Altered Immune-Metabolomic Axis in Overweight and Obesity

Objective:  Obesity affects over one billion people globally and one-third of U.S. adults, with links to increased mortality, diabetes, and cardiovascular disease. Emerging evidence suggests a bidirectional association between obesity and oral health, with individuals with obesity being twice as likely to develop periodontitis. However, the mechanisms underlying this link remain unclear. The oral mucosa, a key barrier site, involves complex host-microbe interactions, and disruptions in this balance can lead to periodontitis. Therefore, it is essential to identify the end-stage small molecules (metabolites) in this environment, and the host immune response to these molecules. This study aimed to explore the oral metabolome-immune axis in obese patients through a cross-sectional case-control study.

Methods:  Unstimulated saliva, subgingival plaque, and gingival tissue biopsies (subset of 11 patients) were collected from 40 periodontally healthy obese (OB), overweight (OW), and normal weight healthy (NWH) adults at the University of Iowa College of Dentistry. Subjects were frequency-matched for age, gender, and ethnicity. Gas Chromatography/Mass Spectrometry identified metabolites, which were annotated using the Small Molecule Pathway Database, with enrichment analysis determining significance. Multiplex assays quantified adipokines and cytokines, with statistical significance assessed using MAsLin2. Sparse Partial Least Squares Regression (sPLS) integrated metabolome and immune data.

Results:  Niche-specific metabolome alterations were observed. Citrulline, Indoleacrylate, Proline, and Isoleucine were enriched in NWH, while metabolites such as adenine, beta-hydroxybutyrate, niacin, and glucose were elevated in OB. Proinflammatory mediators (e.g., MCP-1, TNFa, IL-1b, IL-2, EGF) were significantly enriched in OB and correlated with metabolites linked to periodontitis.

Conclusion:  This study reveals distinct oral metabolome and immune alterations in obese patients, even without clinical periodontal disease. These findings highlight the need for personalized treatment strategies for this population

Supported by: NIDCR R03 DE030527; University of Iowa College of Dentistry Seed Grant

Exploring the Oral Virome in Metabolic Syndrome and Obesity

Objective:  More than one-third of the US adult population suffer from Metabolic Syndrome (MetS) a cluster of risk factors including central obesity, blood pressure elevation, low HDL cholesterol, high triglycerides, and hyperglycemia/insulin resistance. MetS is linked to increased risk for Type 2 Diabetes and Cardiovascular Disease, and emerging evidence for increasing severity of periodontitis, a microbially-induced inflammatory disease. While ongoing work is analyzing microbial populations in this system, no work has examined the viral component of the oral microenvironment and the impact of the increased inflammation in MetS. Thus, this study aimed to explore the role of the oral virome in individuals with MetS and obesity.

Methods:  Fifty-three subjects were recruited in three groups: MetS patients, Metabolically Healthy Obese (MHO) individuals, and Normal Weight Healthy (NWH) controls. Metagenomic sequencing of subgingival plaque was filtered for quality and human reads. Reads were assembled with MEGAHIT, viral contigs and taxonomy predicted with geNomad, and CheckV used to assess quality. Quantitative analysis and visualization were performed with QIIME2, phyloToAST, and MaAsLin2.

Results:  A total of 7139 predicted viral genomes were recovered: 120 complete genomes and 162 High-Quality. Distinct differences were seen in virome composition between MetS, MHO, and NWH groups. Beta diversity (Aitchison, Bray-Curtis) showed significant clustering between NWH and MetS/MHO (p < 0.001, PERMANOVA). MaAsLin2 indicated over 300 viruses to be differentially abundant between all three groups. Finally, a GradientBoosting classifier (1000 trees, 10-fold CV) was able to predict class membership with an average ROC AUC of 0.86.

Conclusion:  The findings suggest that alterations in the oral virome may play a significant role inthe metabolic and inflammatory dysregulation observed in MetS patients and may provide potential targets for therapeutic interventions in MetS and related conditions.

Supported by: NIDCR T90 DE023520, R03 DE030527, R03 DE033993

Strain-Level Metagenomic Insights into Oral Microbiota in Metabolic Syndrome

Objective:  Metabolic Syndrome (MetS) affects over one-third of U.S. adults and is associated with a heightened risk for Type 2 Diabetes, cardiovascular diseases, and periodontitis. MetS patients are twice as likely to develop periodontitis, but the mechanisms behind this link remain unclear. One possible explanation is the disruption of the delicate balance between the oral microbiome and the host immune system. Strain-level analysis is crucial since different bacterial strains, even within the same species, can have distinct functions—some driving inflammation, while others contribute to maintaining homeostasis. This level of granularity is essential to identifying microbial drivers specific to disease, which are often overlooked in broader taxonomic approaches. We aimed to perform a detailed metagenomic strain-level analysis of the subgingival microbiome in MetS patients without periodontitis, in order to uncover microbial signatures that may mediate the connection between metabolic and oral health.

Methods:  We recruited 200 periodontally healthy adults, divided into three groups: MetS, metabolically benign obesity (MBO), and normal-weight controls (NWH). Subgingival plaque samples were collected, microbial DNA was extracted, and genomic sequencing was performed using the BGI DNBSEQ platform. Strain-level profiles were mapped with Woltka, and differentially abundant strains were identified using PERMANOVA and MaAsLin2.

Results:  Principal component analysis revealed significant clustering among the groups based on their metabolic status (p=0.0001, ANOVA). Specifically, virulent strains of Porphyromonas gingivalis (W83, WW2903) and Selenomonas (F0473, W7667) demonstrated a 4-5 log-fold increase in MetS and MBO groups compared to NWH controls (FDR adj p-value < 0.0002).

Conclusion:  Our strain-level metagenomic approach is the first to reveal that MetS and MBO patients, despite lacking clinical periodontitis, possess a subgingival microbial environment that closely resembles the periodontal disease state. This highlights an “at-risk-for-harm” microbiome that may predispose these patients to oral health issues in the future and needs to be validated longitudinally in diverse populations.

Supported by: NIDCR R03 DE030527; University of Iowa College of Dentistry Seed Grant

Metformin Induced Survival of Caenorhabditis elegans on Streptococcus gordonii

Objective:  Mitis group streptococci are opportunistic pathogens that reside in the oral cavity and generate hydrogen peroxide (H2O2) as major contributor to their pathogenesis. Previously, we established that mitis group mediates killing of the nematode Caenorhabditis elegans via H2O2. Further, we have shown pretreatment of worms with metformin mediates protection from streptococcal-derived H2O2 and is attributed to the activation of the oxidative stress response transcription factor SKN-1/NRF2. Using worms expressing SKN-1 fused to green fluorescent protein, we have shown the p38 MAPK pathway and the transcription mediator complex protein MDT-15 are required for the activation of SKN-1 by metformin. Based on the data, we will further evaluate the activation of SKN-1 by p38 MAPK and MDT-15.

Methods:  To determine the activation of the PMK-1 (p38) in response to metformin and the dependence of components ofthe p38 MAPK pathway NSY-1 and SEK-1, we compared the level of phosphorylation of PMK-1 in wild-type (WT) andnsy-1 and sek-1 worms pretreated in the presence or absence of metformin by Western blot. Furthermore, the levels of phosphorylation of NSY-1 were compared in the presence or absence of metformin in WT and nsy-1 worms. Lastly, we compared the levels of phosphorylation of PMK-1 in WT and mdt-15 worms as described above.

Results:  A significant increase in the levels of phosphorylation of PMK-1 was observed in the presence of metformin which was dependent on nsy-1 and sek-1. Furthermore, the levels of phosphorylation of NSY-1 were dependent on the presence of metformin. However, a significant increase of phosphorylation of PMK-1 was observed in mdt-15 compared to WT in the presence or absence of metformin.

Conclusion:  Based on the data we have established that metformin mediated activation of SKN-1 is dependent on the p38 MAPK pathway. Conversely, SKN-1 activation by MDT-15 is independent of PMK-1.

Supported by: NIH R01 AI158429; University of Iowa Faculty Start Up Fund

Functional Assessment of an Oculo- Auriculo-Vertebral Spectrum Associated Variant in Zebrafish

Objective:  Oculo-auriculo-vertebral spectrum (OAVS) is a prevalent craniofacial disorder affecting approximately 1 in 3500 births, characterized by a range of craniofacial differences. Despite its frequency, the genetic mechanisms behind OAVS remain unclear. Whole-genome sequencing of a three-generational family with OAVS identified the PDGFRA gene as having the highest likelihood of pathogenic involvement. This study aimed to investigate the role of PDGFRA in OAVS by using zebrafish as a model. The specific objectives were to perform loss-of-function assays for PDGFRA, assess the resulting phenotypic changes, and determine if introducing human PDGFRA variant mRNA can rescue or alter these developmental defects.

Methods:  Zebrafish pdgfra function was disrupted using morpholino oligonucleotide (MO) knockdown or a genetic knockout approach. For MO injected embryos, Western blot analysis was used to verify reduced pdgfra levels. Following MO or genetic disruption of pdgfra, embryos were examined for craniofacial abnormalities using alcian blue staining to highlight skeletal structures. Unaffected or OAVS-associated human PDGFRA variant mRNA will be introduced to evaluate its potential to restore normal development inpdgfra-deficient embryos. Phenotypic assessments will be conducted between groups.

Results:  Western blot analysis confirmed reduced pdgfra protein in MO-injected embryos. Genetic or MO-induced disruption of pdgfra in zebrafish resulted in craniofacial abnormalities, some overlapping those observed in OAVS, including macrostomia. Ongoing studies are assessing if the introduction of human PDGFRA variant mRNA modifies the developmental defects induced by PDGFRA disruption.

Conclusion:  This study supports previous work identifying a critical role ofPDGFRA in zebrafish craniofacial development, further linking variants identified in this gene to OAVS pathology. Further, mRNA based experiments may determine the exact pathogenicity of the OAVS variant identified. These findings contribute toa better understanding of OAVS and may inform future genetic counseling and therapeutic approaches.

Supported by: University of Iowa College of Dentistry Student Research Program

Functional Shifts of Toxin-Related Microbial Genes in Dental Caries

Objective:  Dental caries affects 3.5 billion adults and 573 million children globally, with oral microbiota playing a central role in development. The molecular mechanisms underlying microbial genes and pathway alterations in healthy, caries-affected, and treated (fluoride and arginine) conditions are not fully elucidated. This study aimed to delineate toxin-related microbial gene expression profiles within the oral microbiome and to determine how these profiles are modulated by caries and therapeutic interventions using high-throughput metatranscriptomics.

Methods:  We used two RNA-seq-based metatranscriptomic datasets: the Carda-Diéguez et al. (2022) (Carda dataset) and the Ev et al. (2023) (Daniela dataset). These datasets included supragingival plaque samples from caries-free, caries-affected, fluoride-treated, and fluoride-arginine-treated individuals. We processed the datasets using the Multi-Organism, Multi-Omics Microbiome Analysis Pipeline (MOMO-MAP) to identify functional gene clusters, which were then cross-referenced to UniRef90 toxin clusters to discover toxin-related genes. We performed differential expression analysis using ANCOM-BC and functional profiling by mapping sequencing reads to InterProScan, TADB3.0 (bacterial toxin-antitoxin database), and VFDB (Virulence factor database) and by machine learning-based gene ranking to identify key clusters using Bootstrap Ensemble Attribute Ranking (BEAR) was employed.

Results:  The overlap between UniRef90 clusters and the MOMO-MAP identified 563 clusters unique to the Carda dataset, 174 unique to the Daniela dataset, and 452 shared by both. While no significant differential expression was observed in the dataset-specific clusters, the Type II toxin-antitoxin system - VapC family toxin showed significant differential expressions across healthy, caries, and intervention groups. Furthermore, functional profiles and BEAR analysis highlighted several toxin-antitoxin-related genes, including antitoxin components from the ribbon-helix-helix domain, the YwqK family antitoxin, and the addiction module toxin RelE (Type II system).

Conclusion:  Our findings advance the understanding of the oral microbiome’s involvement in toxin-antitoxin systems and their potential modulation in dental caries. Identifying key toxin-antitoxin genes, particularly those involved in microbial stress responses and survival, suggests a novel link between these systems and caries pathogenesis, which warrants further experimental validation.

Supported by: James S. and Janice I. Wefel Memorial Research Award

Loss of MEMO1 Results in Hypomineralized Enamel

Objective:  Amelogenesis imperfecta (AI), which affects 1 in 15,000 infants, is a rare genetic disorder characterized by defective enamel formation. Enamel is discolored, pitted, and susceptible to wear, and if untreated, results in tooth pain, sensitivity, increased susceptibility to caries. Notably, 50% of AI cases lack an identified genetic cause—with the genes identified largely represented by enamel matrix proteins—highlighting the need to identify novel genes and cellular processes involved in enamel formation and AI pathogenesis. In previous studies, we identified Mediator of Cell Motility 1 (MEMO1) as a crucial factor in enamel integrity. Loss of Memo1 in ameloblasts of mice resulted in an AI-like phenotype, with enamel prone to wear and breakage. These findings were observed in aged mice, but the early developmental role of MEMO1 in ameloblasts remained unclear. Based on MEMO1’s expression in ameloblasts during embryonic development, we hypothesized that the enamel defects in aged mice were a consequence of a much earlier, developmental and perinatal defect.

Methods:  To test this, we examined ameloblast and enamel progression from the earliest stages of their development. Using our in vivo approach, we conditionally deleted MEMO1 from ameloblasts and analyzed the phenotype of the mutants compared to controls using micro-computed tomography, scanning electron microscopy, and histology.

Results:  Conditional deletion of Memo1 in ameloblasts, in vivo, resulted in significant enamel volume and mineralization defects, relative to controls, and closely resembled defects seen in human AI.

Conclusion:  Ongoing investigations aim to uncover the mechanisms by which MEMO1 regulates enamel mineralization, potentially improving diagnostic strategies and risk assessment for enamel-related mineralization defects.

Supported by: NIDCR R00 DE026823, T90 DE023520, F31 DE034609; University of Iowa College of Dentistry Seed Grant

Novel Prognostic Gene Pairs Derived from Multi-Omics Approaches in OSCC

Objective:  Many OSCC cases are diagnosed at advanced stages, complicating treatment and lowering survival rates, while early detection significantly improves prognosis. This study aims to investigate tumor biology using a multi-omics approach to identify novel prognostic gene pairs that can enhance early detection, improve prognostic accuracy, and support OSCC treatment monitoring.

Methods:  We obtained gene expression (GE) and methylation (ME) data for head and neck squamous cell carcinoma (HNSC) from TCGA GDC portal. Using ICD-10 codes, we extracted oral-specific samples (OSCC, n=337). An ensemble feature selection method was applied to identify differentially expressed genes and differentially methylated CpG sites (Feature-Set 1). Survival analysis on individual genes and CpG sites highlighted those associated with varying survival rates (Feature-Set 2). To enhance prognostic predictions, we conducted pairwise survival analysis separately on both datasets (GE and ME) to identify significant gene pairs/CpG sites from each feature set. Subsequently, we annotated the CpG sites to their corresponding genes. We then analyzed SNPs related to each gene pair in different expression/methylation groups to assess their association with survival outcomes. Finally, network analysis on gene pair sets was performed using two datasets from STRING DB.

Results:  The ensemble feature selection identified 15/1 genes/CpG sites (Feature-Set 1). Survival analysis revealed 3420/14123 significant (p-value < 0.05) genes/CpG sites (Feature-Set 2). Survival analysis of gene pairs/CpG site pairs showed their combined predictive power surpassed that of any single one. An intersection of significant gene pairs and CpG site pairs (annoted to genes) revealed 6 key genes, including DKK1, CPNE8, SYT1, SMAD3, TNXB and DIO3.  SNPs mapped to significant gene pairs exhibited varying patterns in patient subgroups stratified by high or low gene expression/methylation levels of each gene pair. By integrating the gene expression and methylation protein networks, we identified 704 proteins shared between the two networks.

Conclusion:  This OSCC multi-omics analysis identified critical genes with strong prognostic potential, with gene pairs offering enhanced predictive power.

Cross-Repression of Pitx2 and Tfap2a/b Regulates Early Mandibular Lineage Specification

Objective:  The establishment of dental epithelial cell lineage within the mandibular epithelium along the dorsal-ventral axis during dental lamina formation remains a critical unanswered question.

Methods:  To identify potential genes and transcriptional regulatory networks involved in specifying dental epithelial cells within the continuous naive mandibular epithelium, we performed RNA-seq to establish mandibular epithelial gene expression patterns along the dorsal-ventral axis during dental lamina formation (E11.5). We also examined the functions of these domain specific transcription factors (TFs) in Pitx2 general knockout (Pitx2 KOs) andTfap2a/Tfap2b ectodermal double knockout (Tfap2a/b EDKO) mouse models. Additionally, we performed CUT&RUN-seq to identify the downstream targets of Pitx2 and Tfap2a/b respectively.

Results:  We comprehensively identified TFs and signaling pathways differentially expressed along dorsal-ventral axis of mandibular epithelium. With two knockout mouse models: Pitx2 KOs (dental lamina-specific TF) and Tfap2a; Tfap2b EDKOs (ventral surface specific TFs), we found Tfap2a and Tfap2b establish the ventral epithelium identity by upregulating multiple WNT ligands, subsequently activating WNT pathway activity in ventral mesenchyme. Similarly, Pitx2 promotes expression of FGF ligands in dental lamina. CUT&RUN-seq reveal distinct set of downstream targets of Pitx2and Tfap2a/b. In addition, we identified a cross-repression mechanism between Pitx2and Tfap2a/b both in vitro and in vivo, suggesting functional antagonism between these domain-specific TFs. Moreover, we found the cross-repression between Pitx2and Tfap2a/b regulates the underlying mesenchymal SHH and WNT pathway activity, which interface at the site of wild-type incisor formation and define the location of the ectopic incisor.

Conclusion:  These results uncover a previously unknown molecular mechanism involving cross-repression of domain specific TFs, Pitx2 and Tfap2a/b, in establishing and localizing dental epithelial cell lineage along dorsal-ventral axis of the mouse mandible.

Supported by: NIDCR R01 DE033009

Human Adipose Derived Stem Cell Membranes for Bone Regeneration

Background:  Obtaining adipose tissue is a better alternative to harvesting stem cells versus getting the stem cells from bone marrow. Research has shown bone marrow stem cell membranes have some promise in tissue regeneration through coating micro/nanoparticles or marco-porous scaffolds. Stem cell membranes from adipose cells have yet to be investigated for bone tissue engineering and drug delivery. 

Objective:  Study human adipose-derived stem cells (hADSCs) membranes for drug loading capacity and osteogenic potential.

Methods:  The hADSCs (from Lonza) were expanded and cultured for membrane vesicle isolation, and their size and zeta potential were characterized by using Dynamic Light Scattering (DLS). The membrane vesicles were labeled with PKH67. Human bone marrow stem cells (hBMSC, from Lonza) were treated with cell membranes and imaged using confocal microscopy. The membranes were loaded with osteogenic fludrocortisone acetate (FA) through sonication and collected by high-speed centrifuge. After seven days of incubation of membrane +/-FA, alkaline phosphatase (ALP), an early marker of osteogenic differentiation, activity of hBMSCs was quantified following the kit manual. Each sample was run in triplicate.

Results:  The average size of membrane vesicles was 550.75nm, and the zeta potential was -5.93 mV.  hBMSC can effectively take up PKH67-labeled hADSC membranes. Our preliminary test suggested that the hADSC membrane vesicles can barely affect the ALP activity of hBMSCs. Interestingly, we found hADSC membrane vesicles have the capacity to load the osteogenic FA and significantly increases ALP activity compared to control groups after 7 days of culture.

Conclusion:  These results indicate that hADSC membrane vesicles have the potential for bone tissue engineering, but further studies are needed. Collecting hADSC is a less invasive procedure than collecting hBMSC. This finding indicates that hADSC membranes could be used as an alternative new tool for regenerative medicine.

Supported by: University of Iowa College of Dentistry Student Research Program; University of Iowa Faculty Start-Up Funds

Pilot Testing of RNA Based Therapeutic Approaches for Bone Regeneration

Objective:  Currently, BMP2 is delivered via bone graft and must be delivered in high concentrations to be effective.  However, these high concentrations have been shown to result in adverse side effects. The objective of this pilot study is to test the stability and translation efficiency of modified RNA and circular RNA to deliver the therapeutic effects of BMP2 while alleviating negative side effects.

Methods:  Human BMP2 gene was cloned into circular RNA and modified RNA systems that were recently developed to enhance protein production. The circular RNA and modified RNA encoding GFP and BMP2 were transfected into 293T cells and 313T-E1 cells and were used to measure in vitro stability and translation. GFP protein production was visually inspected using fluorescent microscopy, qPCR was used to assess BMP2 circular RNA and modified RNA stability in vitro, and ALP was used to assess protein production and cell differentiation progression.

Results:  Fluorescence measurements of EGFP showed prolonged expression of EGFP over one week, with the highest levels of expression on Day 2 for transfected circular RNA and modified RNA. qPCR results showed minimal degradation of BMP2 of circular RNA and modified RNA over a four-day period, with the highest levels of expression on Day 2.

Conclusion:  This pilot study successfully cloned human BMP2 into circular RNA and modified RNA production plasmids and showed that circular RNA and modified RNA could be efficiently translated in vitro. The cloned BMP2 will be useful in future studies to further investigate RNA expression through additional qPCR and ALP testing. Additional optimization of the transfection protocol could further improve the stability and translation efficiency of BMP2. Long term goals of this study are to develop RNA based therapeutic approaches for bone regeneration in vivo.

Supported by: NIDCR R01 DE033009; University of Iowa College of Dentistry Seed Grant

Lactate Modulates Inflammatory Microenvironment and Bone Metabolism for Bone Regeneration

Objective:  Lactate, long treated as a detrimental metabolite, has recently been recognized for its potential anabolic contribution through immunomodulation in some chronic diseases. Interestingly, recent study indicates patients with osteoporosis exhibit relatively low lactate levels. However, its role in directly regulating bone metabolism remains largely elusive. This study aims to investigate the role of lactate in modulating local inflammation and osteoblastic/osteoclastic differentiation, and thereby to develop a novel bioengineering approach for bone regeneration.

Methods:  Macrophage J774A.1 cells were stimulated with LPS to induce an M1-like phenotype and treated with varying concentrations of lactate. After 24 hours, the cells were observed for morphology changes, and expressions of pro-inflammatory factors, including tumor necrosis factor (TNF-a) and IL-6, were detected using ELISA. Pre-osteoblast MC3T3-E1 cells and mouse primary bone marrow derived stem cells (mBMSC) were treated with lactate for osteogenic differentiation. Alkaline phosphatase (ALP) activity and Alizarin Red staining were quantified on day 7 and 21 days of culture, respectively. Pre-osteoclasts Raw 264.7 cells were incubated with lactate and RANKL, and TRAP staining was performed to evaluate lactate’s effect on osteoclastogenesis.

Results:  We found that lactate, as a main metabolite of glycolysis, demonstrated excellent biocompatibility. ELSIA data indicated that lactate significantly and does-dependently mitigate the expression of TNF-a and IL-6 in J774A.1 cells. Remarkably, ALP activity was increased significantly both for MC3T3-E1 and mBMSC cells. Alizarin Red staining showed that lactate treatment enhanced the mineralization on day 21 compared with the control groups. Furthermore, lactate was able to inhibit RANKL-induced osteoclast formation in vitro.

Conclusion:  Lactate down-regulated the production of pro-inflammatory factors in the LPS-stimulated macrophages. In addition, lactate promoted osteogenic differentiation of both pre-osteoblasts and BMSCs, and inhibited osteoclastogenesis in vitro. Subsequently, we will focus on developing innovative bioengineering technologies to achieve more efficient delivery for potential bone tissue engineering applications.

Supported by: NIH R01 DE029159

MiR-200c Promotes Osteogenesis Synergistically with miR-218

Objective:  We have previously demonstrated that miR-200c can promote osteogenesis and regenerate bone in a critical-sized rat calvaria defect model. However, its potency in regenerating mandibular bone is limited due to less vascularization and stem cell source. MiR-218 has been reported to promote osteogenesis and inhibit osteoclastogenesis. In this study, we aim to characterize whether there are synergistic effects among miR-200c and miR-218 on osteogenesis in order to enhance bone regeneration in the restoration of mandibular defects.

Methods:  Human bone marrow stem cells (hBMSCs) were co-transfected with plasmid encoding miR-200c and miR-218 using biocompatible calcium carbonate (CaCO3)-based nanoparticles and subsequently cultured in osteogenic differentiation medium. Transfection efficiencies of microRNAs and osteogenic biomarkers were quantified using RT-qPCR. Alkaline phosphatase and alizarin red S staining were also used to determine osteogenic differentiation and mineralization of hBMSCs after different treatments.

Results:  CaCO3-based nanoparticles effectively transfect both miR-200c and miR-218 into hBMSCs in vitro and enhance osteogenic differentiation biomarkers. Interestingly, co-transfection of both miR-200c and miR-218 significantly increased miR-200c expression level compared to transfection with miR-200c alone. Additionally, co-transfected cells were more heavily stained on both the alkaline phosphatase and alizarin red S stain.

Conclusion:  Co-transfection ofmiR-200c and miR-218 concurrently promotes miR-200c expression and osteogenic differentiation of hBMSCs in vitro compared to overexpression of miR-200c alone.

Supported by: NIDCR R56 DE026433

Engineering Alpha-Ketoglutarate for Bone Regeneration

Objective:  Alpha-ketoglutarate (AKG), a key Tricarboxylic acid cycle component, has garnered attention for its antiaging properties in various organisms. Our recent study has shown that Dimethyl alpha-ketoglutarate (DMAKG), a cell-permeable derivative of AKG, significantly promotes osteogenic differentiation and bone regeneration in aged mice. However, its cytotoxicity and rapid hydrolysis limit its application. To address these challenges, a new system which can sustainably deliver AKG directly into cells is to be developed.

Methods:  Specifically, we designed and synthesized a novel polyester incorporating an AKG moiety (PAKG) to fabricate nanoparticles (NPs). Preosteoblast MC3T3-E1 cells and mouse bone marrow mesenchymal stem cells (mBMSCs) were cultured with NPs to test the osteoblastic/osteogenic differentiation and the subsequent mineralization. Furthermore, we developed a macro-porous microparticle (MPs) with PLGA and PAKG as scaffold for cell growth/differentiation and delivery. MC3T3-E1 and mBMSCs were seeded onthe porous MPs. ALP activity and mineralization were also evaluated.

Results:  Excitingly, these biodegradable PAKG NPs were highly phagocytosable for nonphagocytic cells (e.g., pre-osteoblasts MC3T3-E1 and primary bone marrow MSCs) while common poly (L-lactic acid) and poly (lactic-co-glycolic acid) MPs (PLLA & PLGA) with similar size fail to do so. In vitro studies demonstrated that PAKG NPs significantly promoted osteoblastic/osteogenic differentiation and subsequent mineralization in different cell types. Thein vitro data also suggested that the chemical components, hydrophilicity, and the size of the particles significantly affected the cytotoxicity and pro-osteogenic activity. Thein vivo studies showed that PAKG-based NPs significantly promote the cranial bone regeneration of mice. Moreover, the osteoblastic/osteogenic differentiation and subsequent mineralization in different cell types were significantly improved bythe PLGA-PAKG porous MPs compared to PLGA porous MPs group.

Conclusion:  In summary, the novel PAKG and the porous PLGA-PAKG MPs scaffold showed capabilities in osteogenic differentiation and bone regeneration and held great potential for broad regenerative medicine.

Supported by: NIDCR R01 DE029159

Predoctoral Dental Students’ Interest in Practice Management at UIowa

Objective:  Seven competencies have been added to dental school accreditation standards to ensure consistent teaching of practice management. While dental students may not fully grasp what the curriculum should include, identifying their interests and determining the best timing for delivering practice management content can provide valuable insights for both students and faculty.

This project aimed to assess predoctoral dental students’ interest in practice management curriculum and to identify factors related to this interest.

Methods:  All predoctoral students (341) at the University of Iowa College of Dentistry were emailed a survey using the online software Qualtrics (September 2024). This cross-sectional study asked currently enrolled dental students (D1-D4) about their overall interest in practice management and specific interests they have in practice management topics.

Results:  Approximately thirty six percent of students participated inthe survey, with similar response rates across all classes. Among respondents, sixty six percent indicated they are most likely to pursue a career in private practice after graduation. Additionally, ninety four percent expressed moderate to strong interest in required practice management courses, while ninety two percent showed similar levels of interest in elective courses. However, ninety percent reported feeling only somewhat to not very knowledgeable about the practice management curriculum offered while in school. Notably, eighty eight percent of respondents had attended “Lunch and Learn” sessions to enhance their understanding of practice management.

Conclusion:  The findings highlight a strong interest among predoctoral dental students in acquiring practice management skills, particularly in areas critical to private practice success, suggesting the need for curricular focus on these competencies.

Supported by: University of Iowa College of Dentistry Student Research Program

Role of Margin Design on Marginal Adaptation and Fracture Strength

Objective:  To evaluate the impact of margin design onthe marginal adaptation and fracture strength of crowns fabricated with polymer-infiltrated ceramic network (PICN) and zirconia-reinforced lithium disilicate (ZRLD).

Methods:  Sixty crowns (n=10) were milled from ZRLD (Vita Suprinity) and PICN (Vita Enamic) CAD/CAM blocks. Each crown was designed from 3D-printed teeth prepared with one of the three following margin designs: light chamfer, heavy chamfer, or modified shoulder. Dual-cure resin cement was used to cement the crowns. Margins were assessed in six standardized areas at baseline with digital scanner (Omnicam, Sirona) and microscope. A score was assigned to each segment based on marginal adaptation (0=closed, 1=open 0.01-0.50mm, 2=open 0.51mm-1.0mm, 3=open >1.0mm). Crowns were then subjected to 500,000 mechanical load cycles to simulate masticatory forces. After cycling, each marginal segment was re-evaluated and rescored. Crowns were then loaded until catastrophic failure to obtain fracture strength (N). Three-way ANOVA was used to statistically analyze marginal adaptation whereas two-way ANOVA was used for fracture strength. After, both were analyzed with Tukey’s test (a=5%).

Results:  All crowns survived the mechanical cycling. After cycling, all margins were significantly more open than before cycling (p<0.001). There was no difference between PICN and ZRLD materials (p=0.49) on marginal adaptation. For PICN, more open margins were attained with heavy chamfer than with light chamfer (p=0.03). PICN showed no difference (p=0.883) in fracture strength among margin designs. Light chamfer in ZRLD crowns provided significantly lower fracture strength compared to other margin designs (p<0.001). Light chamfer attained similar fracture strength for both ceramics (p=0.589). However, ZRLD was superior to PICN for the other two margin designs (p<0.001).

Conclusion:  Within the limitations of this study, we can conclude that light chamfer finishing margins are preferable to PICN rather than heavy chamfer. Nevertheless, light chamfer may provide lower fracture strength for ZRLD ceramics.

Supported by: University of Iowa College of Dentistry Student Research Program

Student Perceptions of a Data-Informed Strategy to Reduce Food Insecurity

Objective:  Food insecurity (FI) is prevalent among post-secondary students including dental students and is associated with negative academic and health outcomes. In 2023 we conducted a study to identify potential strategies to reduce FI among dental students. We have since piloted one of these strategies in Iowa’s College of Dentistry, and our objective was to evaluate dental students’ perception of the strategy.

Methods:  In response to student-suggested grab-and-go breakfast and lunch items, we introduced a Snack Program Resource in the Student Lounge, featuring perishable (e.g., yogurt) and non-perishable (e.g., granola bars) items. An email was sent to all dental students to inform them of the resource. Approximately six weeks post-implementation, a survey was sent to all dental students (n=333) to assess demographics, food security status, perceptions ofthe Snack Program, preferred items, usage frequency, and barriers to access. The survey was open for three weeks, and participants received a $15 grocery gift card as compensation.

Results:  Of the 156 respondents, 58 or 37% identified as FI. Mean age of respondents was 24.7± 3.4 years with 60% female and 82% White. Utilization of the Snack Program Resource showed that 45% never used it, 46% used it 1-2 times per week, 8% 3-5 times per week, and 1% 6-10 times per week. The most preferred items included cheese sticks, protein bars, and mixed nuts. Primary usage times were at lunchtime (33%) and mid-late afternoon (37%). Reasons for not using the resource included not needing it, disliking the choices, being unaware, and being busy. Students appreciated the resource, provided favorable feedback, and offered suggestions for improvement (i.e., greater volume and variety, additional locations).

Conclusion:  The Snack Resource Program is being used, and students shared ideas to further meet their needs.

Supported by: M.W. Finkelstein Teaching Professorship; University of Iowa College of Dentistry Student Research Program

A Retrospective Study of Incomplete Primary Root Canal Treatments

Objective:  Patients may fail to complete primary orthograde root canal treatment (RCT) for various reasons. The disease and patient-based outcomes associated with this treatment have not been extensively studied. This study investigated tooth-specific and patient characteristics linked to RCT initiation without completion in an academic setting.

Methods:  Demographic and clinical data were obtained from axiUm at the University of Iowa College of Dentistry (UICoD) for patients treated between July 2009 and July 2022.

Results:  A total of 599 subjects (one tooth per subject) met the inclusion criteria. The cohort was predominately female (67.8%), mostly white (70.6%), lived within 60 miles of UICoD (64.3%), and were enrolled in the state-sponsored Dental Wellness Plan insurance (55.4%). Of the 599 teeth, 65.8% had incomplete RCT, 19% were not completed due to non-restorability, 5.5% were extracted for unknown reasons, and 4% were extracted due to patient preference following treatment initiation. At the time of the patient’s most recent visit to the UICoD (750.97±1,086.92 days), 38.6% of teeth remained in the mouth, while 37.9% had been extracted or were planned for extraction (23.5%). Pulp necrosis (43.3%) and symptomatic apical periodontitis (58%) were the most common pulpal and periapical diagnoses associated with incomplete RCT. Nearly 62% of teeth with incomplete RCT were molars, and over half (54.4%) were seen in the pre-doctoral clinic.

Conclusion:  Teeth with pain at diagnosis, molars, and limited clinician experience may increase the risk of patients failing to complete treatment.

Supported by: University of Iowa College of Dentistry Student Research Program

Alumni Practice Tendencies for Patients with Special Healthcare Needs

Objective:  Access to adequate dental care is a significant issue, particularly for individuals with special healthcare needs (SHCN). Research shows that many dentists hesitate to treat these patients due to inadequate training. The University of Iowa College of Dentistry established the Geriatric and Special Needs Clinic (GSNC) to enhance dental students’ clinical experience with SHCN patients. This study aims to assess how clinical experience with SHCN patients affects practitioner comfort and practices after graduation.

Methods:  A survey was constructed on RedCap, a secure database approved by the University of Iowa, that included sections such as demographics, education, and practice tendencies regarding SHCN patients. The anonymous survey was distributed to University of Iowa College of Dentistry alumni that graduated from 2013-2022. Alumni were divided into two groups: a control group, which did not receive GSNC training, and a study group, which did. Data was analyzed between the groups to access the relationship between clinical experience treating patients with SHCN and provider comfort and practice tendencies.

Results:  There was a total of 35 surveys completed, indicating a 6% response rate. Among the alumni that received GSNC training, 72% thought their dental school education prepared them well or somewhat well in treating patients with SHCN. All respondents agreed to being comfortable treating patients with mild autism, mild intellectual disabilities, and wheelchair bound patients. In contrast, 36% of alumni disagreed with feeling comfortable treating patients with severe intellectual disabilities or severe autism. Finally, 50% of alumni cited low financial compensation and 43% reported increased treatment time as barriers to seeing SHCN patients.

Conclusion:  Our findings indicate that while alumni thought their education prepared them to treat SHCN patients, additional factors such as condition severity, low reimbursement, and increased treatment time impacted their decision to treat this population.

Supported by: University of Iowa College of Dentistry Student Research Program

Exploring Mucoadhesive Properties: Buccal Films Dissolution and Stability

Objective:  This study aims to develop and evaluate various experimental buccal films for potential oral applications. The goal is to investigate how different polymer compositions and pH modifications impact the films’ adhesion, dissolution, and stability in saliva-like conditions.

Methods:  Multiple prototypes were prepared by varying the concentrations of polymers and other components across four batches. Batch A contained NaOH and a gelling agent, Batch B included NaOH but no gelling agent, Batch C included the gelling agent but no NaOH, and Batch D had neither NaOH nor a gelling agent. These batches were assessed for stability and dissolution in saliva-like solutions, with pH levels also analyzed. The study explored challenges like maintaining stability during storage and optimizing pH.

Results:  The prototype films exhibited distinct variations in adhesion and dissolution profiles based on their composition. Films without NaOH and gelling agents demonstrated optimal adhesion, flexibility, and prolonged retention on mucosal surfaces. They dissolved gradually, ensuring sustained moisture delivery, and maintained a stable pH suitable for oral applications. These findings suggest that such films could offer comfort and functionality for individuals with xerostomia. Further experiments are needed to confirm these results and refine the formulations.

Conclusion:  This research demonstrates the potential of buccal mucoadhesive films for future oral applications, with specific focus on balancing adhesion, dissolution, and stability.  Further research will focus on refining these formulations to enhance their therapeutic potential and explore clinical applications. The findings provide a foundation for continued research into patient-friendly solutions for oral healthcare.

Supported by: University of Iowa College of Dentistry Student Research Program

Exploring the Relationship Between Stress, Diet, Hygiene, and Caries Incidence

Objective:  Chronic exposure to early-life stressors, known as adverse childhood experiences (ACEs), can disrupt brain development and neural pathways, leading to long-term physical and behavioral health issues. This phenomenon, termed “toxic stress,” is partly mediated by elevated cortisol levels and is associated with adverse outcomes such as immune dysfunction, heart disease, obesity, mental health disorders, and premature death. Studies suggest that increased salivary cortisol may correlate with higher levels of cariogenic bacteria and dental caries in children. However, research exploring the relationship between stress, hygiene, and caries incidence remains limited.

Methods:  This study aims to investigate the association between stress, caries, and hygiene in children aged 2–6 undergoing elective surgical procedures at the University of Iowa Stead Family Children’s Hospital. Legal guardians will complete a questionnaire covering topics such as systemic health, oral hygiene habits, diet, socioeconomic status, mental health, and family dynamics. A dental examination will be performed on the children to determine DMFS and plaque levels.

Conclusion:  This study aims to provide valuable insights into the effects of stress, caries, and hygiene on the oral health of children aged 2–6 undergoing minor surgical procedures at the University of Iowa Stead Family Children’s Hospital. By integrating guardian-completed questionnaires on health, oral hygiene, diet, socioeconomic status, mental health, and family dynamics, this research seeks to deepen the understanding of the complex interplay between stress and oral health. Ultimately, it aims to uncover how adverse childhood experiences and toxic stress contribute to oral health disparities. 

Food Insecurity and Caries: Investigating At-Risk Dental Patients

Objective:  This chart review investigated factors related to food insecurity and the relationship between food insecurity and the prevalence of dental caries using data from a Caries Risk Assessments (CRA) tool.

Methods:  This chart review included patients attending the University of Iowa Dental Clinics during June 1, 2021, through May 31, 2024, who completed a caries risk assessment. Food insecurity was assessed through responses to 2 screening questions and categorized into a binary variable. The relationship between food insecurity and independent variables (age, gender, insurance status, BMI as well as select dietary and health status variable) was analyzed using Fisher’s Exact tests.  Dental caries was assessed based on planned dental caries treatments, and its relationship to food insecurity was analyzed with bivariate analyses, using Fisher’s exact test and univariable logistic regression for explanatory variables. Multivariable logistic regression models were developed for basic, advanced, and all caries treatments, employing stepwise AIC selection to determine significant predictors.

Results:  The sample included 4,594 participants, with 9.5% reporting food insecurity.  Those with food insecurity were significantly more likely to have public insurance, eat unstructured meals, consume sugar-sweetened beverages and have a diagnosed mental health condition. 

Logistic regression found that food insecurity was associated with an increased likelihood of caries (all caries), although this was not statistically significant (OR: 1.25, p=0.073). Significant predictors for all caries treatments included frequent snack consumption (OR: 1.33, p=0.002), sugary drink intake (OR: 1.25, p=0.012), higher BMI (OR: 1.02, p<0.001) and mental health diagnoses (OR: 1.18, p=0.031) Logistic regression models for basic and advanced caries were similar; interestingly, food insecurity was not identified as a significant predictor for advanced caries treatments.

Conclusion:  While food insecurity may increase the likelihood of dental caries treatment, frequent snack and sugary drink consumption, higher BMI, and mental health issues are stronger predictors.

Supported by: University of Iowa College of Dentistry Student Research Program

Policy Trends in Annual Benefit Maximums State Medicaid Dental Programs

Objective:  Annual Benefit Maximums (ABMs) are the maximum amount a dental plan will pay for a member’s dental care each year. ABMs are ubiquitous among private dental plans and are increasingly used in state Medicaid programs for adults. ABMs have the potential to restrict access to care for Medicaid beneficiaries whose treatment needs exceed their ABM. The objectives of this study were to 1) examine trends in the presence and amount of ABMs in adult Medicaid dental programs and 2) explore associations between ABM use and other state characteristics.

Methods:  Grey and peer-reviewed literature were searched to collect ABM policy changes as well as other state characteristics. Independent variables were categorized into four domains: Medicaid Policies, Medicaid Population and Spending, Dental Care Access and Oral Health Status, and State Demographic and Geographic characteristics. Bivariate analyses examined associations between the presence of an ABM in 2022 and other state characteristics.

Results:  The number of states with adult dental Medicaid ABMs increased from two in 2003 to 15 in 2022. The average ABM amount increased from $538 in 2003 (n=2) to $1388 in 2022 (n=15). ABM presence was associated with Medicaid reimbursement for children; states with an ABM had higher Medicaid reimbursement than states without an ABM (q=0.034).

Conclusion:  There was a substantial increase in state Medicaid dental programs adopting ABMs in the last 20 years, and ABM amounts have increased. More research is needed to understand the implications of these trends on dental care access for Medicaid beneficiaries.

Supported by: University of Iowa College of Dentistry Student Research Program

Pro- and Anti-Fluoride Messaging on TikTok: Sources and Engagement

Objective:  TikTok has become a prominent platform for disseminating health-related content, including dental care practices, but the accuracy of such information remains uncertain. Fluoride, a key component in oral health, is a topic of controversy on social media, making it crucial to assess how TikTok content aligns with scientific evidence. This study aims to evaluate TikTok creators’ descriptions of fluoride and identify message characteristics potentially influencing user perceptions of fluoride.

Methods:  The first 120 TikTok videos were selected that met the search criteria: provided by TikTok in response to the search string, “fluoride vs. no fluoride”; publicly accessible; and English-language content. Videos were viewed and coded on the following variables: message content; source; fluoride perspective; editing style; length; view rate (views/month); and interaction index (likes/views). Analyses included descriptive statistics, Pearson Chi square, t-tests, and ANOVA.

Results:  Only 22.5% ofthe messaging was evidence-based and only 40.0% was pro-fluoride, even though 54.1% of the sources were health professionals. Evidence-based content was significantly more likely to be produced by health professionals (X2 = 7.18, df = 2, p = 0.028), carry a pro-fluoride message (X2 = 11.91, df = 2, p = 0.003), and be longer (t = 2.16, df = 118, p < .001). Content type did not significantly differ in editing style, view rate or interaction index.

Conclusion:  TikTok’s videos include pro-fluoride and evidence-based content from health professionals, but these are not more popular or engaging than the majority of videos, which are not pro-fluoride and not evidence-based.

Supported by: University of Iowa College of Dentistry Student Research Program

University of Iowa Dental Student Debt and Career Path Implications

Objective:  The Resident Education Deferred Interest Act was introduced into the US Congress in 2023 to assist dental residents with educational debt by allowing them to defer their loan repayments with no interest accrual, until they complete their residency. There is little evidence to date, however, of the effect of student loan debt on dental student’s choice to do a residency, and more broadly, the impact of student loan debt on other career choices after graduation. 

The primary aims of this study are to: 1) investigate how student loan debt influences UI dental students’ practice decisions following dental school, and 2) the potential impact that the ability to defer student loan repayment during postgraduate residency training programs might have on whether to do a residency and/or the type of residency they may choose.

Methods:  An online survey was administered via Qualtrics to all currently enrolled dental students at the University of Iowa during fall 2023-2024. 121 students responded.

Results:  About one in four students were interested in applying for a specialty program.  Over half of all students indicated that their educational debt made it less likely that they would apply for a dental specialty-this was true for 82% of those without a current interest in a dental residency. Future analyses will focus on the factors related to educational debt as it relates to interest in a dental specialty.

Conclusion:  Results of the study are still being analyzed. These results will inform policy makers at both the state and federal level of the impact that deferring dental student loan payments may have on a student’s decision to pursue a residency program.

Supported by: University of Iowa College of Dentistry Student Research Program; M.W. Finkelstein Teaching Professorship

Poverty Simulations: Do Personal Experiences and Empathy Influence Learning?

Objective:  Dental schools use poverty simulations to cultivate empathy for patients facing financial barriers. Research indicates simulations improve attitudes toward poverty, but little research explicitly measures empathy. This study explored associations among personal poverty experiences, empathy, and poverty attitudes before and after a poverty simulation.

Methods:  Dental students (N = 169) completed anonymous surveys at baseline (T1), post-simulation (T2), and at 2-month follow-up (T3). Surveys included personal poverty experiences, a poverty attitudes measure, andthe Interpersonal Reactivity Index (IRI), measuring empathy. Analysis included paired t-tests, Spearman and Pearson correlations, and quantile regression.

Results:  Poverty attitude scores increased at T2 by 9.4 points (p < .001), and at T3 remained 7.9 points (p < .001) higher than baseline. However, the IRI dropped 1.8 points (p < .003) at T2, and remained 2.4 points (p < .001) lower at T3. Personal poverty experiences were associated only with poverty attitudes at T1 (r = 0.16, p < .05). Baseline IRI was not associated with baseline poverty attitudes, but was associated at T2 (r = 0.47, p < .001) and T3 (r = 0.39, p < .001). For every 1-point increase in baseline IRI, poverty attitudes increased 0.15 (0.05, 0.26) points for the 0.25 quantile (p < 0.005) and 0.98 (0.28, 1.7) for the 0.90 quantile (p < 0.006). The T1 to T3 change was significant only for the IRI’s 0.90 quantile: for every 1-point increase in baseline IRI, poverty attitudes changed 0.94 (0.33, 1.5) points (p < 0.003).

Conclusion:  Following a poverty simulation, poverty attitudes improve, but empathy declines. Personal poverty experiences are not associated with empathy or post-simulation attitudes. Baseline empathy is associated with higher post-simulation poverty attitudes, but with changes in poverty attitudes only for those in the 0.25 or 0.90 quantile. Poverty simulations may be more effective for students already higher in empathy.

Poverty Simulations: Poverty Attitude Changes and Poverty Simulation Evaluations

Objective:  Poverty simulations are commonly used to enhance attitudes toward patients facing financial barriers. Research indicates significant improvement in average attitudes toward those in poverty following simulations, but little research has considered whether students benefit uniformly. The objective of this study was to measure the variability in poverty attitude change following poverty simulations and to determine whether any change is associated with evaluations of the simulation.

Methods:  Third-year dental students participating in a poverty simulation in 2022 and 2023 were surveyed at baseline (T1), post-simulation (T2), and 2-month follow-up (T3). Poverty attitudes reflect Likert-scale agreement with 20 statements (1-5 points each), addressing personal responsibility and public policies. Simulation evaluations were measured at T2 and T3 by agreement with 8 statements addressing the simulation’s usefulness and impact on patient care. Data analysis relied on descriptive statistics, paired-samples t-tests, and Pearson correlations.

Results:  Students’ (N = 169) average poverty attitudes were 9.4 points higher at T2 than T1 (p < .001) and 7.9 points higher at T3 than T1 (p < .001). Among these, however, 21.7% reported lower attitude scores at T2 and 34.6% reported lower attitude scores at T3, with a 4-point median decline from T1 in each group. Baseline poverty attitudes did not predict evaluations, but T2 poverty attitudes correlated with T2 (r = 0.33) and T3 
(r = 0.45) evaluations. Also, T3 poverty attitudes were correlated with T2 evaluations (r = 0.35) and T3 (r = 0.44) evaluations (all p < .001).

Conclusion:  Poverty simulations enhance poverty attitudes on average, but potentially worsen attitudes among substantial minorities of students. Those with worse attitudes toward poverty are likely to value the simulation less and may require different teaching strategies.

Medical-Dental Integration at Federally Qualified Health Centers: Facilitators and Barriers

Objective:  To evaluate the status of Medical-Dental Integration (MDI) at Federally Qualified Health Centers (FQHCs) by identifying barriers and facilitators to implementation.

Methods:  An online survey was distributed in November 2023 to medical and dental leadership at FQHCs via the National Network of Oral Health Access listserv. Respondents selected the top three barriers and facilitators to MDI implementation. Descriptive and bivariate analyses were conducted to compare perceptions between medical and dental clinics regarding factors that enhance or hinder MDI implementation.

Results:  There were a total of 31 medical and 180 dental respondents. All medical and 95% of dental respondents reported some form of MDI at their FQHCs. The three most common facilitators were executive leadership support (52% medical and 63% dental respondents), clinical staff willingness to perform MDI services (34% and 44%), and proximity between medical and dental clinics (32% and 30%). The most common barriers were inadequate financial incentives for MDI (22% and 26%), referral process challenges (24% and 19%), and limited time for MDI services during patient visits (22% and 19%). No significant differences were found between medical and dental clinics regarding these factors.

Conclusion:  Over half ofthe medical and dental respondents cited leadership support as a top MDI facilitator. The lack of significant differences in barriers and facilitators suggests these issues universally impact MDI implementation. Future research should explore best practices to overcome challenges and strengthen MDI services at FQHCs.

Supported by: Delta Dental of Iowa Smile Grant; HRSA D88 HP37557

Oral Health Services by FQHC Providers Regarding Early Childhood Caries

Objective:  Early childhood caries (ECC) is a preventable, common condition that causes pain and infection if untreated. Prevention of ECC relies on preventive oral health services (OHS). This study identifies variables associated with federally qualified health center (FQHC) medical providers’ provision of OHS and confidence in delivering these services.

Methods:  A 62-item online survey was developed and distributed to FQHC medical providers nationwide.  Provided OHS scores were summed and dichotomized as “few”=0-4 vs. “many”=5-6.  Confidence in providing OHS was dichotomized as “more confident” vs. “less confident.”  Descriptive and bivariate analyses were conducted (alpha=0.05).

Results:  Eighty surveys were received. Respondents included: Nursing Assistants/Medical Assistants-25%, Nurse Practitioners (NPs)/Physician Assistants (PAs)/Assistant Physicians (APs)-29%, Physicians-30%, and Registered Nurses (RNs)/Licensed Practical Nurses (LPNs)-16%. Sixty-seven percent of respondents had oral health training. Forty-three percent of respondents provided few OHS, while 57% provided many. OHS and confidence in providing OHS varied by clinical role. NPs/PAs/APs and Physicians were more likely than Nursing/Medical Assistants and RNs/LPNs to provide OHS (87% and 86% vs 5% and 33%; p < 0.001) and express more confidence in doing so (55% and 57% vs 11% and 8%; p < 0.001). Providers with oral health training were more likely than those without training to provide oral health education (77% vs. 55%; p=0.041) and evaluate children’s teeth (66% vs. 42%; p=0.046).

Conclusion:  The provision of OHS and confidence varied among providers based on their clinical role and oral health training. Further research is needed to increase OHS provided by non-dental providers.

Supported by: HRSA D88 HP37557, James S. and Janice I. Wefel Memorial Research Award

Impact of Ridge Height and Methods on Mandibular Denture Duplication

Objective:  The accuracy of complete denture duplication is crucial for maintaining the clinical functionality and comfort of mandibular dentures. Ridge height variations and the choice of duplication methods may influence the precision of the duplicated dentures. With advancements in intraoral scanning technologies, understanding their role in achieving accurate duplicates is essential.

Methods:  This study evaluated the effect of different ridge heights (low, medium, and high) and two intraoral scanning systems (TRIOS 5 and PrimeScan) on the accuracy of mandibular complete denture duplication. A total of 3 mandibular dentures representing diffident mandibular ridge heights were designed using Exocad software and fabricated using the SprintRay 3D printer. The reference scans were performed using a laboratory scanner (Ceramill Map 600), ensuring high-resolution baseline data. Each denture was scanned again using both TRIOS 5 and PrimeScan to generate digital models. Trueness and precision were measured by comparing the duplicated scans to the reference scan using Geo Magic software. Analysis focused on four areas: the teeth, cameo surface, intaglio surface, and the overall complete denture. Deviations were analyzed using 3D analysis software to assess linear and volumetric differences.

Conclusion:  Ridge height plays a critical role in the precision of mandibular denture duplication. The use of Exocad software and SprintRay printing ensured standardized denture fabrication, while the Ceramill Map 600 provided robust reference scans. Type of scanners significantly influenced the trueness and precision values.

Biomarker Identification for Oral Cancer Prognosis via Multi-Omics Analysis

Objective:  Oral squamous cell carcinoma (OSCC) has a high mortality rate. Advances in high throughput sequencing now allow for integrated multi-omics analysis, providing deeper insights into disease mechanisms. This study aimed to identify potential biomarkers using network-based integrative analysis and paired-gene survival analysis.

Methods:  Data from 318 head and neck cancer samples (304 cancer, 14 normal) were obtained from the TCGA data portal. Common significant genes from differential gene expression (DEG) and methylation analyses (DMP) were used to construct gene network with iNETgrate, identifying gene modules. Module significance was validated through gene set enrichment analysis (GSEA). Genes were integrated with mutation data for single-gene and paired-gene survival analyses.

Results:  DEG analysis identified 5,459 significant genes (2,979 upregulated, 2,480 downregulated), while DMP  analysis revealed 73,001 significant sites (37,767 hypermethylated, 35,234 hypomethylated). 3,489 common genes formed the fused network, clustered into 11 modules. GSEA of these modules highlighted cancer-related pathways such as WNT signaling and NOTCH signaling. Single-gene survival analysis identified 60 significant genes, while paired-gene analysis revealed 106,804 significant pairs. By examining SNPs of genes selected from the top 10 significant pairs based on paired-gene survival analysis, we identified 9 SNPs across 8 genes associated with the low-survival-rate group and 13 SNPs across 8 genes associated with the high-survival-rate group. However, single-gene survival analysis on these genes identified 7 mutations across 5 genes in the low-survival-rate group and 7 mutations across 4 genes in the high-survival-rate group, highlighting key gene interactions that affect patient outcomes.

Conclusion:  This study combines network-based analysis and pairwise gene interactions to deepen understanding of cancer biology. Integrating gene expression and methylation data identified key gene modules associated with survival outcomes. The paired-gene survival analysis revealed gene pairs linked to prognosis. These findings highlight potential biomarkers and therapeutic targets for personalized cancer treatment.

Microbiome Simulation Using MCMC Sampling

Objective:  Newly developed sequencing technologies have democratized meta-omics sequencing, generating vast amounts of microbiome data and spurring the development of novel statistical methods to explore the associations between human microbiota and diseases. However, the inherent complexity of microbiome data – characterized by sparsity, overdispersion, and compositionality – makes realistic microbiome data challenging yet pivotal for methods development and benchmarking. Existing approaches including using statistical models (Dirichlet-Multinomial distribution, gamma-hypergeometric model, and generalized gamma distribution) and neural networks (MB-GAN and DeepMicroGen) have shown varying degrees of success. This is partly because these models rely on simplified assumptions of the input microbiome data that limit their performance and fail to capture the nuances of microbiome data. Here, we propose using Markov Chain Monte Carlo (MCMC) sampling methods to simulate realistic microbiome data.

Methods:  This method circumvents the need for closed-form gene expression distribution approximations, making the framework for benchmarking statistical tools flexible and elegant. 

Conclusion:  The results demonstrated that the simulated microbiome data generated using the MCMC approach closely resemble real microbiome data, outperforming its competitors. This simulation approach provides a robust framework for evaluating various computational methods, including normalization techniques and differential abundance analyses, to establish best practices for microbiome data analysis.

Supported by: University of Iowa Interdisciplinary Program in Informatics

Optimizing Data Preprocessing for Oral Multi-Omics

Objective:  Oral diseases such as periodontitis pose substantial global public health challenges, with emerging research spotlighting the pivotal role of oral microbes in such conditions. Integrative whole microbiome sequencing - metagenomics, metatranscriptomics, and metaproteomics, facilitates the exploration of functional dynamics by examining taxonomy, gene expression, pathway and protein level profiles within the microbiome. In this context, understanding and mitigating technical biases in multiomics data through effective normalization and batch effect correction are imperative to yield meaningful biological insights. In this study, we performed a comprehensive comparative analysis of prevalent normalization methods and batch effect correction strategies to determine the most effective technique.

Methods:  A systematic review was conducted to compare widely used methods such as TMM (Trimmed Mean of M-values), Arcsine, MinMax, Blom, Inverse Rank, Upper Quantile and RLE (Relative Log Expression) present in contemporary microbial data analysis literature. Subsequently, these methods were utilized on standardized, re-analyzed multiomics datasets from the study by Belstrom et al., which were made publicly available. The statistical measures for each dataset were used to evaluate the central tendencies of the datasets. The abundance values from metagenomics, expression profiles from metatranscriptomics, and protein intensity data from metaproteomics were then utilized to identify the most effective normalization and batch effect correction methodologies for oral multiomics data. This process emphasizes the importance of tailored methods for complex data in microbial ecology. Such an approach provides a comprehensive perspective onthe temporal dynamics and current technological advances in oral microbiome research. By comparing the outcomes, we pinpointed the most appropriate techniques for oral multi-omics data analyses, highlighting the necessity for specialized approaches to navigate the intricacies of dataset analysis in microbial studies.

Results:  Our comparative analysis provides crucial insights into optimizing data preprocessing strategies for the accurate interpretation of each type of multiomics data, thereby enhancing our understanding of microbial influences on oral health and disease.

Supported by: James S. and Janice I. Wefel Memorial Research Award

Interim Therapeutic Restoration Eligibility among Nursing Home Residents with Caries

Objective:  Nursing home residents experience a high burden of dental disease and substantial barriers to dental care. Interim Therapeutic Restorations (ITR), provided by dental hygienists in community-based settings, have the potential to increase access to caries management services for patients faced with these barriers. The aim of this study was to assess the eligibility of ITR treatment for carious teeth among nursing home residents in a state where ITR was not in the scope of practice for hygienists in community-based settings. 

Methods:  Study subjects included 25 dentate nursing home residents who participated in a broader study about a Virtual Dental Home (VDH) demonstration program. Tooth-level presence of untreated caries was assessed. Carious teeth were assessed for ITR eligibility based on lesion and margin accessibility, lesion depth, and restorability. Additionally, data were collected about residents’ medical conditions, cooperation, mobility, and urgency of treatment needs.

Results:  Among subjects (n=25), most were very cooperative (80%), 40% needed help with dental chair transfers, and 28% had urgent treatment needs. Overall, 92% (n=23) (n=133 teeth; mean:5.3; range:0- 15) of subjects had untreated caries. Among subjects with untreated caries, 87% (n=20) (n=80 teeth; mean:3.2; range:0-12) had a lesion that was eligible for ITR based on study criteria. Among the 80 ITR-eligible teeth, 49% (n=39) were anterior teeth, 19% (n=15) were premolars and 32% (n=26) were molars. For teeth that were ineligible for ITR, the most common reasons were inaccessible lesions and lesions that were too large for ITR. 

Conclusion:  Among subjects with untreated caries in this study, the vast majority (87%) (60% of teeth with untreated caries) had at least one tooth that could have been treated with ITR in the nursing home if allowed by state scope of practice. ITR can be used to treat caries and improve access to care for populations with care barriers.

Supported by: Delta Dental of Iowa Foundation

Reprogramming of Fibroblasts Into Dental Mesenchymal Cells by Transcription Factors

Objective:  Interactions between epithelium and mesenchyme give rise tothe development of ectodermal organs, like hair and teeth. In the initiation stage of early tooth development, dental mesenchymal cells have been found an essential role. The project aims to screen a combination of transcription factors to induce human fibroblast cells into dental mesenchymal.

Methods:  There are many dental mesenchymal transcription factors that have been found in odontogenesis. Among them, we designed and produced lentivirus of the genes identified potentially essential in the dental mesenchymal development. We also performed CRISPR experiments to construct transgenic BJ cells, a type of human fibroblast, with fluorescence reporters on two main dental mesenchymal markers, PAX9 and MSX1(PAX9:  EGFP; MSX1:  mCherry). To narrow down the candidates, we will then employ the transgenic BJ cells in the screening for the combination of tissue factors.

Results:  Over 20 tissues factors have been marked promising and prepared lentivirus. In constructing PAX9:  EGFP; MSX1:  mCherry knock-in reporters fibroblasts, we have optimized the procedure for electroporation in BJ cells. AmaxaV solution with program code EN150 and EH100 in the 4D-Nucleofector System was found highest transfection efficiency. We also designed & cloned 3 sgRNAs targeting PAX9 and tested these 3 sgRNAs for CRISPR knock-in efficacy using HEK293 cells.

Conclusion:  The current results show that BJ cells exhibit appropriate transfection efficiency and could be used as cell line candidate in the project. We have also optimized CRISPR mediated knock-in protocol with HEK293 cells and will try this protocol in BJ cells. The result of this research will be of value in the tooth regeneration study in the future.

Supported by: NRSA R90 DE024296

Evaluating Dentists’ Perceived Administrative Burden in Iowa’s Medicaid Program

Objective:  Access to dental services for Medicaid recipients relies substantially on dentists’ participation in state Medicaid programs. However, many private practice dentists choose not to participate, often citing administrative challenges such as excessive paperwork, payment delays, and shifting regulatory requirements. While these barriers are well-documented, few studies have examined the perceived administrative burden across dental managed care plans within the same state. This research aims to evaluate the factors associated with Iowa private practice dentists’ perceptions of the administrative burden of the Medicaid Dental Wellness Plan (DWP) among dentists participating in the state program’s two dental plans.

Methods:  Secondary data from the 2021 Survey of Iowa Private Practice Dentists resulted in 564 responses from 1,256 mailed surveys (46% response rate), with 497 general dentists included in the final dataset for analysis. The 34-question paper survey collected data on dentist characteristics, practice attributes, and perceptions of administrative burdens. Statistical analyses, including descriptive, bivariate, and multivariable logistic regression, were performed to evaluate relationships between perceived administrative burden and variables such as dentist age, gender, practice busyness, and role within the practice.

Results:  Preliminary findings show low participation levels among Iowa dentists in the DWP for both managed care organizations, with 27% of dentists accepting new adult patients with Plan 1 and 7% accepting new adult patients with Plan 2. About half of participating dentists in both plans reported the overall administrative burdens as a major burden. Further analyses will evaluate the relationship of the other independent variables with the level ofthe perceived administrative burden.

Conclusion:  Administrative burden is a key factor influencing Medicaid dentist participation in Iowa’s DWP, with variations observed across managed care plans. Reducing these burdens could encourage better provider participation and optimize access to Medicaid services for enrollees.

Molecular Mechanisms Underlying Dentin-Pulp Complex Responses to Bacterial By-Products

Objective:  To explore the molecular mechanisms underlying dentin-pulp complex responses to caries progression by studying the gene expression of matrix metalloproteinases (MMP1, 2, 3, 8, 9, 13, and 20) and tissue inhibitors of metalloproteinases (TIMP1, 2, 3, and 4) by dental pulp stem cells (DPSCs) stimulated with bacterial by-products.

Methods:  Pure isolates ofStreptococcus mutans (SM) andLactobacillus rhamnosus (LR) were cultured on blood agar for 48 hours, then transferred to Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum (FBS) and 1% MEM vitamins. After 24 hours, bacterial filtrates were prepared from filter-sterilized supernatants and used to stimulate DPSCs. Lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Bacillus subtilis served as control bacterial components. DPSCs were treated with/without 20% bacterial filtrates, 10 µg/mL LPS, or 15 µg/mL LTA for 24 hours. Gene expression of MMPs and TIMPs was quantified by RT-qPCR, using GAPDH as the reference gene. Linear mixed models were used to assess statistically significant differences in expression profiles (α = 0.05).

Results:  Both LPS and LTA treatments significantly upregulated MMP1, MMP3, and MMP13 expression compared to controls (p<0.001). LPS specifically increased MMP8 expression (p = 0.006), while LPTA upregulated MMP2 (p<0.001). LR bacterial filtrate led to significant upregulation of MMP1 and MMP13 and downregulation of MMP8, whereas SM filtrate induced a significant increase in MMP13 expression only (p<0.05). TIMP1 and TIMP3 expression remained unaffected across all treatments, but LPS upregulated TIMP4 (p=0.030) and downregulated TIMP2 (p=0.036). None of the treatments stimulated MMP9 or MMP20 expression in DPSCs.

Conclusion:  The expression of MMPs and TIMPs in DPSCs is modulated by specific bacterial by-products, with distinct regulatory effects observed. These findings underscore the potential roles of MMPs and TIMPs in the dentin-pulp complex’s responses to different bacterial components during caries progression.

Supported by: NIDCR K08 DE029490

HIV-Associated Salivary Immune Dysregulation Despite Antiretroviral Therapy

Objective:  Despite advances in antiretroviral therapy (ART), Human Immuno-deficiency Virus (HIV) poses a significant public health challenge, particularly in sub-Saharan Africa. HIV infection leads to profound immune dysfunction, increasing susceptibility to both systemic and oral diseases, such as periodontal disease and dental caries. Perinatal HIV transmission, which can occur during pregnancy, delivery, or breastfeeding, raises concerns about immune dysregulation in children. This study investigates whether perinatal HIV exposure, with or without infection, affects salivary immune profiles in children.

Methods:  Saliva samples were collected from 75 randomly-selected children aged 4–11 at the University of Benin Teaching Hospital in Nigeria. The children were divided into three groups: 5 perinatally-infected with HIV (PHIV+), compared to age- or sex-matched? 40 HIV-exposed but uninfected (HEU) and 30 unexposed and uninfected (HUU). Salivary cytokine profiles were analyzed using the Milliplex 48-cytokine panel, which includes markers of inflammation such as IFN-γ, IL-6, TNF-a, and IL-8. Kruskal-Wallis nonparametric tests were used to compare cytokine levels across the three groups.

Results:  The PHIV+ group exhibited significantly elevated levels of 29 out of 48 cytokines associated with increased inflammation and immune cell recruitment, including IFN-γ, IL-6, IL-10, Eotaxin, FGF-2, Fractalkine, IL-2, IL-5, IL-12, IL-15, IL-17A, IL-27, RANTES, and TGF-a, when compared to the HEU group. A subset of 15 cytokines, including IL-8, M-CSF, TNF-a, VEGF-A, and IL-22, was significantly higher in the PHIV+ group compared to both the HEU and HUU groups. No significant differences were observed between the HEU and HUU groups.

Conclusion: HIV infection in children, rather than perinatal exposure alone, is associated with persistent salivary immune dysregulation, characterized by elevated pro-inflammatory cytokines. This immune dysregulation despite ART, which controls systemic viral load, suggested unresolved localized immune disturbances in the oral environment. Further research is needed to address the long-term oral health effects in children living with HIV.