-analyzable data.

In-vitro cytocompatibility and growth factor content of GBR/GTR membranes.

OBJECTIVE: To assess the cytocompatibility of five commercially available xenogenic barrier membranes used for oral regenerative procedures and to determine the growth factor content of these membranes in-vitro. METHODS: Human mesenchymal stem cells (hMSCs) and immortalized periodontal ligament stem cells (PDL-hTERTs) were used to determine the cytocompatibility of xenogenic barrier membranes made of collagen (Biogide, BG, Geistlich Pharma AG, Switzerland; Biomend, BM, Zimmer Biomet, USA; Osseoguard OG, Zimmer Biomet, USA; OssixPlus, OX, Datum Dental, Israel) or extracellular matrix (ECM) (Dynamatrix, DM, Keystone Dental, USA) and of their eluates obtained by washing. Cells were cultured with previously washed and unwashed membranes (n=4) and in the medium used for washing (eluate). Cell proliferation at 3 days (eluates) and at 7 days (membranes) was assessed using the WST-1 cell proliferation kit. Growth factor content of the membranes was measured using multiplex ELISA. RESULTS: The eluate of BG and BM significantly inhibited proliferation of hMSCs, whereas DM and OX showed stimulating effects. The highest impact was observed for DM, its eluate doubled the cell proliferation of adherent cells when compared to the control (p<0.001). The eluate of OG did not influence eluate cell cultures (p>0.05). The presence of membranes had different impact on hMSCs and PDLs. hMSCs seem to be more resistant to the inhibitory effects of BG, OG and BM. hMSCs are only affected by OX, which actually stimulates hMSCs when the specimens are not washed previously. PDLs however proliferate significantly less once they are placed into culture with BM and OG as well as BG-not washed. Once BG is washed no inhibitory effect on PDLs was observed, however overall the washing of membrane samples prior to the placement into the cell culture did hardly have any effect on the outcome. The strongest inhibition of proliferation was shown with the BM and OG membrane in PDL-hTERTs (p<0.001). Growth factor contents were quite similar quantitatively and qualitatively among the tested membranes with concentrations in the range of 50-500 pg/ml. Intriguingly DM contained considerably higher amounts of bFGF with up to 8000 pg/ml. SIGNIFICANCE: The collagen membranes cross-linked with aldehydes show poor outcomes in PDLs while the collagen membrane cross-linked with polysaccharides generally shows promising results similar to the ECM-membrane DM in both membrane and eluate tests. The findings may be due to various factors, especially differences observed in composition, processing and bFGF content.

The severity of human peri-implantitis lesions correlates with the level of submucosal microbial dysbiosis.

AIM: To cross-sectionally analyse the submucosal microbiome of peri-implantitis (PI) lesions at different severity levels. MATERIALS AND METHODS: Microbial signatures of 45 submucosal plaque samples from untreated PI lesions obtained from 30 non-smoking, systemically healthy subjects were assessed by 16s sequencing. Linear mixed models were used to identify taxa with differential abundance by probing depth, after correction for age, gender, and multiple samples per subject. Network analyses were performed to identify groups of taxa with mutual occurrence or exclusion. Subsequently, the effects of peri-implant probing depth on submucosal microbial dysbiosis were calculated using the microbial dysbiosis index. RESULTS: In total, we identified 337 different taxa in the submucosal microbiome of PI. Total abundance of 12 taxa correlated significantly with increasing probing depth; a significant relationship with lower probing depth was found for 16 taxa. Network analysis identified two mutually exclusive complexes associated with shallow pockets and deeper pockets, respectively. Deeper peri-implant pockets were associated with significantly increased dysbiosis. CONCLUSION: Increases in peri-implant pocket depth are associated with substantial changes in the submucosal microbiome and increasing levels of dysbiosis.

The association between coeliac disease and periodontitis: Results from NHANES 2009-2012.

AIM: To investigate whether coeliac disease (CD) was associated with periodontitis among a nationally representative sample of US adults. MATERIALS AND METHODS: The National Health and Nutrition Examination Survey (NHANES) 2009-2012 enrolled 6,661 subjects with full-mouth periodontal examination and serological testing for antitissue transglutaminase (tTg) and antiendomysial (EMA) antibodies. CD was defined as (i) self-reported physician diagnosis while on a gluten-free diet; or (ii) tTg levels >10.0 U/ml and positive EMA results. Positive serology without self-reported diagnosis was defined as undiagnosed CD (UdxCD). Periodontitis was defined according to the CDC/AAP definition. Multivariable linear and logistic models were used to regress the mean probing depth (PD) or attachment loss (AL) outcomes across CD categories (none, diagnosed and undiagnosed). RESULTS: The prevalence of moderate/severe periodontitis and diagnosed/undiagnosed CD was 40% and 0.74%, respectively. Mean AL was lower among those with CD although results were not statistically significant (p = .67). The odds of periodontitis among individuals with diagnosed and undiagnosed CD were: 0.5(0.22, 1.16) and 0.62(0.1, 3.75), respectively. Mean PD levels among those without CD or with diagnosed or undiagnosed CD were 1.49 ± 0.02, 1.36 ± 0.11 and 1.31 ± 0.11 (p = .03). CONCLUSION: CD is associated with modestly lower levels of mean PD but was not associated with mean AL or periodontitis. Larger studies are necessary to enhance precision and strengthen conclusions.

Restoring lost gingival pigmentation in the esthetic zone: A case report.

BACKGROUND AND OVERVIEW: There is insufficient literature on the lack of oral pigmentation in the esthetic zone. The aim of this case report was to illustrate the potential impact of loss of gingival pigmentation in the esthetic zone, describe its surgical treatment, and discuss the limited literature on this topic. CASE DESCRIPTION: An African American woman with high smile line had localized loss of gingival melanin pigmentation as a complication after implant failure and attempted guided bone regeneration in site 8. A highly pigmented free gingival graft was collected from the facial-attached gingiva of the maxillary posterior teeth and placed onto the previously de-epithelialized recipient bed in the maxillary front. Some pigmentation of the graft was preserved and was visible a few weeks after surgery; some pigmentation recovered over time. At 6 months after surgery, the patient was satisfied with the esthetics. Complete recovery of pigmentation took 12 months, at which time the patient was ready to proceed with the final prosthetic work. CONCLUSIONS AND PRACTICAL IMPLICATIONS: Gingival pigmentation can be restored using a free gingival graft from a highly pigmented area. When surgical procedures are performed in such cases, loss of gingival pigmentation should be part of the informed consent. However, further research, including histology, is needed.

Cytotoxicity of post and core composites as a function of environmental conditions.

OBJECTIVES: In the revised version of ISO 7405 there are so far no detailed recommendations concerning temperature and humidity during specimen production for light curing and chemically setting dental materials. The main objective of the present study was to observe if different environmental conditions during specimen production influence cytotoxicity and degree of conversion of four post and core composite materials and to investigate if cytotoxicity of post and core materials is influenced by their corresponding bonding substances. METHODS: Specimens of four different post and core composite materials (LuxaCore - Dual, Core X-Flow, Flow White and MultiCore Flow) were produced in a climate test chamber at 23°C/50% relative humidity or 37°C/95% relative humidity and were dual-cured or self-cured, with or without their corresponding bonding substances. Specimens were added to cell cultures immediately after production or after preincubation for 7 days. Specimens were incubated with L-929 fibroblasts for 72h and cell numbers determined by a flow cytometer. FTIR spectroscopic measurements of post and core materials were performed at the same temperature conditions as for the cytotoxicity assay (23°C or 37°C). RESULTS: Dual-cured specimens of all post and core composites exhibited less cytotoxicity under both environmental conditions than self-cured specimens. All self-cured specimens manufactured at 37°C/95% showed less cytotoxicity than specimens produced at 23°C/50%. All dual-cured specimens showed similar cytotoxicity at both environmental conditions. After 7 days of preincubation most dual-cured specimens produced at 23°C/50% showed less cytotoxicity than self-cured specimens (with the exception of Flow White). Compared to fresh specimens, 7-day aged specimens of most materials showed reduced cytotoxicity. Materials already showing low cytotoxicity as fresh specimens did not further reduce their cytotoxicity after 7 days of preincubation. For dual-cured materials the degree of conversion was higher compared to self-cured materials. SIGNIFICANCE: Different temperatures during specimen production have an impact on cytotoxicity and degree of conversion of dual-curing composite materials. Detailed recommendations for standardization concerning environmental conditions during specimen production are required.

Circulating endothelial progenitor cells in periodontitis.

BACKGROUND: Several biologically plausible mechanisms have been proposed to mediate the association between periodontitis and atherosclerotic vascular disease (AVD), including adverse effects on vascular endothelial function. Circulating endothelial progenitor cells (cEPCs) are known to contribute to vascular repair, but limited data are available regarding the relationship between cEPC levels and periodontitis. The aims of this cross-sectional study are to investigate the levels of hemangioblastic and monocytic cEPCs in patients with periodontitis and periodontally healthy controls and to associate cEPC levels with the extent and severity of periodontitis. METHODS: A total of 112 individuals (56 patients with periodontitis and 56 periodontally healthy controls, aged 26 to 65 years; mean age: 43 years) were enrolled. All participants underwent a full-mouth periodontal examination and provided a blood sample. Hemangioblastic cEPCs were assessed using flow cytometry, and monocytic cEPCs were identified using immunohistochemistry in cultured peripheral blood mononuclear cells. cEPC levels were analyzed in the entire sample, as well as in a subset of 50 pairs of patients with periodontitis/periodontally healthy controls, matched with respect to age, sex, and menstrual cycle. RESULTS: Levels of hemangioblastic cEPCs were approximately 2.3-fold higher in patients with periodontitis than periodontally healthy controls, after adjustments for age, sex, physical activity, systolic blood pressure, and body mass index (P = 0.001). A non-significant trend for higher levels of monocytic cEPCs in periodontitis was also observed. The levels of hemangioblastic cEPCs were positively associated with the extent of bleeding on probing, probing depth, and clinical attachment loss. Hemangioblastic and monocytic cEPC levels were not correlated (Spearman correlation coefficient 0.03, P = 0.77), suggesting that they represent independent populations of progenitor cells. CONCLUSION: These findings further support the notion that oral infections have extraoral effects and document that periodontitis is associated with a mobilization of EPCs from the bone marrow, apparently in response to systemic inflammation and endothelial injury.

Polymerization shrinkage kinetics of dimethacrylate resin-cements.

OBJECTIVES: To determine polymerization shrinkage-strain (S(Y)) and shrinkage-stress (S(Z)) of six resin-cements and to compare their performance with the aid of degree of conversion (DC) data. METHODS: Variolink 2 (VL2), Multilink Automix (MA), Multilink Sprint (MS, all Ivoclar-Vivadent), Nexus 2 (NX2), Maxcem (MX, both Kerr) and RelyX Unicem (RX, 3M-Espe) were investigated. MS, MX and RX were self-adhesive; others require a bonding-agent. All measurements were conducted at 23 degrees C for 60min (n=5), except 80min for RX, with materials self-cured only (sc) and dual-cured (dc); NX2 and VL2 were additionally light-cured only (lc). S(Y) was measured by the bonded-disk method [Watts DC, Cash AJ. Determination of polymerization shrinkage kinetics in visible-light-cured materials: methods development. Dent Mater 1991;7(4):281-7; Watts DC, Marouf AS. Optimal specimen geometry in bonded-disk shrinkage-strain measurements on light-cured biomaterials. Dent Mater 2000;16(6):447-51]; S(Z) by the Bioman instrument [Watts DC, Satterthwaite JD. Axial shrinkage-stress depends upon both C-factor and composite mass. Dent Mater 2008;24(1):1-8 [Epub October 24, 2007]; Watts DC, Marouf AS, Al-Hindi AM. Photo-polymerization shrinkage-stress kinetics in resin-composites: methods development. Dent Mater 2003;19(1):1-11]. Light-cure was achieved by QTH at 500mW/cm(2). The respective DCs were measured under the same conditions by FTIR-ATR spectroscopy. Data were analyzed by One-Way ANOVA plus Bonferroni test, and by t-test, at p<0.05. RESULTS: DC by self-curing was less than the DC by dual-curing, for all cements. Shrinkage-strain ranged from 1.77 to 5.29% and shrinkage-stress from 3.36 to 10.37MPa. NX2 and VL2 were not significantly different, when light-cured only. Except for RX, sc and dc shrinkage-strain varied maximally by 0.4%. MX showed the highest S(Y), RX the lowest. When sc, RX initially expanded by <0.5% (t approximately 5min). For most materials, S(Y) correlated with their filler loading. The highest stress with sc was exerted by MX, and when dc by MS, which was not statistically different from MX. SIGNIFICANCE: Shrinkage data of resin-cements are of intrinsic clinical importance. Self-cure, despite a lower DC, did not necessarily result in a lower S(Y) compared to dual-cure. S(Y)-rate and S(Z) development depend upon cure mode and S(Y) upon filler fraction.