Are healing abutments being reused in periodontics residency programs in the United States? A survey-based study.

PURPOSE: Recent studies indicate that reusing healing abutments (HAs) may pose a risk of biomaterial cross contamination among patients. The intent is to investigate whether postgraduate periodontics residency programs in the United States are reusing dental implant HAs and determine if there is a standardization in the decontamination and sterilization protocol of used HAs. METHODS: An electronic survey consisting of-seven multiple choice and/or short answer questions pertaining to the re-use of HAs among postdoctoral periodontics programs was sent to program directors of 57 accredited dental schools in the United States via an online survey system (Qualtrics). Three follow-up remainder emails were sent to programs that did not respond after over a 6-month period. Data were analyzed using descriptive statistics. RESULTS: Of the 57 postdoctoral periodontics program directors contacted, only 14 responded with three programs (3/14, 21%) reported reusing HAs. Approximately, 46% stated their residents place dental implants in two stages, while ∼54% stated they used a one-stage protocol indicating varied time exposure of HA to the oral cavity. Even in a two-stage protocol, the extended time HA remained in situ varied from 4 weeks to 6 months. Each program reusing HAs employed a distinct decontamination approach highlighting a notable lack of standardization in practices. CONCLUSION: The findings from our study suggest that a minority of residency programs in the United States are reusing HAs. However, the limited number of responses leaves uncertainty regarding whether our findings underestimate the prevalence of this practice and accurately reflect the reality. Among those re-using HAs, there seems to be a lack of standardization in their decontamination, potentially leading to cross-contamination of residual biomaterial among patients.

Assessment of Detoxification Strategies for Used Dental Implant Healing Abutments: Macroscopic and Biological Implications.

PURPOSE: Dental implant manufacturers recommend healing abutments (HA) be used for single-patient use; however, reuse on multiple patients following decontamination and sterilization is common. This study aims to evaluate four decontamination strategies utilizing enzymatic agents, available in most clinical settings, to determine the level to which biomaterial can be removed in a group of previously used HA (uHA). Secondly, to determine the degree to which the decontaminated HA are capable of inducing an inflammatory response in-vitro compared to new, never used HA. MATERIALS AND METHODS: Fifty HA were collected following 2-4 weeks of intraoral use and distributed randomly into 5 test groups (Group A-E; n = 10/group). Group A: Enzymatic cleaner foam + Autoclave; Group B: Ultrasonic bath with enzymatic cleaner + Autoclave; Group C: Prophy jet + Enzymatic cleaner foam + Autoclave; Group D: Prophy jet + ultrasonic bath with enzymatic cleaner + Autoclave; Group E: Prophy jet + Autoclave. Ten new, sterile HA served as controls (Group "Control"). Residual protein concentration was determined by a Micro BCA protein assay while HA from each group were stained with Phloxine B and macroscopically examined for the presence of debris. To examine the inflammatory potential, human primary macrophages were exposed to HA and supernatant levels of 9 cytokines/chemokines profiles were analyzed using a multiplex bead assay. RESULTS: All test groups presented with differences in the degree of visual decontamination compared to Controls, with Groups D and E displaying the most effective surface debris removaland reduced protein concentration. Of the detoxification strategies, Groups D and E removed the greatest biomaterial while least effective was Group A. However, compared to Controls, multiplex assays revealed high levels of inflammatory cytokine secretion up to 5 days from all Test Groups (A-E) irrespective of the decontamination method used. CONCLUSION: Our study found that compared to new, never used HA, decontamination of uHA utilizing enzymatic cleaners failed to reestablish inert HA surfaces and prevent an inflammatory immune response in-vitro. Clinicians should not reuse HA even after attempts to decontaminate and sterilize HA surfaces.

Dynamic Changes in Macrophage Polarization during the Resolution Phase of Periodontal Disease.

Periodontal inflammation is largely governed by infiltration of myeloid cells, in particular macrophages. Polarization of Mφ within the gingival tissues is a well-controlled axis and has considerable consequences for how Mφ participate in inflammatory and resolution (tissue repair) phases. We hypothesize that periodontal therapy may instigate a pro-resolution environment favoring M2 Mφ polarization and contribute towards resolution of inflammation post-therapy. We aimed to evaluate the markers of macrophage polarization before and after periodontal therapy. Gingival biopsies were excised from human subjects with generalized severe periodontitis, undergoing routine non-surgical therapy. A second set of biopsies were excised after 4-6 weeks to assess the impact of therapeutic resolution at the molecular level. As controls, gingival biopsies were excised from periodontally healthy subjects, undergoing crown lengthening. Total RNA was isolated from gingival biopsies to evaluate pro- and anti-inflammatory markers associated with macrophage polarization by RT-qPCR. Mean periodontal probing depths, CAL and BOP reduced significantly after therapy and corroborated with the reduced levels of periopathic bacterial transcripts after therapy. Compared to heathy and treated biopsies, higher load of Aa and Pg transcripts were observed in disease. Lower expression of M1Mφ markers (TNF-α, STAT1) were observed after therapy as compared to diseased samples. Conversely, M2Mφ markers (STAT6, IL-10) were highly expressed in post-therapy as opposed to pre-therapy, which correlated with clinical improvement. These findings corroborated with murine ligature-induced periodontitis and resolution model, comparing the respective murine Mφ polarization markers (M1 Mφ: cox2 , iNOS2 and M2 Mφ: tgm2 and arg1 ). Our findings suggest that imbalance in M1 and M2 polarized macrophages by assessment of their markers can provide relevant clinical information on the successful response of periodontal therapy and can be used to target non-responders with exaggerated immune responses.

Downregulation of miRNA-26 in chronic periodontitis interferes with innate immune responses and cell migration by targeting phospholipase C beta 1.

AIM: To evaluate the potential role of miR-26 family members in periodontal pathogenesis by assessing innate immune responses to periopathic bacteria and regulation of cytoskeletal organization. MATERIALS AND METHODS: Expression of miR-26a-5p and miR-26b-5p was quantified in gingival biopsies derived from healthy and periodontally diseased subjects before and after non-surgical (scaling and root planing) therapy by RT-qPCR. Global pathway analysis and luciferase assays were performed for target identification and validation. Cytokine expression was assessed in miR-26a-5p transfected human oral keratinocytes upon stimulation with either live Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans or Pg lipopolysaccharide (LPS). Wound closure assays were performed in cells transfected with miR-26a-5p, while the impact on cytoskeletal organization was assessed by F-actin staining. RESULTS: miR-26a-5p and miR-26b-5p were downregulated in diseased gingiva and restored 4-6 weeks post-therapy to levels comparable with healthy subjects. Target validation assays identified phospholipase C beta 1 as a bona fide novel target exhibiting antagonistic expression pattern in disease and post-therapy cohorts. miR-26a-5p transfected cells secreted higher levels of cytokine/chemokines upon stimulation with periopathogens and demonstrated impaired cell migration and cytoskeletal rearrangement. CONCLUSIONS: Downregulated miR-26a-5p levels in periodontal inflammation may interfere with key cellular functions that may have significant implications for host defence and wound healing.

Association between ridge morphology and complexity of implant placement planning in the posterior mandible.

STATEMENT OF PROBLEM: Uncertainties regarding the 3D ridge morphology of the posterior mandible can greatly increase the risk of surgical complications during dental implant placement. By using cone beam computed tomography (CBCT) imaging and a computer-guided implant treatment software program before any invasive procedure, it is possible to assess ridge morphology and understand the surgical complexity and risk level. PURPOSE: The purpose of this radiological clinical study was to evaluate a large series of CBCT images to evaluate ridge shape variations along posterior mandibular edentulous regions and to clarify their associations with the level of implant planning complexity. MATERIAL AND METHODS: One hundred and twenty CBCT files were analyzed retrospectively for a total 240 hemimandibular sites. Images of each edentulous region of the mandibular first and second premolar and first and second molar sites were evaluated in the sagittal plane. Ridge morphology and implant planning complexity per site were assessed. Categorical variables were presented as number of events and percentages. The chi-square test was used to compare the categorical variables (P=.05). RESULTS: Of 491 partially edentulous mandibular sites, 235 were on the right, and 256 were on the left. Forty-two sites had a distal adjacent tooth, while 266 sites had no distal adjacent tooth. The sagittal bone sections demonstrated oblique (40.53%), straight (31.77%), S-shape (24.24%), hourglass (2.44%), and basal bone (1.02%) ridge morphologies. Implant complexity was deemed straightforward in 66.19% of sites, while 31.6% were identified as advanced and 2.54% as complex. When ridge morphology was evaluated from straight to basal-round bone shape, the implant complexity followed the same trend of change from a straightforward to complex procedure (P=.001) for edentulous second and first molar regions. No significant differences were noted at edentulous second premolar sites (P=.063). The missing second molar sites with oblique morphology were predicted to have 60.9% straightforward complexity, and first molar sites with oblique morphology had 55.8% straightforward implant complexity. Second premolars with straight ridge morphology had 71.7% straightforward complexity, whereas first premolars with the same shape had 92.5% straightforward implant complexity. CONCLUSIONS: Careful evaluation of sagittal CBCT images can provide significant clinical information regarding ridge shape and anticipated surgical complexity before and at the time of implant placement. Surgical complexity is greatest at the most posterior mandibular edentulous sites, and extra attention and caution should be exercised during the surgical planning phases of implant surgery.

Peri-implant disease education and diagnosis in the pre-doctoral curriculum.

PURPOSE: The purpose of this study was to assess how pre-doctoral periodontal programs in the United States of America are educating their dental students regarding the management of peri-implant diseases and secondarily, to determine if a current standard of teaching exists. METHODS AND MATERIALS: Electronic surveys were distributed to pre-doctoral program directors across 57 dental schools in the United States via a secure online survey system. The survey consisted of 19 questions pertaining to curriculum structure involving didactic and clinical management of peri-implant diseases. RESULTS: A total of 25 program directors (44%) responded, and data were analyzed using descriptive statistics. The results indicated a lack of standardization of pre-doctoral didactic and clinical curriculum among dental schools. CONCLUSIONS: Data pooled from 25 pre-doctoral periodontal programs in the United States show that there is currently no standardization in the dental school curriculum related to the didactic and clinical management of peri-implant diseases. The development of standardized content is recommended to assist program directors in assessing and enhancing educational experiences for dental students on the management of peri-implant diseases.

Human herpesvirus-encoded MicroRNA in host-pathogen interaction.

Human herpesviruses (HHV) are ubiquitous, linear dsDNA viruses that establish lifelong latency, disrupted by sporadic reactivation. HHV have evolved diverse ingenious mechanisms to evade robust host defenses. Incorporation of unique stem loop sequences that generate viral microRNAs (v-miRs) exemplifies one such evolutionary adaptation in HHV. These noncoding RNAs can control cellular and viral transcriptomes highlighting their ability in shaping host-HHV interactions. We summarize recent developments in functional characterization of HHV-encoded miRNAs in shaping the outcome of host-pathogen interaction. Non-immunogenic dissemination of v-miRs through exosomes confer added advantage to HHV in incessant modulation of host microenvironment. This review delineates the mechanistic role of v-miRs in facilitating viral persistence and tropism by targeting genes associated with cellular (apoptosis, angiogenesis, cell migration, etc.) and viral life cycle (latency, lytic and reactivation). Burgeoning evidences indicate plausible association of v-miRs in various immune-mediated diseases (nasopharyngeal carcinoma, neurological disorders, periodontal diseases, etc.) and herpesvirus-related malignancies indicating their broad-spectrum impact on host cellular pathways. We propose to exploit tisssue and systemic levels of v-miRs as diagnostic and prognostic markers for cancers and immune-mediated diseases. Therapeutic targeting of v-miRs will advance the promising outcomes of preclinical discoveries to bedside application.

Evidence-Based Clinical Outcomes of Immediate and Early Loading of Short Endosseous Dental Implants: A Meta-analysis.

PURPOSE: Short dental implants serve as a valuable alternative for patients with limited bone height. Immediate or early provisionalization facilitates a more physiologic environment for the gingival tissues to be modeled. The purpose of this meta-analysis was to systematically review and evaluate the implant survival and marginal bone loss with immediate and early loading protocols of short dental implants (≤ 6 mm). MATERIALS AND METHODS: A literature search (electronic and manual) was conducted to identify studies with a focused PICO question: "In patients with short dental implants, does loading time affect treatment outcomes?" Studies using an immediate or early loading protocol for restoration of short implants with a mean follow-up of at least 1 year, and refraining from the use of advanced surgical procedures (sinus floor elevation, bone augmentation), were included. After evaluating patient selection and outcome reporting biases, a meta-analysis was conducted to assess implant survival and bone loss for studies fulfilling the inclusion criteria. Bone loss differences between immediate and early loading protocols were evaluated by Student t test, and Spearman correlation analysis was used to analyze the trends between crown-to-implant (C/I) ratio and bone loss. RESULTS: A total of 396 studies with patients receiving short implants (≤ 6 mm) with immediate or early prosthetic loading protocols were identified. For the 7 included studies, the pooled implant survival rate for 322 implants with a follow-up ranging from 1 to 10 years (5 years) was 91.63% (95% CI: 88% to 94%), with a mean bone loss effect estimate of 0.52 ± 0.1 mm (z = 3.07, P < .002). The differences observed in the mean bone loss for studies using immediate loading as opposed to early loading were not statistically significant. A moderate but significant positive correlation was observed between the C/I ratio and mean bone loss levels (r = 0.67, P = .02). CONCLUSION: Short implants with immediate or early loading protocols have satisfactory long-term treatment prospects with satisfactory implant survival rates and minimal bone loss.

Used dental implant healing abutments elicit immune responses: A comparative analysis of detoxification strategies.

PURPOSE: To determine if healing abutments (HA) can be "decontaminated" using four strategies available in clinical settings and compare the detoxification efficacy by quantifying residual biomaterial and capacity to elicit an inflammatory response in-vitro. MATERIALS AND METHODS: Forty HA collected from subjects following intraoral use were randomly distributed into four test groups (A-D): A: autoclave only, B: ultrasonic bath plus autoclave, C: prophy-jet plus autoclave, and D: Scrub sponge plus autoclave. New, sterile HA: group E (Control). Residual protein concentration was determined by Micro BCA assay and stained with Phloxine B for macroscopic examination. HA were placed in human CD14+ monocyte derived-macrophage (mo-Mφ) cultures and supernatant collected at 4, 24, 48, and 5 days to analyze cytokine profiles using multiplex bead assay. RESULTS: Test groups showed visible differences in "decontamination" levels compared to control. Groups C and D showed most effective debris removal and lowest residual protein concentration. Multiplex assay showed marked induction of pro-inflammatory cytokines by groups A and B and to a significantly lower level by groups C and D. CONCLUSION: HA were not entirely "decontaminated" using common methods available relative to new, sterile HA and were capable of stimulating an immune response.

Impaired cell migration and structural defects in myeloid cells overexpressing miR-30b and miR-142-3p.

Macrophages (MΦ) and dendritic cells (DC) play a fundamental role in shaping immune responses by sensing a plethora of Pathogen Associated Molecular Patterns (PAMPs), phagocytosis and antigen presentation to T lymphocytes. These important biological processes require efficient cell movement and an intact cellular morphology for dynamic interaction. The role of microRNAs (miRs) in this regard, however, is not well understood. In the present study, we show that miR-30b and miR-142-3p regulate migration and morphology of MΦ and DC. Transient overexpression of miR-30b and miR-142-3p attenuates migration and these cells display unique morphological deformities observed under electron microscopy. In addition, miR-142-3p overexpression in MΦ impaired phagocytosis of FITC-conjugated latex beads using live microscopy imaging. Interestingly, live cell imaging and F-actin staining revealed marked changes in the cell polarity and actin polymerization status, respectively. To identify miR-142-3p regulated pathways, we profiled global transcriptome changes in miR-142-3p or control mimic transfected DC. Expression of several genes were differentially altered by miR-142-3p and were associated with pathways related to cell movement, cell adhesion, and cytoskeletal rearrangement. Bioinformatics analysis identified a significant subset of downregulated genes with one or more predicted miR-142-3p binding sites in their 3'UTR strongly suggesting direct post-transcriptional impact of these miRNAs on multiple transcripts. Using dual luciferase assays, novel miR-142-3p binding sites were validated for three genes (Vinculin, Dab2 and Skap2) directly associated with cytoskeletal rearrangement and cell movement. In summary, our results show that miR-30b and miR-142-3p are regulators of myeloid cell cytoskeletal homeostasis and morphology.

Quantity and Quality of Intraoral Autogenous Block Graft Donor Sites with Cone Beam Computed Tomography.

PURPOSE: The autogenous bone block graft is regarded as the gold standard material due to reported osteoconductive, osteoinductive, and osteogenic properties. Various intraoral donor sites for autogenous block grafts are presented in the literature. The aim of this study was to radiographically evaluate the maximum dimensions, volume, and bone quality values of these sites. MATERIALS AND METHODS: According to the inclusion criteria, 50 cone beam computed tomography (CBCT) images from 50 subjects were evaluated. The maximum length, width, height, and volume of autogenous regions where block grafts could be harvested were measured. Radiographic bone quality was calculated by using Hounsfield units derived from CBCT (CBCT-HU). RESULTS: The mean age of 50 subjects (19 men and 31 women) was 55.84 ± 15.9 years. In this study, the symphysis was the largest potential donor site (3.14 ± 1.05 cm3), while maxillary tuberosity was the smallest (0.53 ± 0.34 cm3). These results correlated with bone density values, where the symphysis retained the highest values (937.31 ± 160.59 CBCT-HU) and the maxillary tuberosity had the lowest values (360.87 ± 141.48 CBCT-HU). CONCLUSION: Intraoral bone blocks have restrictions due to surrounding vital anatomical structures. The surgeons should consider these vital structures using accurate CBCT evaluation. The volume and density of the maximal bone harvest from the symphysis was statistically higher in comparison with ramus, palatal, and maxillary tuberosity bone blocks.

In-depth morphological evaluation of tooth anatomic lengths with root canal configurations using cone beam computed tomography in North American population.

OBJECTIVE: This study aimed to assess the association between tooth size and root canal morphology by using CBCT analysis. METHODOLOGY: In this retrospective study, tooth anatomic lengths (crown and root lengths, buccolingual and mesiodistal dimensions) of 384 patients were assessed and correlated with Vertucci's root canal morphology classification. Data was analyzed for gender-related differences using the independent sample t-test, ANOVA, and the Pearson's correlation for a possible relation between anatomic lengths and canal morphology. RESULTS: The maxillary first and second premolars showed a greater predilection for Type IV and II variants, respectively, while the mandibular first premolar showed a greater predilection for Type II canal system. The root canal system of the mandibular second premolar showed maximal diversity (47% Type I, 30% Type II, and 20% Type III). The dimensions were greater in men regardless of tooth type. The most significant relation (p<0.05) between the anatomic size and canal morphology was observed in the maxillary first premolars, followed by the mandibular canines (buccolingual dimension) and the lower second premolars (crown length). Negative correlations existed between the crown length and the patient's age for the anterior teeth and mandibular second premolar (r=-0.2, p<0.01). CONCLUSIONS: The most common canal formation for anterior teeth was the Type I. The anatomic lengths had the strongest influence on the canal configuration of the maxillary first premolar, with Type IV being the most common root canal system. The mandibular second premolars showed maximal diversity in the canal classification terms and had a significant correlation with their crown lengths. CLINICAL RELEVANCE: The complex relationship between the canal morphology and anatomic tooth sizes need meticulous awareness and recognition during endodontic procedures, in conjunction with the demographic variabilities.

In-depth morphological evaluation of tooth anatomic lengths with root canal configurations using cone beam computed tomography in North American population

Abstract Objective This study aimed to assess the association between tooth size and root canal morphology by using CBCT analysis. Methodology In this retrospective study, tooth anatomic lengths (crown and root lengths, buccolingual and mesiodistal dimensions) of 384 patients were assessed and correlated with Vertucci's root canal morphology classification. Data was analyzed for gender-related differences using the independent sample t-test, ANOVA, and the Pearson's correlation for a possible relation between anatomic lengths and canal morphology. Results The maxillary first and second premolars showed a greater predilection for Type IV and II variants, respectively, while the mandibular first premolar showed a greater predilection for Type II canal system. The root canal system of the mandibular second premolar showed maximal diversity (47% Type I, 30% Type II, and 20% Type III). The dimensions were greater in men regardless of tooth type. The most significant relation (p<0.05) between the anatomic size and canal morphology was observed in the maxillary first premolars, followed by the mandibular canines (buccolingual dimension) and the lower second premolars (crown length). Negative correlations existed between the crown length and the patient's age for the anterior teeth and mandibular second premolar (r=−0.2, p<0.01). Conclusions The most common canal formation for anterior teeth was the Type I. The anatomic lengths had the strongest influence on the canal configuration of the maxillary first premolar, with Type IV being the most common root canal system. The mandibular second premolars showed maximal diversity in the canal classification terms and had a significant correlation with their crown lengths. Clinical Relevance The complex relationship between the canal morphology and anatomic tooth sizes need meticulous awareness and recognition during endodontic procedures, in conjunction with the demographic variabilities.

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.

The Role of Implant-Tooth Distance on Marginal Bone Levels and Esthetics.

PURPOSE: The peri-implant bone and mucosa architecture contribute to the health and esthetics of single-tooth dental implants. The implant-tooth distance (ITD) has been regarded as a key determinant of their outcomes. This study was conducted to determine the relationship between ITD and peri-implant bone, mucosa, and pink esthetic scores (PES) for anterior single-tooth implants. MATERIALS AND METHODS: For 44 dental implants with a microthread conical abutment interface design placed in 38 participants, periapical radiographs and photographs were evaluated at 1 and 4 years to assess interproximal bone levels and PES. RESULTS: Mean mesial and distal marginal bone level change over 4 years was 0.20 ± 1.00 mm and 0.20 ± 0.74 mm, respectively. In this cohort there was no relationship between ITD and interproximal bone changes or papilla fill at 4 years; however, marginal bone changes influenced PES score-the smaller the ITD, the lower the PES (P < .001). Alone, ITD did not influence marginal bone levels or papilla in this cohort. CONCLUSION: These results imply a complex relationship between ITD, marginal bone levels, and PES scores for single-tooth implants.

Dysregulation of human miRNAs and increased prevalence of HHV miRNAs in obese periodontitis subjects.

AIM: To evaluate human and herpesvirus-encoded microRNA (miRNA) expression in healthy and diseased gingiva of obese and non-obese subjects and compare the impact of localized and systemic inflammation on human miRNA profiles. MATERIAL AND METHODS: Healthy and inflamed gingival biopsies were collected from obese and non-obese subjects. Human and herpesvirus miRNA expression was quantified using quantitative PCR. Predicted targets of dysregulated miRNAs were identified using bioinformatics analysis, validated by dual luciferase assays and their expression assessed in healthy and diseased tissues. RESULTS: Our results show differential expression of miRNAs in both diseased groups compared to healthy counterparts. MMP-16 is identified as a novel target of miRNAs altered in disease. Expression analysis of genes predicted as target of differentially expressed miRNAs show significant changes in disease compared with healthy tissues. Finally, quantitation of four herpesvirus-derived viral miRNAs show that the expression and prevalence of herpesvirus miRNAs in diseased gingiva of obese subjects. CONCLUSION: Our findings show that miRNA (both cellular and virus) expression is differentially responsive to local and systemic inflammation. Some of these miRNAs can modulate key cellular genes with direct consequences on inflammatory pathways suggesting their impact on oral tissue transcriptome and functions.

Herpesviruses and MicroRNAs: New Pathogenesis Factors in Oral Infection and Disease?

The oral cavity incessantly encounters a plethora of microorganisms. Effective and efficient oral innate and adaptive immune responses are incumbent to maintain healthy mucosa. A higher prevalence of Human Herpesviruses (HHV), a family of large enveloped DNA viruses, has been reported in multiple oral inflammatory diseases suggesting their involvement in disease progression. However, the viral components contributing to oral disease remain obscure. MicroRNAs (miRNA) are non-protein coding, single stranded ribonucleic acid (RNA) molecules that post-transcriptionally regulate diverse messenger RNAs. Thus, miRNAs can control large repertoire of biological processes. Changes in miRNA expression are associated with various oral infections and diseases. Cellular miRNAs can act as pro- or anti-viral factors and dysregulation of host miRNA expression occurs during herpesviruses infection. This strongly suggest a critical role of cellular miRNAs in host-herpesvirus interaction. Interestingly, HHV also encode multiple miRNAs (called viral miRNAs) that may play key role in host-pathogen interaction by modulating both host biological pathways and controlling viral life cycle. Recent studies from our laboratory have identified viral miRNAs (v-miRs) in diseased oral tissue biopsies and demonstrate their immunomodulatory roles. This review discusses the association of miRNAs (both host and viral) and herpesviruses in the pathogenesis of oral inflammatory diseases.

MicroRNAs and immunity in periodontal health and disease.

MicroRNAs (miRNAs) are critical regulators of the host immune and inflammatory response against bacterial pathogens. In the present review, we discuss target genes, target gene functions, the potential regulatory role of miRNAs in periodontal tissues, and the potential role of miRNAs as biomarkers and therapeutics. In periodontal disease, miRNAs exert control over all aspects of innate and adaptive immunity, including the functions of neutrophils, macrophages, dendritic cells and T and B cells. Previous human studies have highlighted some key miRNAs that are dysregulated in periodontitis patients. In the present study, we mapped the major miRNAs that were altered in our reproducible periodontitis mouse model relative to control animals. The miRNAs that were upregulated as a result of periodontal disease in both human and mouse studies included miR-15a, miR-29b, miR-125a, miR-146a, miR-148/148a and miR-223, whereas miR-92 was downregulated. The association of individual miRNAs with unique aspects of periodontal disease and their stability in gingival crevicular fluid underscores their potential as markers for periodontal disease progression or healthy restitution. Moreover, miRNA therapeutics hold great promise for the future of periodontal therapy because of their ability to modulate the immune response to infection when applied in conjunction with synthetic antagomirs and/or relatively straightforward delivery strategies.

In silico prediction of cellular gene targets of herpesvirus encoded microRNAs.

Herpesviruses have evolved to encode multiple microRNAs [viral miRNAs (v-miRs)], a unique feature of this family of double stranded DNA (dsDNA) viruses. However, functional role of these v-miRs in host-pathogen interaction remains poorly studied. In this data, we examined the impact of oral disease associated v-miRs viz., miR-H1 [encoded by herpes simplex virus 1 (HSV1)] and miR-K12-3 [encoded by Kaposi sarcoma-associated herpesvirus (KSHV)] by identifying putative targets of viral miRNAs. We used our published microarray data (GSE107005) to identify the transcripts downregulated by the v-miRs. The 3' untranslated region (UTR) of these genes were extracted using BioMart tool on Ensembl and subjected to RNA:RNA interaction employing RNA Hybrid. We obtained hundreds of potential and novel miR-H1 and miR-K12-3 binding sites on the 3'UTR of the genes downregulated by these v-miRs. The information can provide likely regulatory mechanisms of the candidate v-miRs through which they can exert biological impact during herpesvirus infection and pathogenesis.

Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses.

Prevalence of the members of herpesvirus family in oral inflammatory diseases is increasingly acknowledged suggesting their likely role as an etiological factor. However, the underlying mechanisms remain obscure. In our recent miRNA profiling of healthy and diseased human tooth pulps, elevated expression of human herpesvirus encoded viral microRNAs (v-miRs) were identified. Based on the fold induction and significance values, we selected three v-miRs namely miR-K12-3-3p [Kaposi sarcoma-associated virus (KSHV)], miR-H1 [herpes simplex virus 1 (HSV1)], and miR-UL-70-3p [human cytomegalovirus (HCMV)] to further examine their impact on host cellular functions. We examined their impact on cellular miRNA profiles of primary human oral keratinocytes (HOK). Our results show differential expression of several host miRNAs in v-miR-transfected HOK. High levels of v-miRs were detected in exosomes derived from v-miR transfected HOK as well as the KSHV-infected cell lines. We show that HOK-derived exosomes release their contents into macrophages (Mφ) and alter expression of endogenous miRNAs. Concurrent expression analysis of precursor (pre)-miRNA and mature miRNA suggest transcriptional or posttranscriptional impact of v-miRs on the cellular miRNAs. Employing bioinformatics, we predicted several pathways targeted by deregulated cellular miRNAs that include cytoskeletal organization, endocytosis, and cellular signaling. We validated three novel targets of miR-K12-3-3p and miR-H1 that are involved in endocytic and intracellular trafficking pathways. To evaluate the functional consequence of this regulation, we performed phagocytic uptake of labeled bacteria and noticed significant attenuation in miR-H1 and miR-K12-3-3p but not miR-UL70-3p transfected primary human Mφ. Multiple cytokine analysis of E. coli challenged Mφ revealed marked reduction of secreted cytokine levels with important roles in innate and adaptive immune responses suggesting a role of v-miRs in immune subversion. Our findings reveal that oral disease associated v-miRs can dysregulate functions of key host cells that shape oral mucosal immunity thus exacerbating disease severity and progression.