Periodontal disease is a chronic inflammation of the gingiva and periodontium that leads to progressive destruction and irreversible damage to the supportive structures of the teeth. It affects nearly half of the United States population and is a particular risk factor in adults older than 65 years of age. Oral microorganisms assemble in plaque as a polymicrobial biofilm and Porphyromonas gingivalis, an important secondary colonizer in oral biofilms, has been implicated in periodontal disease. Although the protective functions of various salivary molecules such as antimicrobial proteins have been delineated, lipids present in saliva and on the oral mucosa have been largely ignored and there is growing evidence that the role of lipids in innate immunity is more important than previously realized. In fact, recent studies suggest that sphingoid bases and fatty acids, which exhibit potent broad spectrum antimicrobial activity against a variety of bacteria and fungi, are likely important innate immune molecules involved in the defense against oral bacterial and fungal infections. However little is known about their spectrum of activity or mechanisms of action. In addition, the effects of these lipids that are endogenous to the oral cavity have not been explored against oral bacteria. In this study I hypothesized that oral mucosal and salivary lipids exhibit dose-dependent antimicrobial activity against P. gingivalis and alter cell morphology and metabolic events. To test this hypothesis, I first examined the effects of two fatty acids: sapienic acid and lauric acid, and three sphingoid bases: sphingosine, dihydrosphingosine, and phytosphingosine, against a variety of gram-positive and gram-negative bacteria including P. gingivalis. Using broth microdilution assays to determine minimum inhibitory and minimum bactericidal concentrations, I show that antimicrobial activity against bacteria is dose-dependent, lipid specific, and microorganism specific. Kill kinetics were also variable across each bacteria-lipid combination. Upon examination of select bacteria-lipid combinations via scanning and transmission electron microscopy, different morphologies were evident across all treatments, demonstrating differential activity of each lipid for a particular bacterium as well as for each bacterium across different lipids. In addition, all sphingoid bases and fatty acids were taken up and retained in association with P. gingivalis cells and could be extracted along with bacterial lipids and separated using thin layer chromatography. Using a combination of two-dimensional in-gel electrophoresis and Western blots followed by mass spectroscopy and n-terminus degradation sequencing, I show that sapienic-acid treatment induces a unique stress response in P. gingivalis, as evidenced by the ability of P. gingivalis to upregulate a set of proteins involved in fatty acid biosynthesis metabolism and energy production, protein processing, cell adhesion, and virulence. Finally, utilizing flow cytometry and confocal microscopy, I assessed the effects of oral antimicrobial lipids against a representative host cell and describe oral lipid concentrations that are both antimicrobial to P. gingivalis cells and non-cytotoxic to the representative host cells tested. Combined, these data strongly suggest that sphingoid bases and fatty acids found within the saliva and on oral mucosa likely do contribute to the innate antimicrobial activity of saliva, mucosal surfaces, and skin and this dose-dependent activity is both lipid specific and bacteria specific. This information adds to current knowledge of the innate functions of endogenous lipids in the oral cavity. With bacterial resistance to current antibiotics increasing, the exploration of new antimicrobial agents is important and these lipid treatments may be beneficial for prophylactic treatments or therapeutic intervention of infection by supplementing the natural immune function of endogenous lipids on skin and other mucosal membranes.

Sphingoid bases (sphingosine, dihydrosphingosine and phytosphingosine) have been recently found in the oral cavity where they may serve to fortify innate immunity against commensals and periodontal pathogens. In fact, sphingoid bases have potent antimicrobial activity against Gram- positive and Gram- negative bacteria including oral pathogens like Porphyromonas gingivalis. It is not known whether these lipids are cytotoxic or alter the chemokine and cytokine responses of human dendritic cells, a finding important to their future potential as a therapeutic for treatment of periodontal disease. Objectives: The objective of this study was to determine the effects of sphingoid bases on the cytotoxicity and cytokine responses of human myeloid dendritic cells. Methods: Dendritic cells were treated with sphingoid bases (0.2-80.0 μM) for 16 hours in the presence or absence of 0.02 μM hemagglutinin B, a nonfimbrial adhesin of P. gingivalis used as a pro-inflammatory stimulus. The cytotoxicity of the inocula and its ability to induce the production of chemokines and pro-inflammatory cytokines was determined after 16 hours. Results: Higher concentrations of sphingoid bases were cytotoxic (e.g., 40.0-80.0 μM), but physiologic concentrations of sphingoid bases (e.g., 0.2-20.0 μM) were not. At 5, 10, or 20 μM, sphingosine did not enhance or attenuate any HagB-induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. At 5 or 10 μM, neither phytosphingosine nor dihydrosphingosine enhanced or attenuated any HagB- induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. Conclusion: Sphingoid bases exhibit dose-dependent cytotoxicity and cytokine responses against human myeloid dendritic cells. But at physiologic concentrations sphingoid bases appear to be safe and efficacious at the doses needed to prevent or treat microbial infections in the oral cavity.

Chlorhexidine mouth rinses remove dental plaque from teeth, preventing dental caries, dental decay, and more serious systemic infections. Tooth discoloration due to extrinsic staining is the most prominent side effect of regular chlorhexidine use. Decreasing the concentration of chlorhexidine reduces staining area and severity. Staining can also be prevented and treated by tooth bleaching, a common cosmetic dental procedure that diminishes extrinsic staining by oxidizing chromagens adsorbed onto the tooth surface. In the proposed trial, common bleaching agents will be investigated for their efficacies in preventing chlorhexidine staining. Results of the trial could further the development of a chlorhexidine mouth rinse that is suitable for long term use.

Previous studies suggest that Medicaid-enrolled children have difficulties accessing dental care, which can lead to untreated dental disease, poor oral health, and compromised overall health status. While Medicaid-enrolled children with a chronic condition (CC) encounter additional barriers to dental care, most relevant studies on dental utilization fail to adopt risk adjustment methods. As such, the impact of CC status and CC severity on access to dental care for Medicaid-enrolled children is poorly understood. The main objectives of this dissertation were to: 1) compare dental utilization for Medicaid-enrolled children with and without a CC; 2) assess the relationship between CC severity and dental utilization; and 3) identify the other factors associated with dental utilization. The 3M Clinical Risk Grouping (CRG) Methods were applied to enrollee-level data from the Iowa Medicaid Program (2003-2008) to identify children with and without a CC and to classify children with a CC into a CC severity level. Three outcome measures were developed: 1) access to an annual dental visit; 2) use of dental services under general anesthesia (GA); and 3) time to the first dental visit after initial enrollment into the Medicaid program. We used multiple variable logistic regression models and survival analytic techniques to test our study hypotheses. Compared to Medicaid-enrolled children without a CC, those with a CC were more likely to have had an annual dental visit and earlier first dental visits. Having a CC was an important determinant of dental utilization under GA for older but not for younger Medicaid-enrolled children. In terms of CC severity, Medicaid-enrolled children with more severe CCs were less likely to have had an annual dental visit and more likely to have utilized dental services under GA. CC severity was not associated with the rate at which the first dental visit took place. Not residing in a dental Health Professional Shortage Area, previous use of dental care, and previous utilization of primary medical care were all positively associated with dental utilization. Identifying and understanding the determinants of access to dental care is an important first step in developing clinical interventions and policies aimed at improving access to dental care for all Medicaid-enrolled children. Future work should focus on identifying the socio-behavioral determinants of as well as the clinical outcomes associated with access to dental services for vulnerable children.

Background P. gingivalis, a non-motile, rod-shaped, anaerobic, Gram-negative bacterium is one of the principal sources of periodontal disease. It possesses a number of potential virulence factors thought to be important in the disease process including 5 hemagglutinins (Hag). One of these is HagB. It is a well characterized nonfimbrial adhesin expressed on the surface of P. gingivalis. HagB is very pro-inflammatory and induces robust chemokine and cytokine responses in vitro and in vivo. Since the chemokine and cytokine responses seen from single cells grown in tissue culture often are not representative of the chemokine and cytokine profiles seen in clinical samples or biopsy specimens, we devised a co-culture model of keratinocytes, dendritic cells, and T-cells to test the hypothesis that chemokine and cytokine responses from co-cultured cells would be more representative of responses seen in clinical samples from individuals with periodontal disease than single cell models. Methods and materials HagB was prepared by cloning hagb of P. gingivalis (1.4 kb) into the vector pQE31 (QIAGEN Inc., Valencia, CA); expressed in E. coli M15(pREP4)pQE31-TX1; and isolated from E. coli lysates by affinity chromatography using a Ni-charged resin (Profinity IMAC Resin, BioRad, Hercules, CA) and examined by SDS-PAGE. Co-culture models were treated with 10 µg/ml HagB (Test) or 10 µg/ml HagB diluent (Control). At 64 hours the supernatants were collected. Chemokine and cytokine biomarkers GM-CSF, CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), IL-1α, IL-6, IL-8, TNFα, IL-12(p40), and VEGF responses were determined using Milliplex immunoassays. HagB responses were corrected by subtracting the constitutive responses detected in supernatants incubated with HagB diluent. Statistical differences among groups were determined on Log10 transformed biomarker concentration using JMP 10 (version 10.0, SAS, CAR; NC). Results Buffers (e.g. HagB diluent) did not induce a chemokine or cytokine response, however there was a gradual increase in chemokine and cytokine responses from cells at 64 hours. These were subtracted from HagB induced responses. Responses generally fell in 2 groups; in one group containing VEGF, IL-12(p40), IL-6, RANTES and GM-CSF, there were no significant differences among groups (p>0.05). In another group containing IL-1α, IL-8, MIP-1α, MIP-1β and TNF-α, there were significant differences among groups (p< 0.05). Interestingly these resulting responses fell in 2 categories- GM-CSF, IL-12, IL-1α, IL-6 were less than 25pg/ml and IL-8, MIP-1α, MIP-1β, RANTES, TNFα and VEGF were more than 25pg/ml. Some responses were driven by a particular cell type e.g. GM-CSF produced by dendritic cells, RANTES produced by T- cells and VEGF produced by T cells. There were similar responses in HagB-induced IL-8, MIP-1β, MIP-1α and TNFα responses by dendritic cells + keratinocytes and dendritic cells + keratinocytes + T cells. Conclusions Co-culture models can more realistically produce chemokine and cytokine responses to agonists than individual cultures of cells, which is important for predicting and assessing novel therapeutic treatments of periodontal disease.

Porphyromonas gingivalis produces proteases capable of degrading cytokines, host heme proteins, and some antimicrobial peptides. In this work, I show that P. gingivalis culture supernatants fully or partially degrade human neutrophil peptide alpha-defensins and human beta-defensins after 30 minutes. This observation suggests that proteases from P. gingivalis degrade defensins and this activity could abrogate defensin-related innate immune functions.

Antimicrobial peptides are among the repertoire of host innate immune defenses. In mucosal immunity, the health-disease balance can be greatly modulated by the interplay between host immune factors and colonized microflora. Microbial ecology within dental plaque is constantly shaped by environmental factors present within the oral cavity. Several antimicrobial peptides are detected in saliva and their bactericidal activities against oral bacteria, including Streptococcus mutans, the primary etiologic agent of dental caries, have been clearly demonstrated. However, the role of these antimicrobial peptides in S .mutans ecology and host caries experience is not well-defined. We hypothesized that various strains of S. mutans possess different inherent susceptibility/resistance profiles to host salivary antimicrobial peptides and that host-specific quantities of these peptides may influence plaque colonization by particular S. mutans strains. S. mutans strains from subjects with variable caries experience were tested for susceptibility to a panel of antimicrobial peptides, including HNP-1-3, HBD-2-3 and LL-37, revealing that the susceptibilities of S. mutans to these peptides were strain-specific. S. mutans strains from high caries subjects showed greater resistance to these peptides at varying concentrations than those from caries-free subjects. In addition, when combinations of these peptides were tested, they showed either additive or synergistic interaction against S. mutans. Determinations of the salivary levels of these peptides showed that their concentrations were highly variable among subjects with no correlation to host caries experience. However, positive relationships between the salivary concentrations of HNP-1-3 and MS in dental plaque were found. Additionally, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. An analysis of the salivary peptide concentrations and the susceptibility profiles of S. mutans strains showed that S. mutans strains obtained from subjects with higher concentration of HNP-1-3 in saliva appeared to be more resistant to HNP-1. Collectively, our findings showed that salivary antimicrobial peptides affect S. mutans ecology by restricting the overall growth of this bacterium within the oral cavity and that their activity may help select resistant strains of S. mutans to colonize within dental plaque. The relative ability of S. mutans to resist host salivary antimicrobial peptides may be considered a potential virulence factor for this species.

The objective was to determine if a correlation exists between Streptococcus mutans and DMFT in the oral cavity. This study examined the feasibility of microbial screenings as an additional caries predictor tool. The sample included 108 participants (ages 18-25) in low, moderate, and high socioeconomic groups. Subjects were selected from one dental clinic and one college in Virginia. Subjects were assessed for DMFT, salivary and plaque bacterial load, and CRA. Salivary load positively correlated with the DMFT. Plaque bacterial load and CRA negatively correlated with DMFT. Comparison of salivary bacterial load among economic levels showed higher bacterial loads with lower economic level only. Twice the observed values were found in the low socioeconomic level with CRA. DMFT and economic level showed differences between economic levels. Microbial screenings may be a useful, additional tool in determining caries risk in oral hygiene programs for all income populations.

Aim: The aim of this study is to determine the varying levels of salivary statherin production in patients with varying levels of risk for diabetes. The goal is to identify a causal relationship and thus, statherin could be used as a preliminary biomarker for identifying patients with diabetes. Materials and Methods: Saliva from 47 participants were collected in order to quantify the levels of statherin production via western blot analysis. Participants were also asked to fill out self-reported questionnaires regarding risk factors for type 2 diabetes. The questionnaire consisted of 7 questions regarding age, sex, history of diabetes, hypertension, level of physical activity, and weight class. Each individual factor as well as total risk for type two diabetes was compared to levels of salivary statherin levels via the unpaired t-test and one way ANOVA testing. Results: Risk factors for type two diabetes such as; age, sex, history of diabetes, hypertension, level of physical activity, and weight class showed no correlation to levels of salivary statherin secretion. All risk factors combined as a total risk level for type two diabetes also did not show a correlation to levels of salivary statherin secretion. Conclusions: It can be concluded in this study that salivary statherin protein does not show a correlation for risk or status of type two diabetes. Salivary statherin does not act as a useful biomarker for detection of type two diabetes.

Objectives: Tooth replacement therapy using endosseous implants has become an essential component of contemporary dental practice. While a plethora of factors determine clinical success, the bucco-lingual and apico-coronal dimensions of the peri-implant mucosa play an important role in both esthetics and the maintenance of peri-implant health. Studies, most of which treat mucogingival defects in the natural dentition, comparing acellular dermal matrix (ADM) and autologous subepithelial connective tissue grafts (sCTG) have shown similar clinical outcomes. The purpose of this non-inferiority trial is to determine the clinical efficacy of ADM in the augmentation of peri-implant mucosa thickness (PMT) as compared to an autologous sCTG in human adults. Methods: Twenty healthy adults treatment planned for a single tooth implant restoration in need of simultaneous peri-implant mucosa augmentation at the time of implant placement were recruited on the basis of an eligibility criteria. Patients were randomly assigned to the control group (autologous sCTG), or the experimental group (ADM allograft). Clinical measurements of mucosal thickness at the site were made with a periodontal probe and an endodontic spreader at baseline and 16 weeks post-op. These measurements were made by a masked, calibrated examiner. Gingival health, oral hygiene, wound healing and patient reported outcomes were also obtained. Mann-Whitney U tests were used to compare the mean mucosal thickness changes between the groups. Results: The mean gain in PMT was approximately 1.5mm in the control group and 0.8mm in the experimental group. When measured at 1, 3 and 5mm apical from the CEJ, only the 3mm site exhibited a difference between the groups that approached statistical significance (control: 2.08 ± 0.80mm, test: 0.83 ± 1.37mm, Mann Whitney U = 10.00, p=0.05). Changes in keratinized mucosa width, healing index and patient reported outcomes were generally similar between the two groups. Conclusions: Within the limitations of this study, both autologous sCTG and ADM appear to be adequate materials to augment PMT without sacrificing other relevant clinical parameters and/or patient related outcomes.