Epigenetic reprogramming in periodontal disease: Dynamic crosstalk with potential impact in oncogenesis.

Periodontitis is a chronic multifactorial inflammatory disease associated with microbial dysbiosis and characterized by progressive destruction of the periodontal tissues. Such chronic infectious inflammatory disease is recognized as a major public health problem worldwide with measurable impact in systemic health. It has become evident that the periodontal disease phenotypes are not only determined by the microbiome effect, but the extent of the tissue response is also driven by the host genome and epigenome patterns responding to various environmental exposures. More recently there is mounting evidence indicating that epigenetic reprogramming in response to combined intrinsic and environmental exposures, might be particularly relevant due its plasticity and potential application towards precision health. The complex epigenetic crosstalk is reflected in the prognosis and progress of periodontal diseases and may also lead to a favorable landscape for cancer development. This review discusses epigenomics modifications focusing on the role of DNA methylation and pathways linking microbial infection and inflammatory pathways, which are also associated with carcinogenesis. There is a more clear vision whereas 'omics' technologies applied to unveil relevant epigenetic factors could play a significant role in the treatment of periodontal disease in a personalized mode, evidencing that public health approach should coexist with precision individualized treatment.

Effect of pore size in bone regeneration using polydopamine-laced hydroxyapatite collagen calcium silicate scaffolds fabricated by 3D mould printing technology.

OBJECTIVE: The pore size of the scaffold is a critical factor in repairing large bone defect. Here, we investigated the potential of bone regeneration using novel nanocomposite polydopamine-laced hydroxyapatite collagen calcium silicate (HCCS-PDA) scaffolds with two different pore sizes, 250 and 500 µm. SAMPLES/SETTING: A total of 12 male Sprague-Dawley rats were implanted with HCCS-PDA scaffold with pore size of either 250 or 500 µm into surgically created critical-sized defect (CSD). METHODS: HCCS-PDA scaffolds were fabricated using mould printing technique. The effect of pore size on mechanical strength of the scaffolds was assessed by compression testing. After seeding with rat mesenchymal stem cells (rMSCs), the scaffolds were implanted, and new bone formation was evaluated using microCT and histomorphometric analysis after 8 weeks. RESULTS: MicroCT and histology analysis demonstrated restricted peripheral new bone formation in either dural or periosteal side and limited new bone formation in the 250 µm pore scaffold. Conversely, the 500-µm pore scaffold showed more penetration of new bone into the scaffold and greater bone regeneration in the rat CSD. CONCLUSION: Based on our results, which demonstrated improved new bone formation in 500 µm pores scaffold, we can conclude that effective scaffold pore size that induces osteointegration and bone regeneration is around 500 µm for HCCS-PDA nanocomposite scaffold.

Comparing Initial Wound Healing and Osteogenesis of Porous Tantalum Trabecular Metal and Titanium Alloy Materials.

Porous tantalum trabecular metal (PTTM) has long been used in orthopedics to enhance neovascularization, wound healing, and osteogenesis; recently, it has been incorporated into titanium alloy dental implants. However, little is known about the biological responses to PTTM in the human oral cavity. We have hypothesized that, compared with conventional titanium alloy, PTTM has a greater expression of genes specific to neovascularization, wound healing, and osteogenesis during the initial healing period. Twelve subjects requiring at least 4 implants in the mandible were enrolled. Four 3 × 5mm devices, including 2 titanium alloy tapered screws and 2 PTTM cylinders, were placed in the edentulous mandibular areas using a split-mouth design. One device in each group was trephined for analysis at 2 and 4 weeks after placement. RNA microarray analysis and ingenuity pathway analysis were used to analyze osteogenesis gene expression and relevant signaling pathways. Compared to titanium alloy, PTTM samples exhibited significantly higher expressions of genes specific to cell neovascularization, wound healing, and osteogenesis. Several genes-including bone morphogenic proteins, collagens, and growth factors-were upregulated in the PTTM group compared to the titanium alloy control. PTTM materials may enhance the initial healing of dental implants by modifying gene expression profiles.

Genome-wide association study of biologically informed periodontal complex traits offers novel insights into the genetic basis of periodontal disease.

Genome-wide association studies (GWAS) of chronic periodontitis (CP) defined by clinical criteria alone have had modest success to-date. Here, we refine the CP phenotype by supplementing clinical data with biological intermediates of microbial burden (levels of eight periodontal pathogens) and local inflammatory response (gingival crevicular fluid IL-1ß) and derive periodontal complex traits (PCTs) via principal component analysis. PCTs were carried forward to GWAS (∼2.5 million markers) to identify PCT-associated loci among 975 European American adult participants of the Dental ARIC study. We sought to validate these findings for CP in the larger ARIC cohort (n = 821 participants with severe CP, 2031-moderate CP, 1914-healthy/mild disease) and an independent German sample including 717 aggressive periodontitis cases and 4210 controls. We identified six PCTs with distinct microbial community/IL-1ß structures, although with overlapping clinical presentations. PCT1 was characterized by a uniformly high pathogen load, whereas PCT3 and PCT5 were dominated by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, respectively. We detected genome-wide significant signals for PCT1 (CLEC19A, TRA, GGTA2P, TM9SF2, IFI16, RBMS3), PCT4 (HPVC1) and PCT5 (SLC15A4, PKP2, SNRPN). Overall, the highlighted loci included genes associated with immune response and epithelial barrier function. With the exception of associations of BEGAIN with severe and UBE3D with moderate CP, no other loci were associated with CP in ARIC or aggressive periodontitis in the German sample. Although not associated with current clinically determined periodontal disease taxonomies, upon replication and mechanistic validation these candidate loci may highlight dysbiotic microbial community structures and altered inflammatory/immune responses underlying biological sub-types of CP.

Resonance frequency analysis as a predictor of early implant failure in the partially edentulous posterior maxilla following immediate nonfunctional loading or delayed loading with single unit restorations.

OBJECTIVES: To assess the ability of baseline resonance frequency analysis (RFA) measurements to predict early implant failure in the posterior maxilla and to evaluate potential correlations between this measurement with Hounsfield units, bone quality variables, and implant dimension. MATERIALS AND METHODS: This prospective randomized study involved 46 SLActive Straumann implants placed in the posterior maxillae of 21 subjects. Each patient received at least one control (delayed loading) and one experimental (immediate nonfunctional loading) implant. Each site was evaluated with presurgical computer-assisted tomography (CT) scans, histomorphometric analysis of bone cores, and subjective determination of bone quality. Baseline implant stability quotients (ISQ) were determined by RFA measurements made at the time of fixture placement. Pearson's correlation analysis and Spearman's test were used to identify statistically significant correlations within the resultant data. Receiver operating characteristic (ROC) analysis was used to determine whether baseline ISQ values can accurately predict early implant failure. RESULTS: The mean baseline ISQ values for the two groups were 66.8 (experimental) and 66.2 (control). The 12-month survival rates were 86.4% (experimental) and 100% (control). There were no statistically significant correlations between baseline ISQ values and early implant failure, bone quality variables, or implant dimension. ROC analysis showed that baseline ISQ values cannot predict early implant failure. CONCLUSION: Baseline RFA measurements were not able to predict early failure of immediately loaded implants placed in the posterior maxilla and therefore should not be used to determine whether an implant is a candidate for immediate nonfunctional loading in this region of the mouth.

Maintaining barrier function of infected gingival epithelial cells by inhibition of DNA methylation.

BACKGROUND: Infection and inflammation induce epigenetic changes that alter gene expression. In periodontal disease, inflammation, and microbial dysbiosis occur, which can lead to compromised barrier function of the gingival epithelia. Here, we tested the hypotheses that infection of cultured human gingival epithelial (HGEp) cells with Porphyromonas gingivalis disrupts barrier function by inducing epigenetic alterations and that these effects can be blocked by inhibitors of DNA methylation. METHODS: Primary HGEp cells were infected with P. gingivalis either in the presence or absence of the non-nucleoside DNA methyltransferase (DNMT) inhibitors RG108, (-) epigallocatechin-3-gallate (EGCG), or curcumin. Barrier function was assessed as transepithelial electrical resistance (TEER). DNA methylation and mRNA abundance were quantified for genes encoding components of three cell-cell junction complexes, CDH1, PKP2, and TJP1. Cell morphology and the abundance of cell-cell junction proteins were evaluated by confocal microscopy. RESULTS: Compared to non-infected cells, P. gingivalis infection decreased TEER (P < 0.0001) of HGEp cells; increased methylation of the CDH1, PKP2, and TJP1 (P < 0.0001); and reduced their expression (mRNA abundance) (P < 0.005). Pretreatment with DNMT inhibitors prevented these infection-induced changes in HGEp cells, as well as the altered morphology associated with infection. CONCLUSION: Pathogenic infection induced changes in DNA methylation and impaired the barrier function of cultured primary gingival epithelial cells, which suggests a mechanism for systemic consequences of periodontal disease. Inhibition of these events by non-nucleoside DNMT inhibitors represents a potential strategy to treat periodontal disease.

MicroRNA Expression Profiles in External Cervical Resorption.

INTRODUCTION: External cervical resorption (ECR) has been challenging for its diagnosis, prevention, and treatment. Its etiology and pathogenesis are largely unknown. This study characterized microRNA (miRNA) expression patterns of human tissues from ECR lesions and identified potential messenger RNA targets and pathways. METHODS: Granulomatous tissues from ECR (n = 5) and their adjacent nonaffected asymptomatic gingival connective tissues (n = 5) were collected. Similarly, chronic periodontitis (CP) and control samples were collected (n = 3). Quantitative reverse transcription polymerase chain reaction array analysis compared the expression profiles of 88 miRNAs between diseases. Differentially expressed miRNAs were identified using the Student t test. Bioinformatics for messenger RNA (miRWalk) and KEGG pathway analyses were performed to identify predicted target genes and biological/cellular functions and signaling pathways. RESULTS: Three miRNAs (miR-20a-5p, miR-210-3p, and miR-99a-4p) were significantly down-regulated and 1 miRNA (miR-122-5p) was significantly up-regulated in ECR (P < .05). One up-regulated and 1 down-regulated miRNA reached the significance threshold in CP. A comparison of miRNA expression in ECR and CP identified 3 differentially expressed miRNAs, indicating differences in disease pathobiology. Inflammation-associated Wnt, PI3K-Akt, mitogen-activated protein kinases signaling, and bone formation-associated transforming growth factor beta pathways were identified and predicted to be modulated by differentially expressed miRNAs in both ECR and CP. Biological processes unique to each disease entity were identified, such as T- and B-cell receptor signaling pathways, osteoclast differentiation, and extracellular matrix-receptor interaction for CP. Glycosaminoglycan biosynthesis, mineral absorption, and insulin signaling pathways for ECR were identified. CONCLUSIONS: This proof-of-principle in vivo study indicated that ECR has both common and unique miRNA expression profiles in comparison with CP, which are predicted to target genes regulating inflammation, immunity, and metabolism of mineralized tissues.

Sterilization effects on ultrathin film polymer coatings for silicon-based implantable medical devices.

Novel biomaterials for medical device applications must be stable throughout all stages of preparation for surgery, including sterilization. There is a paucity of information on the effects of sterilization on sub-10 nm-thick polymeric surface coatings suitable for silicon-based bioartificial organs. This study explores the effect of five standard sterilization methods on three surface coatings applied to silicon: polyethylene glycol (PEG), poly(sulfobetaine methacrylate) (pSBMA), and poly (2-methacryloyloxyethyl phosphorylcholine) (pMPC). Autoclave, dry heat, hydrogen peroxide (H2 O2 ) plasma, ethylene oxide gas (EtO), and electron beam (E-beam) treated coatings were analyzed to determine possible polymer degradation with sterilization. Poststerilization, there were significant alterations in contact angle, maximum change resulting from H2 O2 (Δ - 14°), autoclave (Δ + 15°), and dry heat (Δ + 23°) treatments for PEG, pSBMA, and pMPC, respectively. Less than 5% coating thickness change was found with autoclave and EtO on PEG-silicon, E-beam on pSBMA-silicon and EtO treatment on pMPC-silicon. H2 O2 treatment resulted in at least 30% decrease in thickness for all coatings. Enzyme-linked immunosorbent assays showed significant protein adsorption increase for pMPC-silicon following all sterilization methods. E-beam on PEG-silicon and dry-heat treatment on pSBMA-silicon exhibited maximum protein adsorption in each coating subset. Overall, the data suggest autoclave and EtO treatments are well-suited for PEG-silicon, while E-beam is best suited for pSBMA-silicon. pMPC-silicon was least impacted by EtO treatment. H2 O2 treatment had a negative effect on all three coatings. These results can be used to determine which surface modifications and sterilization processes to utilize for devices in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2327-2336, 2018.

GWAS for Interleukin-1ß levels in gingival crevicular fluid identifies IL37 variants in periodontal inflammation.

There is no agnostic GWAS evidence for the genetic control of IL-1ß expression in periodontal disease. Here we report a GWAS for "high" gingival crevicular fluid IL-1ß expression among 4910 European-American adults and identify association signals in the IL37 locus. rs3811046 at this locus (p = 3.3 × 10-22) is associated with severe chronic periodontitis (OR = 1.50; 95% CI = 1.12-2.00), 10-year incident tooth loss (≥3 teeth: RR = 1.33; 95% CI = 1.09-1.62) and aggressive periodontitis (OR = 1.12; 95% CI = 1.01-1.26) in an independent sample of 4927 German/Dutch adults. The minor allele at rs3811046 is associated with increased expression of IL-1ß in periodontal tissue. In RAW macrophages, PBMCs and transgenic mice, the IL37 variant increases expression of IL-1ß and IL-6, inducing more severe periodontal disease, while IL-37 protein production is impaired and shows reduced cleavage by caspase-1. A second variant in the IL37 locus (rs2708943, p = 4.2 × 10-7) associates with attenuated IL37 mRNA expression. Overall, we demonstrate that IL37 variants modulate the inflammatory cascade in periodontal disease.

Diffusive Silicon Nanopore Membranes for Hemodialysis Applications.

Hemodialysis using hollow-fiber membranes provides life-sustaining treatment for nearly 2 million patients worldwide with end stage renal disease (ESRD). However, patients on hemodialysis have worse long-term outcomes compared to kidney transplant or other chronic illnesses. Additionally, the underlying membrane technology of polymer hollow-fiber membranes has not fundamentally changed in over four decades. Therefore, we have proposed a fundamentally different approach using microelectromechanical systems (MEMS) fabrication techniques to create thin-flat sheets of silicon-based membranes for implantable or portable hemodialysis applications. The silicon nanopore membranes (SNM) have biomimetic slit-pore geometry and uniform pores size distribution that allow for exceptional permeability and selectivity. A quantitative diffusion model identified structural limits to diffusive solute transport and motivated a new microfabrication technique to create SNM with enhanced diffusive transport. We performed in vitro testing and extracorporeal testing in pigs on prototype membranes with an effective surface area of 2.52 cm2 and 2.02 cm2, respectively. The diffusive clearance was a two-fold improvement in with the new microfabrication technique and was consistent with our mathematical model. These results establish the feasibility of using SNM for hemodialysis applications with additional scale-up.

Preliminary Diffusive Clearance of Silicon Nanopore Membranes in a Parallel Plate Configuration for Renal Replacement Therapy.

Silicon nanopore membranes (SNMs) with compact geometry and uniform pore size distribution have demonstrated a remarkable capacity for hemofiltration. These advantages could potentially be used for hemodialysis. Here, we present an initial evaluation of the SNM's mechanical robustness, diffusive clearance, and hemocompatibility in a parallel plate configuration. Mechanical robustness of the SNM was demonstrated by exposing membranes to high flows (200 ml/min) and pressures (1,448 mm Hg). Diffusive clearance was performed in an albumin solution and whole blood with blood and dialysate flow rates of 25 ml/min. Hemocompatibility was evaluated using scanning electron microscopy and immunohistochemistry after 4 hours in an extracorporeal porcine model. The pressure drop across the flow cell was 4.6 mm Hg at 200 ml/min. Mechanical testing showed that SNM could withstand up to 775.7 mm Hg without fracture. Urea clearance did not show an appreciable decline in blood versus albumin solution. Extracorporeal studies showed blood was successfully driven via the arterial-venous pressure differential without thrombus formation. Bare silicon showed increased cell adhesion with a 4.1-fold increase and 1.8-fold increase over polyethylene glycol (PEG)-coated surfaces for tissue plasminogen factor (t-PA) and platelet adhesion (CD41), respectively. These initial results warrant further design and development of a fully scaled SNM-based parallel plate dialyzer for renal replacement therapy.

The Novel ASIC2 Locus is Associated with Severe Gingival Inflammation.

An increasing body of evidence suggests a significant genetic regulation of inflammatory response mechanisms; however, little is known regarding the genetic determinants of severe gingival inflammation (GI). We conducted a genome-wide association study of severe GI among 4077 European American adults, participants in the Dental Atherosclerosis Risk In Communities cohort. The severe GI trait was defined dichotomously using the 90th percentile of gingival index ≥2 extent score. Genotyping was performed with the Affymetrix 6.0 array platform and an imputed set of 2.5 million markers, based on HapMap Phase II CEU build 36, was interrogated. Genetic models were based on logistic regression and controlled for ancestry (10 principal components), sex, age, and examination center. One locus on chromosome 17 met genome-wide statistical significance criteria-lead single nucleotide polymorphism (SNP): rs11652874 [minor allele frequency=0.06, intronic to ASIC2 (acid sensing ionic channel-2, formerly named ACCN1); odds ratio=2.1, 95% confidence interval=1.6-2.7, p=3.9×10-8]. This association persisted among subjects with severe periodontitis and was robust to adjustment for microbial plaque index. Moreover, the minor [G] allele was associated with higher levels of severe GI in stratified analyses among subsets of participants with high load of either "red" or "orange" complex pathogens, although this association was not statistically significant. While these results will require replication in independent samples and confirmation by mechanistic studies, this locus appears as a promising candidate for severe gingival inflammation. Our findings suggest that genetic variation in ASIC2 is significantly associated with severe gingival inflammation and the association is plaque-independent.

Salivary biomarkers in a biofilm overgrowth model.

BACKGROUND: The purpose of this study is to determine whether baseline salivary inflammatory biomarkers could discriminate between different clinical levels of disease and/or detect clinical changes over a 3-week stent-induced biofilm overgrowth (SIBO) period. METHODS: A total of 168 participants were enrolled in a 21-day experimental gingivitis investigation and grouped according to clinical measures of periodontal status of health and diseased individuals representing each of five biofilm gingival interface (BGI) periodontal groups: 1) health, all probing depth (PD) <3 mm and bleeding on probing (BOP) <10%; 2) gingivitis, all PD <3 mm and BOP ≥10%; 3) periodontitis (P)1, ≥1 site with PD >3 mm and BOP ≤10%; 4) P2, ≥1 site with PD >3 mm and BOP >10% but ≤50%; and 5) P3, ≥1 site with PD >3 mm and BOP >50%. Stents were used to prevent plaque removal during brushing over one maxillary and one mandibular posterior dental sextant for 21 days. Clinical periodontal parameters and unstimulated saliva were collected at screening, baseline, and each week during SIBO. Saliva samples were assessed for levels of 13 different biomarkers by multiplex immunoassay. RESULTS: Higher salivary levels of interleukin (IL)-1ß, matrix metalloproteinase (MMP)-3, MMP-8, MMP-9, and neutrophil gelatinase-associated lipocalin (NGAL) were found in diseased groups compared with the healthy group at baseline. Conversely, higher IL-1 receptor antagonist (ra) levels were found in healthy patients at baseline. In addition, during SIBO, MMP-1, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 levels increased across all participant groups. A stepwise linear regression model using all salivary biomarkers demonstrated that, at baseline, increased IL-1ra (P = 0.004) and IL-6 (P = 0.009) were significantly associated with change in PDs during SIBO. CONCLUSIONS: In summary, this investigation supports salivary levels of IL-1ra and IL-6 as potential indicators for PD changes during induced gingival inflammation. In addition, participants from the BGI-P3 group (severe periodontitis) demonstrated elevated baseline levels of IL-1ß, MMP-3, MMP-8, MMP-9, and NGAL compared with the other study groups, strengthening the relevance of participants' biologic phenotype on expression of salivary biomarkers.