Abstracts

Teddy Dong

Department: Oral and Maxillofacial Surgery

Faculty Mentors: Dr. Jasjit Dillon, Dr. Thomas Dodson, Dr. Andrea Burke

Objectives: Gender bias has been recognized in the process of candidate selection for medical residency programs. Letters of recommendation have a critical role in applications for residency programs. This study examined the presence of gender bias in recommendation letters for Oral and Maxillofacial Surgery residency candidates.

Methods: The investigators implemented a cross-sectional study and analyzed letters of recommendation entered in the American Dental Education Association Postdoctoral Application Support Service (PASS) for 2021 to 2022 applicants to the University of Washington School of Dentistry Oral and Maxillofacial Surgery. Qualitative text analysis, quantitative text mining, and topic modeling were performed with software including Dedoose, QDA Miner, and RStudio.

Results: There were 784 letters of recommendation for 153 applicants (74.8% male) analyzed. Of 784 letter writers, 69% were male, 31% chairs, 20% deans, and 8% professors. The average word count was 394. Letter writers for female compared with male applicants had a higher average word count (female = 400, Male= 375, p= 0.714). Female letter writers tended to write LORs with higher average word count. Both male and female letter writers managed to write longer letters for female OMS residency candidates than for male candidates. There was no significant difference in quantitative text mining and topic modeling.

Conclusions: Letters of recommendation for Oral and Maxillofacial Surgery residency generally advocate for the applicant. Male and female applicants are described similarly, and this study did not find any significant differences in the description of applicants. Future studies involving more application cycles and academic institutions are recommended to validate the absence of gender influence on the letters of recommendation in the specialty of Oral and Maxillofacial Surgery.

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund

Bita Fathipour

Department: Pediatric Dentistry

Faculty Mentor: Dr. Ana L. Seminario

Objectives: In Kisumu (West Kenya) and on a global scale, the issue of inadequate oral health is a significant public health concern. Oral health literacy (OHL) plays a pivotal role in the burden of oral diseases. Enhancing OHL is a challenge that must be addressed to increase access to dental care. Our team conducted a study with the following objectives: (1) Identify the existing OHL resources, (2) Identify barriers and facilitators to improving OHL among patients and providers.

Methods: A qualitative study was conducted in Kisumu, including 20 key informant interviews with a sample of dentists (n=1), clinical officers (n=3), nurses (n=2), nutritionists (n=3), mentors (n=3), and parents (n=8). Participants who consented were recruited via purposive and snowball sampling from county HIV clinics. Based on latest literature review of the topic, provider interviews included 15 questions covering topics such as patient education, caries risk assessment, anticipatory guidance, teach-back, gaps in professional training, and recommendations on improving OHL. Patient interviews included 10 questions assessing knowledge and barriers and enablers to achieving good oral health.

Results: Less than 1% of providers utilize teach-back, caries risk assessment, and anticipatory guidance. From the perspective of healthcare providers, obstacles to enhancing OHL include a lack of training (91%), time constraints (100%), and inadequate access to educational resources (100%). Providers recommended using educational materials in plain language, continuing medical education courses, and formal training as potential facilitators. Among the patients, 38% noted language as a barrier to OHL, while 75% recommended health talks, and 25% using posters as facilitators.

Conclusions: Results revealed major gaps in current OHL practices in Kisumu. The results detail specific provider and patient recommendations for OHL training and resources to improve OHL communication. These findings underscore the need to translate research into action by developing a toolkit of OHL resources for providers and community organizations.

Supported By: Ms. Janet Matthews and UW Timothy A. DeRouen Center for Global Oral Health

Vijaya Bharathi Jawaharlal

Department: DECOD (Dental Education in the Care of Persons with Disabilities)

Faculty Mentor: Dr. Kimberly Espinoza

Objectives: 1. Outline how to use interpretative phenomenological analysis (IPA) as a qualitative research method. 2. Describe the lived experiences of special care dentists who emphasize the care of adults with developmental disabilities, including how these providers “make sense” of their professional pathways. 3. Outlines “the essence” of special care dentists who focus on the care of adults with developmental disabilities.

Methods: Interpretative phenomenological analysis (IPA) is a qualitative research method from the field of psychology with origins in both phenomenology (lived experiences) and hermeneutics (sense-making). Special care dentists with at least 40% of practice/time focused on the care of adults with developmental disabilities were interviewed, and the results were analyzed using IPA methods.

Results: We will describe the lived experiences of special care dentists who emphasize the care of adults with developmental disabilities, including how these providers “make sense” of their professional pathways. We will outline “the essence” of special care dentists in these pathways.

Conclusions: Understanding the lived experiences and sense-making of this subpopulation of special care dentists highlights the essential nature of professional identity formation within the field of special care dentistry and what it means to “be a special care dentist” with a focus on the care of adults with developmental disabilities. The results can help address the workforce shortage in special care dentistry by understanding what motivates providers to enter and stay in the field.

Supported By: NA

Divya Kannappan

Department: Oral Medicine

Faculty Mentor: Dr. Milda Chmieliauskaite

Objectives: The objective of this study is to understand stakeholder perspectives on the need for modifying the existing DC/TMD pain drawing, and suggestions for modification.

Methods: A purposively selected group of sixty-two individuals that have published/worked on pain drawings within the last 10 years were invited by e-mail to participate in an electronic survey with closed-ended and open-ended questions. Questions elicited their agreement or disagreement with statements about the need for changes to the DC/TMD pain drawing, the drawing’s ability to accurately capture pain locations, and proposed changes. Open ended questions solicited suggestions for improving the DC/TMD pain drawing. The survey was available to invited participants until saturation of new ideas was reached. A consent was available at the start of the survey and selecting yes and continuing to the next page implied consent. The study was determined to be exempt by the University of Washington Institutional Review Board.

Survey results were exported to an Excel spreadsheet and summarized using descriptive statistics. Three individuals independently reviewed the open-ended responses and summarized the themes and counted the frequency of unique individuals mentioning a theme. Results were compared between raters for agreement.

Results: Nineteen individuals completed the survey, for a 30% response rate. Their ages ranged from 36 to 67 years old (median age 58) with 7 female and 11 male respondents. Participants were predominantly orofacial pain specialists (n=17) and worked in an academic setting (n=18). The respondents worked in Europe (n=12), North America (n=4), Central/South America (n=2) and Asia (n=1). Seventy three percent (n=14) strongly or somewhat agreed that the DC/TMD pain drawing could be improved by modifications. Seventy-eight percent (n=15) strongly or somewhat agreed that the current DC/TMD pain drawing allows individuals to accurately indicate all oral pain locations. And 89% (n=17) somewhat or strongly agreed that the current DC/TMD pain drawing allow the patient to accurately indicate all head, neck and facial pain locations. Three proposed modifications were presented to participants including a visual representation of the instructions to which three quarters of participants somewhat or strongly agreed (n=14). Sixty eight percent (n=13) strongly or somewhat agreed to adding a new three quarter view of the left and right face, and 95% strongly or somewhat agreed too adding a back of the head/neck view (n=18).

Free ended responses were categorized into one of three areas and included; general instrument considerations, anatomical considerations, and instructions/administration. General instrument considerations included ideas that the drawing should be designed in such a way as to support quantification and data analysis for research. A sentiment to keep the instrument simple for the patient. And to digitalize the instrument and use 3-D views.

The most salient anatomical considerations were to add a large frontal and back view of the face/head extending to the neck and shoulder (i.e upper trapezius).

The most salient considerations for the instructions were to instruct the patient to delineate painful areas associated with the chief complaint from other surrounding pains. Create administration instructions and provider verifications to increase the validity of the drawing. And for the instructions to delineate a time frame for the drawing (e.g pain in the last 30 days) consistent with other DC/TMD instruments.

Conclusions: The majority of stakeholders that participated in this study agree that the existing DC/TMD pain drawing captures oral and head/neck pain locations accurately yet believe that minor modifications may improve the instrument. The strongest support was for additional views of the head/neck highlighting the importance of evaluating these structures in patients with TMDs. Select suggested modifications to the drawing and its instructions/administration will be created and tested with end users using cognitive interviews as the next step in this work.

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund

Harnoor Kaur Mahal

Department: DeRouen Center for Global Oral Health

Faculty Mentor: Dr. Ana Lucia Seminario

Objectives:

Objective 1: Conduct informational interviews about nutritional education and resource availability with caregivers of children aged 2-5 years old in Kisumu, Kenya, particularly focusing on those who may or not be impacted by HIV.

Objective 2: Conduct informational interviews with healthcare providers to determine the nutritional literacy programs available and implemented for families with children under 5.

Objective 3: Analyze the information collected and create a strategic framework to improve any existing disparities in knowledge between families and healthcare providers of children aged 2 to 5 years old. The goal is to foster improved communication and understanding within this vital demographic.

Methods:

Research Methodology: This qualitative study employed 1-on-1 interviews utilizing a standardized set of questions. The interview questions were formulated based on the “National Guidelines for Health Diets and Physical Activity” document from the Republic of Kenya and the “Situational Analysis of Children & Women in Kenya” published by UNICEF. Provider interviews encompassed both a “community assessment” and a “government assessment.”

Data Analysis Method: Thematic Analysis was employed, involving processes such as familiarization, coding to identify patterns/themes, and the identification of recurring themes within the data. Research interviews were recorded using both audio and video recordings, and thematic analysis was applied during the data analysis phase.

Study participant demographics:

Sample Size: The study included 5 providers and 5 caregivers of patients aged 2-5 years.

Locations: Conducted in rural areas, specifically hospitals and clinics outside of Kisumu, Kenya.

Provider Job Titles: Participants included Chief Clinical Officers, Nutritionists, and Dentists.

Interview Length: The duration of interviews ranged from 15 minutes to 1 hour.

Results: This study identified key themes encompassing various aspects of nutrition and healthcare, including:

• Clinical Diagnosis for Nutrition, involving parameters such as MUAC Score, Z Score, Nutritional Assessment, and CBC tests. Healthcare providers at all clinics in the study sample showed a comprehensive understanding of these diagnostic tools. Moreover, these tools were frequently employed within the clinic setting for diagnosing children aged 2-5 years old.
• Nutritional Counseling, which encompasses services related to malnutrition, education in maternity wards, and resources shared with patients at community clinics. These findings, such as the use of F-75 and F-100 powder for severe malnourishment, shed light on the availability of nutritional resources and suggest potential strategies for improving nutritional understanding among caregivers.
• Local Cultural Beliefs, exploring the impact of family diet, local beliefs, and cultural foods on nutritional practices. These insights contribute to the appreciation of cultural factors, such as Ugali and Strong Tea, in shaping nutritional practices.
• Barriers, including factors such as socioeconomic limitations, poverty, lack of education/awareness, and environmental influences of nutritional health. One caregiver shared “How will knowing the egg is good for my child help me if I cannot even afford to purchase the egg?” Understanding these barriers is crucial to creating a framework that employs accessibility and creates sustainable grassroots solutions to help impoverished communities.
• Provider Education, examining the role of government trainings/protocols and sensitization programs in enhancing healthcare providers’ knowledge. Healthcare providers shared various programs offered by the government and highlight the effectiveness of sensitization in enhancing healthcare providers’ knowledge. Access to education varied from clinic to clinic. Some clinics did not have access to the internet or digital resources, while others had computers and digital databases. Most healthcare officials showed print medium books and binders with printed resources to patients as the main form of education.
• Caregiver Education, highlighting the correlation between education levels, poverty, awareness, and socioeconomic capacity in caregivers. There was a stark contrast in nutritional understanding between caregivers of high socioeconomic capacity and education levels, and those on the opposite spectrum. These findings underscore the importance of caregiver education and the ability to afford nutritional resources.

Conclusions: The study revealed disparities in knowledge between healthcare providers and caregivers of children aged 2-5 years. Healthcare providers demonstrated a comprehensive understanding of the nutritional guidelines established by the Kenyan government, along with basic nutritional analysis for pediatric patients. Conversely, caregivers exhibited varying levels of knowledge, influenced by factors such as their highest education attained, local cultural beliefs, and socioeconomic constraints.

Caregivers with a college-level education and those residing in single-family homes demonstrated a solid grasp of national nutritional guidelines, implementing them effectively. On the other hand, caregivers with elementary education levels and/or living in polygamous households showed a noticeable decline in awareness of nutritional guidelines and struggled with the implementation of nutritious diets for children.

To address these educational disparities among caregivers, a framework has been devised to provide additional resources for families. This includes the distribution of educational handouts covering topics such as proper child feeding practices and nutritional guidelines to community clinics. Furthermore, initiatives to enhance nutritional accessibility and sustainability for families are proposed, such as the implementation of a “vertical garden” system. This sustainable resource, created using “shoe carriers,” requires only soil, water, and seeds, making it an affordable and viable option for low-income households.

Supported By: DeRouen Center for Global Oral Health and Janet Mathews (Donor for the Kenya Project)

Brian Merrill

Department: Restorative Dentistry

Faculty Mentor: Dr. Yen-Wei Chen

Objectives: This study aims to investigate the effects of preparation design on the marginal gap formation and progression in lithium disilicate onlay restorations on molar teeth.

Methods: Four adhesive overlay designs were selected for testing: 1. Tabletop onlay with butt joint margins; 2. Circumferential hollow-ground bevel; 3. Rounded shoulder with 2 mm axial wall height; 4. Full crown (rounded shoulder with 4 mm axial wall height). Extracted virgin molars were prepared by 2 dental students and refined by a prosthodontist, and lithium disilicate restorations were designed and milled with a CAD/CAM workflow. Samples were cemented using a self-adhesive cement, then subjected to mechanical loading for 1.2 million cycles. Optical Coherence Tomography (OCT) was used to evaluate marginal integrity before loading, then every 300,000 cycles.

Results: The milled butt joint restoration at baseline showed a 200 µm internal discrepancy between the preparation and onlay margins after cementation, but no external interfacial gap due to cement sealing the gap. Current data suggests no significant difference between preparation groups and marginal gap progression.

Conclusions: The other groups exhibited a superior initial fit, implying that a CAD/CAM workflow may not be the optimal choice for fabricating butt joint onlays. Further testing and revised measuring methods are needed to determine if preparation influences marginal gap formation.

Supported By: Dentway CAD/CAM Related Activities Fund, Department of Restorative Dentistry

Bi Nguyen

Department: Orthodontics

Faculty Mentor: Dr. Zi-Jun Liu

Objectives: The spatial relationships between oropharyngeal structures ensure proper adaptations in functions of the oropharynx such as swallowing. However, synchronized and coordinated dynamics of the oropharyngeal structures are not well described. This study aims to analyze the spatial changes of the soft palate (SP), tongue base (TB), epiglottis (EP), and pharyngeal wall (PW) during swallowing episodes.

Methods: Eight 7–8-month-old Yucatán minipigs were used. X-ray video fluoroscopic was recorded during unrestrained feeding sessions with pig chow pellet mixed with barium sulfate suspension. X-ray video clips that showed swallowing episodes were digitized and traced using Tracker 6.1.1 software (OSP, USA). An X-Y coordinate was set up to trace structural and inter-structural distance changes during sporadic swallowing episodes. Then swallowing episodes were traced frame by frame and digitized by measuring each point of the oropharyngeal structures in relation to the static X-Y coordinate.

Results: During swallowing, TB and EP showed larger caudal-dorsal directional movements than those in SP. In contrast, PW movements occurred in the rostral-ventral direction. Among the four traced oropharyngeal structures, TB showed the largest distance changes (2.94 ± 1.28mm) followed by PW, EP, and SP. The inter-structural spatial relation between each structure showed the largest distance changes between EP-PW (2.66 ± 0.66mm) and EP-TB distance (2.29 ± 0.84mm). The smallest inter-structural distance change was seen between SP-TB (1.64 ± 0.58mm) even though no significance was detected. Overall, changes in swallowing episodes were significantly larger than those during chewing (p<0.05, chewing analysis was carried out elsewhere).

Conclusions: The larger movement of TB in coordination with EP plays major roles in swallowing function. Understanding of these spatial relationships in different oropharyngeal structures during normal swallowing provides the baseline for potential mechanisms of various swallowing disorders such as dysphagia.

Supported By: NIH/NIDCR R01 DE028864

Delbert Oxborrow

Department: Oral and Maxillofacial Surgery

Faculty Mentor: Dr. Ivy Lin

Objectives: The practice of dentistry produces waste contributing to climate change and negative health outcomes. Reducing waste is critically important, but there has been minimal emphasis on sustainability in the profession, and little research has been done on climate action in dentistry. Waste audits can help understand the type and amount of waste produced to inform waste reduction strategies. The objective of this study is to conduct a waste audit and analyze the results to identify areas of potential waste reduction and make recommendations on how to reduce that waste.

Methods: We conducted a waste audit of the garbage produced during a typical 4-hour pre-clinical simulation exercise for operative dentistry and fixed prosthodontics. The exercise was conducted in the pre-clinical simulation lab by 72 second year dental students. These exercises are part of a standard curriculum with a uniform format in terms of supplies used and participants and therefore generate a consistent amount of waste, making this data representative of the typical waste generated in the clinic.

Results: We found 894 gloves and 70 masks discarded making an average of 12.42 gloves and 0.97 masks used per student. In total 26,636 g of waste was discarded. Plastic was the largest contributor (57.5%) followed by benchtop paper (19.6%) then impression materials (6.5%). Plastic items included empty tubes of polyvinyl siloxane (PVS) impression material, empty jars of putty components, plastic measuring cups, and plastic wrap packaging.

Conclusions: Based on these findings, our recommendations to reduce waste are categorized into policy changes, alternative purchasing of sustainable materials, and increasing awareness through education. Specific suggested actions include relaxing glove and gown requirements during certain laboratory simulations; replacing single use items with reusable or bulk option; increasing use of intraoral scanners; and posting informational signage with examples of what can be recycled.

Supported By: NA

Amr Abouzeid

Department: Restorative Dentistry

Faculty Mentors: Dr. Van Ramos, Dr. Daniel Chan, Dr. Sumita Jain

Objectives: This in vitro study assessed the effect on surface roughness of a liquid ceramic system (MiYO) on 3Y TZP compared with polished 3Y TZP before and after thermocycling. In addition, two types of bacteria were cultured on each specimen and the growth of each was counted (CFU). Also, the change in color of MIYO-stained 3Y zirconia after repeated firing was investigated.

Methods: Thirty disk-shaped specimens of 3Y monolithic zirconia were CAD-CAM milled from the same STL file. Specimens were divided into three equal groups. Group PM (3 mol% yttria TZP, colored with liquid ceramic, polished). Group PZ (3 mol% Yttria TZP, polished). Group M (3 mol% yttria TZP & colored with liquid ceramic, as fired) Surface roughness was measured using a profilometer for all groups. All groups were subjected to artificial aging: 5000 cycles of thermocycling (5°C-55°C, dwell time 20 sec) Specimens were cleaned and disinfected before applying a defined amount of either Streptococcus mutans or Porphyromonas gingivalis. Discs were incubated for 5 days. Results were evaluated using Ra values compared between groups before and after aging. CFU count was determined and compared. Data analysis: T-test, 1-way ANOVA, and Tukey’s HSD test at α = 0.05. Specimens in groups PM, and M were cleaned, and base color was measured using a spectrophotometer, then all specimens were subjected to an extra three firing cycles and Delta E was measured and compared between each cycle.

Results: There was a statistically significant difference in CFU count among all groups. Group PZ showed the lowest CFU count followed by group PM then group M. There was a statistically significant difference in surface roughness (Ra) between the PZ and M groups before and after aging. There was no statistically significant difference between groups PZ and PM before and after thermocycling. All surface roughness measurements were below 200 nm (0.2 µm- standard for smoothness of restorative materials). Two-way ANOVA with repeated measures revealed a statistically significant difference in delta E after repeated firing.

Conclusions: 1. There was a correlation between surface roughness and CFU count. The polished zirconia group showed lower surface roughness and CFU count followed by the polished MiYO group and then the MiYO as fired group.
2. Thermocycling increased surface roughness among all groups.
3. The number of CFUs depends on surface roughness and type of bacteria; Streptococcus mutans had greater CFU count than Porphyromonas gingivalis.
4. Depending on the predictability threshold, repeated firing of MiYO may be perceived.

Supported By: Department of Restorative Dentistry

Jenna Castillo

Department: Sociology

Faculty Mentor: Dr. Donald Chi

Objectives: Cystic fibrosis (CF) is the most common life-limiting autosomal recessive disease in Caucasians, affecting nearly 40,000 children and adults in the United States. Previous studies have shown oral bacteria may infect the lungs in individuals with CF, which underscores the need to prevent dental caries and gingivitis to avoid further complications such as airway inflammation. This qualitative study examines behavioral and social factors associated with oral diseases in
individuals with CF to identify patient-centered strategies for oral disease prevention.

Methods: Beginning in October 2022, virtual interviews were conducted with twenty-four participants between 12 and 30 years old who enrolled in CF studies at Seattle Children’s Hospital, University of North Carolina, and University of Alabama at Birmingham. Interviews for participants under 18 years old were conducted with a parent or guardian. Interview questions inquired about dietary habits, experiences with CF medical care teams, oral health behaviors, and support systems. Interviews were in English and were audio-recorded, transcribed, and thematically analyzed in Dedoose. A grounded-theory coding approach was used to analyze interviews such that codebooks were systematically and iteratively revised based on emerging themes. Researchers reviewed transcripts using the codebook to meet agreement and reliability.

Results: Participants shared social and behavioral factors affecting dental health such as lack of energy, time, and motivation. They reported frequent or occasional history of cavities and inconsistent dental visits partially due to limited appointments during the COVID-19 pandemic. Moderate to high sugar-sweetened beverage consumption was reported. Most participants described good rapport and high levels of trust with their care team, and reported receiving emotional support and medication reminders from family and friends.

Conclusions: With ongoing data collection, this qualitative study will provide insight on modifiable behavioral risk factors that are amenable to interventions and provider recommendations, and reveal feasible intervention delivery modes for individuals with CF.

Supported By: NIH

Azeez Fashina

Department: Oral Health Sciences

Faculty Mentor: Dr. Robert A. Cornell

Objectives: To identify possible cells and signals that play important roles in primary mouth formation and propose their possible roles in human craniofacial malformations.

Methods: Using the zebrafish as a model, we created several transgenic lines of fishes that allowed us to kill, pause the growth, and monitor different cell lines that are associated with primary mouth formation. The effects of these cells were viewed and analyzed using several novel imaging techniques and immunohistochemistry.

Results: Though an important part of future oro-pharyngeal communication, the foregut endoderm is not an important structure in the invagination of the stomodeum/mouth. Loss of periderm results in no stomodeal invagination and no primary mouth formation. The arrival of the periderm at the primary mouth domain is possibly a key event that is required for the breakdown of the buccopharyngeal membrane to result in a mouth-foregut communication.

Conclusions: In humans, virtually nothing is known about the molecular control of primary mouth formation, but several craniofacial syndromes have been associated with persistent buccopharyngeal membrane as well as cleft lip and palate. Using a zebrafish model, we present data suggesting that the periderm is required for formation of mouth opening. Further understanding of how it does this could provide better insight into the cause and prevention of craniofacial anomalies including cleft lip and palate.

Supported By: NIH-NIDCR R90 DE021984

Hakan Gem

Departments: Oral Medicine, Fred Hutchinson Cancer Center

Faculty Mentors: Dr. David Dean, Dr. Armin Rashidi

Objectives: Gut microbiota are a source of pathogens in immunocompromised patients. While many studies have focused on the gut microbiota, few have investigated the link between gut and oral microbiota in patients receiving induction chemotherapy for AML. The aim of our study is to fill this knowledge gap by characterizing the microbiota in longitudinal salivary, supragingival plaque, and fecal samples of patients receiving inpatient chemotherapy for AML.

Methods: 31 patients were enrolled in this prospective, single-center study at the University of Washington Medical Center. Baseline stool, saliva, and plaque samples were collected during the first week of hospitalization followed by weekly sample collections for 5 weeks or discharge from the hospital, whichever occurred first. Samples were collected in 95% ethanol and immediately stored in -80°C until DNA extraction. After extraction, samples underwent shotgun metagenomic sequencing targeting 10M paired-end reads per sample. Alpha diversity was quantified by Shannon index, beta diversity by Bray-Curtis dissimilarity, and MetaPhlAn4 was used for taxonomic assignment. Inpatient time was divided into three intervals: admission to day 10, day 11-20, and day 21 and later.

Results: Median age was 64 (range 34-82) and median hospitalization length was 19 days (range 6-31). 111 samples (stool: 39, saliva: 36, plaque: 36) from 16 patients (9 male, 7 female) have been sequenced to date. 15 patients were newly diagnosed and 1 had relapsed/refractory disease. Chemotherapy regimens included CLAG-M (n=10), ATRA+ATO (n=3), Vyxeos (n=1), Venetoclax + Azacitidine (n=1), and intermediate-dose Ara-C (n=1). The three most frequently used antibiotics were cefepime (n=14), IV vancomycin (n=12), and levofloxacin (n=11). Neutropenic fever occurred in 13 patients, and documented infections included bacteremia (n=8), skin/soft tissue (n=5), intestinal (n=4), urinary tract (n=4), and pneumonia (n=1).

Four bacterial species isolated from blood cultures of patients with fever were detected by sequencing 1-2 weeks before bacteremia: Gemella Haemolysans in saliva, plaque, and stool, Streptococcusmitis in saliva and stool, Rothia mucilaginosa in saliva and plaque, and Citrobacter freundii in stool. Microbiota diversity from the three sites showed markedly different dynamics: plaque microbiota diversity remained stable through all three intervals with levels as high as fecal microbiota in intervals 1 and 2, and higher than both fecal and salivary microbiota in interval 3. Salivary microbiota diversity also remained relatively stable only experiencing a slight reduction at interval 3, but it showed the lowest overall diversity among all three sites. In contrast, fecal microbiota diversity was highest among all sites in intervals 1 and 2 but dropped to the lowest in interval 3. With all 3 intervals combined, salivary microbiota had the lowest diversity (median 1.9, range 0.04-3.9) followed by plaque (median 2.7, range 0.2-4.3; P=0.01) and then fecal (median 2.9, range 0.3-3.7; P=0.048). The three sites largely segregated on hierarchical clustering using species abundances, however, microbiota in several low-diversity samples from different sites were observed within the same patient: Veillonella parvula and Rhodotorula mucilaginosa were detected in all 3 sites, Veillonella dispar was detected in stool and plaque, and Streptococcus parasanguinis was detected in stool and saliva. Each overlap was present in 5 or more patients.

Conclusions: This preliminary shotgun sequencing analysis of longitudinal salivary, plaque, and fecal microbiota in AML patients offers two novel findings: 1) whereas diversity of fecal microbiota declines over time with exposure to antibiotics, diversity of plaque microbiota remains stable. 2) while healthy adults show no overlap between oral and gut microbiota, low-diversity fecal samples from AML patients contain species of their own oral microbiota, particularly at later time points. These data suggest ectopic colonization of the gut by oral flora which may contribute to gut dysbiosis. Future mechanistic studies of plaque biofilms may reveal community-protective mechanisms that sustain oral plaque microbial diversity and contribute oral flora migration to the gut. Mitigating gut dysbiosis in AML patients by manipulating plaque biofilms could be a novel approach to improve patient outcomes.

Supported By: Innovation Grant from Kuni Foundation

Claire Mills

Department: Fred Hutchinson Cancer Center

Faculty Mentor: Dr. Slobodan Beronja

Objectives: Investigate a mechanism of tumor formation and recurrence in oral squamous cell carcinoma (OSCC) called “field cancerization,” where normal-appearing cells around tumors contain genetic alterations that prime cells to become cancerous, driving recurrence.

I hypothesize that cancer field cells have unique genetic and molecular features that influence stem cell renewal and predispose cells to cancer. I aim to 1) investigate the genetic, epigenetic, and transcriptional characteristics driving normal field cells to become cancerous and 2) test the genetic alterations necessary to transform precancerous cells to cancer using an orthotopic patient-derived xenograft (PDX) model.

Methods: Aim 1: Genetic analyses of fresh patient tissue sample sets (tumor, normal-adjacent field, and healthy contralateral tissues) were performed using whole genome sequencing and single-cell transcriptional and epigenetic sequencing.

Aim 2: Precancerous cell line cells were transduced with lentiviruses to over-express different oncogenes. Cells were transplanted orthotopically into immunodeficient mouse tongues to test for tumor-forming potential. Controls include cancer cell line cells (positive) and healthy oral keratinocyte cell line (negative).

Results: In my ongoing analysis of six patient sample sets, I have observed that normal-adjacent field cells display genetic changes, notably in gene expression and copy number, compared to healthy tissue. These changes alter critical stem cell regulators, including Notch signaling pathway genes.

Underscoring the role of stem cell renewal in tumorigenesis, of the genes tested, overexpression of known stem cell regulator HRAS in precancerous cells induced tumor transformation in vivo.

Conclusions: I have found that stem cell renewal plays an integral role in OSCC progression. I will expand my analysis to additional patient samples to study biomarkers of field cancerization and validate the role of stem cell regulation in OSCC progression. My long-term goal is to determine how the field is genetically primed for tumor formation and recurrence and identify novel mechanisms to prevent recurrence.

Supported By: NIH/NIDCR T90 DE021984

Priti Mulimani

Departments: Oral Health Sciences, Mechanical Engineering

Faculty Mentors: Dr. Tracy Popowics, Dr. Nathan Sniadecki

Objectives: The periodontal ligament (PDL)-bone enthesis is a critical transition zone that facilitates stress distribution and functional adaptation. The interplay between key cell types at this junction determines mechanotransduction and signaling. Cellular events occurring at the PDL-bone enthesis remain under-investigated, partly due to the absence of 3-dimensional (3D) in vitro models. The objective of our investigation was to engineer a 3D in vitro periodontal tissue construct model with embedded PDL-bone cellular enthesis.

Methods: We used the suspended tissue open microfluidic patterning (STOMP) system to create a spatially patterned tissue construct between two silicone posts. Two cell types were used – a) PDL cells obtained from extracted premolars of healthy patients, and b) osteoblastic cells obtained by treating PDL cells with osteogenic induction medium. PDL cells mixed with 4 mg/ml type I rat tail collagen were pipetted in the center of the STOMP microfluidic channel. Next, osteoblasts in collagen mixture were pipetted on either sides of the PDL cell-collagen mixture to surround tips of the posts. After incubating patterned tissues for 48 hours at 37 °C, the microfluidic channel templates were removed. Thus, our technique produced a free-standing, osteoblast-PDL-osteoblast 3D in vitro tissue construct, suspended between two posts.

Results: Analysis with microscopy detected formation of a zigzag region resembling cellular enthesis between PDL and osteoblastic cells on either sides. Immunofluorescence staining demonstrated aligned PDL cells similar to in vivo architecture. Based upon deflection of silicone posts, we noted the tissue constructs (n=6) generated a force of around 439 +/- 65.7 uN.

Conclusions: We have developed a novel 3D in vitro PDL model with a cellular enthesis constituted by PDL and osteoblastic cells. Our model provides a potentially valuable platform to investigate cell biomechanics, mechanotransduction and tissue behaviour at the junction of disparate cell types. This model has significant translational potential for periodontal therapeutic and regenerative applications

Supported By: NIH/NIDCR R03 DE029827, NIH/NIDCR R90 DE02305

Derrick Tang

Department: Oral Health Sciences

Faculty Mentor: Dr. Fritzie Arce-McShane

Objectives: Complex orofacial sensorimotor behaviors, such as feeding, are driven by primary motor cortex (M1) and primary somatosensory cortex (S1). Sensory afferents from richly innervated oral structures, e.g., teeth, palate, and tongue, send tactile and proprioceptive information to the cortex, enabling precisely-controlled and rapidly-adjusting motor efferents to the tongue and jaw. How the cortex encodes self-touch between the tongue and the oral structures during feeding behavior has not yet been described. The loss of fidelity in sensory encoding in both aging and disease may underlie sensorimotor dysfunctions such as dysphagia and dysarthria.

Methods: We recorded neuronal activity using chronically-implanted microelectrode arrays in four cortical regions: rostral M1 (rM1), caudal M1 (cM1), Area 3a/3b, and Area 1/2 of a rhesus macaque. Simultaneously, we tracked 3D movements of the tongue using bilateral video radiography while the monkey engaged in natural feeding behavior. We then used a series of nerve blocks designed to remove tactile feedback only. We trained a self-optimizing K-Nearest Neighbor (KNN) classifier to predict lingual-palatal contacts from neural data with the hypothesis that lingual-palatal contacts are represented by similar patterns of neural activity.

Results: Accuracy of classifying lingual-palatal contacts was above chance levels in each cortical region both naturally and under nerve block conditions for the most commonly occurring contact states. Classification accuracy based on spiking activity of M1 neurons always outperformed classification using S1 neurons. Most strikingly, decoding accuracy increased by about 25% in both S1 regions after nerve block was administered.

Conclusions: The high decoding accuracy of the KNN classifier suggests that the ensemble activity in both M1 and S1 represents lingual-palatal contact states well. Although absence of tactile feedback from oral mechanoreceptors reduced activity overall in S1, the information encoded more tightly corresponded with contact states, suggesting alternative compensatory pathways aid in representation of contact states during feeding.

Supported By: NIH/NIDCR T90 DE021984

Katherine Tang

Departments: Materials Science and Engineering, Oral Health Sciences, Restorative Dentistry

Faculty Mentors: Dr. Dwayne Arola, Dr. Hai Zhang

Objectives: The objective of this study was to determine whether there are changes in the composition and mechanical properties of tooth dentin in Type 1 and Type 2 diabetic mice compared to nondiabetic controls.

Methods: Mice were bred by our collaborators at the UW Diabetes Institute. At 12 weeks after inducing diabetes (or similar age for the controls), the mice were terminated and mandibles were extracted. The mandibles were mounted in epoxy and the first molars were assessed for chemical composition through Raman Spectroscopy and mechanical properties using nanoindentation and vickers testing. Acquisitions were taken at varying regions of the tooth. Statistical analysis was conducted through two-way ANOVA with respect to diabetic status and location of the tooth.

Results: It was found that both Type 1 and Type 2 diabetic mouse dentin is different compositionally and mechanically compared to the nondiabetic controls. The diabetic mouse dentin exhibited changes in mineral-to-collagen ratios and a reduction in hardness.

Conclusions: Diabetes does indeed have an effect on the composition and mechanical properties of tooth dentin.

Supported By: Colgate

Kimia Imani

Department: Oral Health Sciences

Faculty Mentor: Dr. Donald Chi

Objectives: Many veterans are ineligible for dental care through the Veterans Affairs (VA) healthcare system because of the 100% disability rating requirement. This study evaluated the association between veterans’ disability rating and dental treatment complexity, and veterans’ disability rating and dental treatment costs for veterans in a non-profit organization that provides dental care to low-income veterans ineligible for VA dental care.

Methods: The analysis was based on 2014-2020 cross-sectional data from veterans across 30 states enrolled in E4V, who received dental treatment and had complete disability rating data (N=167). To assess the relationship between disability rating, the types of dental treatments received (preventive care, basic restorative care, and major restorative care), and the cost of treatment, logistic and linear regression models were used.

Results: The mean age of veterans was 48.3±17.6 years and most (85%) were male. The average disability rating percentage was 39.0±34.5, ranging from 0 to 90. Almost all veterans (94.0%, n=157) received preventive care services, 70.7% (n=118) received basic restorative care, and about 62% (n=103) received major restorative care. The mean cost of dental treatment was 2,885.0 US dollars. Higher disability rating was not significantly associated with more complex dental treatment (preventive care: p=.069; basic restorative care: p=.17; major restorative care: p=.86) or greater dental treatment costs (p=.47).

Conclusions: A higher disability rating was not associated with more complex or higher-cost dental treatments for low-income veterans ineligible for VA dental care.

Supported By: NIH-NIDCR T90 DE021984, University of Washington Dr. Douglass L. Morell Dentistry Research Fund.

Sydney Kim

Department: Biochemistry

Faculty Mentor: Dr. Hannele Ruohola-Baker

Objectives: The ultimate goal of regenerative dentistry is successful growth of a tooth, including all the organizational complexity of its dentin and enamel hard tissues. To this end, our lab is investigating the differentiation of induced pluripotent stem cells (iPSCs) into secretory odontoblasts and ameloblasts, but validation of this differentiation is currently limited by methods that sacrifice the cells. On the basis of two recent papers that introduce an RNA-based gene marker for live cells, I investigated the application of this technology into plasmids for our use in iPSC differentiation into dental cells.

Methods: Plasmids used in the aforementioned papers were obtained from Addgene. Molecular cloning using PCR amplification and double-stranded DNA gBlocks was employed to create plasmids targeting the housekeeping gene GAPDH, the odontoblast-identifying gene dentin sialophosphoprotein (DSPP), and the ameloblast-identifying gene ameloblastin (AMBN). iPSCs grown to 40-50% confluence were transfected with the plasmid targeting GAPDH using Lipofectamine 3000 and observed for fluorescence after 24 hours.

Results: No GFP fluorescence was observed in iPSCs transfected with the GAPDH-targeting plasmid, which indicated that the plasmid did not work as a GAPDH marker as intended. There was also negligible BFP fluorescence observed, indicating that the transcript itself was not expressed in the transfected cells.

Conclusions: Although the plasmid transfection did not work to indicate the target gene in iPSCs, the experiment will be repeated with the HEK293 cell line, which was used in both papers to validate the RNA-based gene marker. This negative result highlights the fact that in order to be used in iPSC cells undergoing differentiation, this marker sequence must be inserted into an iPSC cell line at the genome level. In the future, creating such a novel iPSC cell line will allow customization to show fluorescence for any specific targeted gene, allowing researchers to identify whether differentiation has occurred without sacrificing the cells. Long term, this ability can allow us to streamline the differentiation process towards creating regenerative dental cells.

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund, NIH/NIDCR T90 DE021984

Joshua Lim

Department: Oral Health Sciences

Faculty Mentor: Dr. Donald Chi

Objectives: To estimate the (a) prevalence of and (b) associations between topical fluoride hesitancy and opposition among caregivers of children.

Methods: We administered an 85-item survey to caregivers from November 2020 to September 2021 (N=1,135). The survey included a 20-item topical fluoride hesitancy measure measuring 5 domains of topical fluoride hesitancy and a question about topical fluoride opposition. From this measure, we created five domain-specific scores of topical fluoride hesitancy (none/moderate/high for each domain); a score reflecting any topical fluoride hesitancy (no/yes to any of the five domains); and a topical fluoride hesitancy severity score (total number of moderate/high responses to the five domains; range 0-5). We ran confounder-adjusted logistic regression models to evaluate associations between topical fluoride hesitancy scores and opposition, defined as any degree of opposition to fluoride on a scale of 0 to 10 (no=0; yes≥1). Statistical analyses were performed in 2023.

Results: The final analyses included 1,042 caregivers. The mean caregiver age was 42.0 years (SD: 8.3), 78.7% identified as woman, and 55.9% identified as white. General hesitancy was reported by 82.9% of caregivers. Domain-specific hesitancy prevalence (moderate or high) was 81.3% for the necessity domain, 31.3% for chemicals, 19.5% for harm, 30.1% for uncertainty, and 25.2% for distrust. With respect to severity, 14.7% of caregivers reported moderate/high hesitancy for all 5 domains, 7.7% for 4, 6.8% for 3, 9.3% for 2, 43.9% for 1, and 17.6 did not report moderate/high hesitancy on any domain. Opposition (≥1) was reported by 39.1% of caregivers. Significant positive association were found between all hesitancy measures and opposition.

Conclusions: Topical fluoride hesitancy and opposition are positively associated but not identical. Additional studies are needed to help caregivers make fluoride-related decisions.

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund

Akashdeep Mahal

Department: Oral Health Sciences

Faculty Mentor: Dr. Jonathan Y. An

Objectives: Age leads to diminished maintenance of cellular and repair processes and is the cause of many diseases. Thus, aging can be viewed as an important risk factor to understand and extend lifespan. Specific inhibition of mTOR via rapamycin, an FDA- approved drug with immune modulating properties, has been shown to also extend both lifespan and dental health span metrics leading to an increase in off-label use. One of the common side effects of rapamycin use reported in literature are changes in the oral tissues, specifically increased mouth ulcers. We completed a secondary data analysis collected via survey and past dental records, on off-label rapamycin users and evaluated past dental radiographs using Overjet, a new platform that uses artificial intelligence to assess radiographs for bone loss and tooth decay.

Methods: Survey data was collected from 505 participants including 333 off-label rapamycin users and 172 non-rapamycin users was analyzed for descriptive of the demographics, oral health outcomes, and Rapamycin usage parameters. For off-label rapamycin users, this study only employed analysis on the subset that has taken rapamycin for at least 90 days at the time of collecting survey data. Past dental radiographs were analyzed by Overjet.

Results: Of the oral health changes experienced by rapamycin users, mouth ulcers were among the most common and most were intermittent and disappeared with continued use. Importantly, mouth ulcers did not stop them from continuously taking rapamycin. Measuring bone loss quantitation in past dental radiographs using Overjet showed that participants taking rapamycin had either no changes or improvement in their bone loss.

Conclusions: In self-reported surveys, rapamycin use has been linked to improved periodontal health, decreased caries presentation, and decreased sensitivity. Past dental radiographs show either no loss or improvement in alveolar bone after rapamycin treatment. Overall, our analysis demonstrates that the oral health side effects are minimal and never severe enough for participants to consider avoiding rapamycin use. Further data from clinical trials will be needed to understand the nature of the ulcer presentation of low-dose rapamycin use.

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund, Longevity Impetus Grant from Norn Group.

Anastasia Williams

Department: Biochemistry

Faculty Mentors: Dr. Blair Zhao, Dr. Hannele Ruohola-Baker

Objectives: The main research objective of this project is to further characterize the Ang1/Tie2 pathway using computationally designed high affinity protein minibinders to understand how receptor valency regulates angiopoietin signaling. We will dissect the mechanism of activation of the two already identified Tie2 complexes and evaluate whether Tie2-mb recruits (1) α5β1 that may upregulate cell migration and stabilize blood vessels7 and/or (2) tight junction molecules (claudin5 and ZO1) to stabilize cell to cell interaction to promote vascular integrity.

Extra: The previous characterization of the Ang1/Tie2 complexes were done with synthetic ligands that displayed F-domains. The laboratory previously utilized 2D protein sheets at micron-size to visualize Tie2 clusters and other associating components. They found that Tie2 indeed forms two types of Tie2 complexes: Tie2-claudin5 and Tie2-α5β1. The main research objective for this project is to further characterize these two complexes using newly AI-designed de novo protein-tools to dissect two critical questions in regulation of blood vessel stability by the Angiopoietin-Tie2 pathway to describe the mode of function of the co-receptor, α5β1 integrin, and the mechanism of tight junction recruitment to regulate vascular stability.
To continue understanding the pathway, we used high affinity designed protein mini-binders to bind and activate Tie2 receptors only. The Institute of Protein Design at the University of Washington was able to computationally design high-affinity protein binders from structural information alone. The benefits of minibinders include being small (around 60 amino acids), selective, stable, specific and bind to their target with nanomolar to picomolar affinity10. In the future, minibinders have a large potential for therapeutic functions due to the fact that they are cheaper and easier to produce than natural ligands. We will determine if high valency Tie2-mb that clusters only Tie2 receptors is sufficient for pAKT activation and evaluate whether Tie2-mb alone is able to form and recruit the two complexes: α5β1and/or tight junction molecule (Claudin 5).

Methods: The Institute of Protein Design (IPD) was able to come up with a Tie2-mb protein design using RF diffusion and sequenced a protein that showed clear binding to the Tie2 target. We started by performing the Golden Gate Assembly reaction to insert the Tie2-mb gene into an already designed plasmid (LM1426). Once the final plasmid with the gene of interest was constructed, it was transformed into E. Coli (DH5a and BL21). We also sequenced the final plasmid to verify that the gene of interest was indeed included. Once the protein was expressed in E. coli, the protein was isolated through Ni-NTA affinity chromatography and purified via size exclusion chromatography.

Now that we had the desired isolated and purified protein, we tested to see if high valency Tie2-mb clusters Tie2 receptors only and if it is sufficient for pAKT activation. This was shown by fusing Tie2-mb with SpyTag to allow conjugation with SpyCatcher-fused multivalent scaffolds. We conjugated (linked) the minibinder to the scaffold using the SpyTag/SpyCapture system. The Tie2-mb was designed by IPD to be genetically fused to SpyTag and the SpyCatcher was attached to the H8 scaffold done by previous HRB Lab experiments. We combined the two components together to allow the SpyTag to form a spontaneous amide bond to the SpyCatcher which locks the two partners together via the Lys and Asp residue side chains11. A Coomassie gel was run and showed successful conjugation efficiency. Now that we have the Tie2-mb attached to the multivalent scaffold, we can test whether Tie2-mb at 8 valency is sufficient to activate pAKT.

Results: Due to the time restraints of the SURF program, I was not able to finish this project in entirety and the HUVEC stem cells and western blot results are still in progress. I was able to still successfully sequence the final plasmid to verify that the gene of interest was included. Expressed, isolated and purified the protein of interest. And the Coomassie gel was run and showed successful conjugation efficiency.

The last step that I didn’t have time to perform was adding the mini-binders to HUVEC cells & looking for pAKT or pERK activation. To evaluate the activity of Tie2-mb conjugated at different valency activity, we will analyze their signaling profiles in human umbilical vein endothelial cells, or HUVECs. 16 hours serum-starved HUVECs will be treated with Tie2-mb scaffolds for 15 mins before protein lysate collection for analysis using Western Blot to evaluate pAKT activation. We will analyze in detail other helical scaffolds used previously (H3 and H8), to dissect whether geometric steric effect presentation of these multivalent minibinders. By conjugating Tie2-mb to a micron-scale 2D protein sheet, we will evaluate whether Tie2-mb alone is able to form/recruit the two complexes: α5β1 integrin and/or tight junction molecules. The conjugated Tie2mb-sheet complex will also be tagged with GFP to visualize the clustering of Tie2 receptors. Serum-starved HUVECs were treated with 20nM of Tie2-mb sheet for 30 minutes before PFA fixation for immunofluorescence staining using antibodies for Tie2, claudin-5, and α5 integrin. The stained cells were analyzed using confocal and super-resolution OMX microscopy.

I predict that we would have seen the scaffold present binders at cluster 6 or 7 Tie2 receptors. That would have been evident in a good increase of pAKT on a western blot. This is important because activation of pAKT correlates with Tie2 signaling pathways by Ang1 which promotes endothelial cell/endothelial progenitor cell migration and protects the cells from apoptosis8. There is still a chance that we have something else like H3 (clusters 3 Tie2 receptors) where pERK gets activated too. pERK correlates to Ang2 and causes vascular destabilization and sensitizing blood vessels9. If we don’t see pAKT or pERK activation then we might wonder whether the mini-binders bind at all.

Conclusions: This study will dissect the molecular basis that drives Tie2-mediated cell migration and vascular stability. The studies presented herein aim to understand how valency regulates angiopoietin signaling, and how to stimulate vascular repair and stability. This project ties back to dentistry by trying to make stable blood vessels for tooth organoids. Understanding how to stimulate vascular stability is important in dentistry due to the fact that the vascular system in a tooth is responsible for nutrient supply, waste removal and support overall health. Whether it be to regenerate teeth or to save an existing tooth, there would need to be a nutrient source to supply the overall survival and growth.

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund

Nelson Wong

Department: Oral Health Sciences

Faculty Mentor: Dr. Jonathan Y. An

Objectives: Periodontal disease is a worldwide burden that reduces the oral health quality of individuals and becomes more prevalent as we age. We have previously demonstrated that targeting mTOR, which is downstream of PI3K, can attenuate periodontitis by reducing inflammation and PI3K signaling.

Methods: Here, we determine if the directly targeting PI3K isoforms, a and y, will have similar effects as inhibiting mTOR. Quantitative Real-Time PCR analysis was used to determine the expression of PI3K isoforms and inflammation markers expressed during periodontitis. MicroCT (mCT) imaging was completed on the maxilla to measure periodontal bone loss changes in aged mice treated with specific inhibitors for PI3K-a and PI3K-g.

Results: Our findings show that aged and sex specific changes occur in PI3K isoform expression, inhibition of PI3K-a and PI3K-g is dependent on oral tissue type and caused cross-reactive responses, and in the aged alveolar bone the elevated levels of IL1-a was decreased with both PI3K inhibitors, but specifically PI3K-g inhibition also lead to decrease in MMP13 and NFATc1. Preliminary mCT analysis shows less periodontal bone loss in PI3K-g inhibitor treated aged females.

Conclusions: Our findings demonstrate the important role of mTOR and PI3K during age and periodontal disease, and the potential effectiveness of specific PI3K inhibitors to attenuate periodontal diseas

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund, VitaDAO Longevity Fund

Ke’ale Louie

Department: Seattle Children’s Research Institute

Faculty Mentor: Dr. Lisa Maves

Objectives: Duchenne muscular dystrophy (DMD) is a congenital disorder characterized by progressive weakness and skeletal muscle fibrosis. Specialized muscles (e.g., craniofacial and cardiac) become affected in advanced disease, despite the long-term administration of palliative therapeutics aimed at preventing appendicular lesions and further functional deficits. The need to prevent late-occurring lesions in specialized craniofacial muscles raises the interest in identifying pharmacologic treatments targeting different muscle groups during development. Our objective in performing a large-scale screen of epigenetic compounds is to identify those affecting muscle biology prior to lesion development.

Methods: Using an established zebrafish model of DMD (i.e., sapje), embryos were exposed to either individual compounds or pools of 8-10 epigenetic compounds from 24-96 hours post fertilization. Embryos were scored for trunk muscle lesion severity and rescue via birefringence microscopy. Phenotypic rescue was reported relative to control DMSO-exposed DMD mutant siblings.

Results: We validated the phenotypic rescue potential at 96hours post fertilization of trichostatin A (TSA), a histone deacetylase inhibitor known to improve the zebrafish DMD model. Other histone deacetylase inhibitors conferred varying degrees of rescue at 96hours post fertilization and reflect the therapeutic potential for novel compounds within this class. Our ongoing investigations will identify the therapeutic window and mechanistic pharmacology of these and other epigenetic small molecules that improve the zebrafish DMD model.

Conclusions: These results create a promising platform for discovery regarding the early development, regulation, and management of this incurable childhood disease.

Supported By: NIH/NIDCR T90 DE021984

Shreyas Punacha

Department: Oral Health Sciences

Faculty Mentor: Dr. Fritzie Arce-McShane

Objectives: To study the effect of aging on stereotypic tongue movements during swallows. We also study the corresponding cortical correlates, and how these were affected by aging and loss of sensation.

Methods: We use high-resolution biplanar video-radiography to track three-dimensional tongue movements and large-scale multi-electrode cortical recordings to capture neural signals in the primary motor (MIo) and somatosensory (SIo) areas of the orofacial cortex of rhesus macaques while they are engaged in natural feeding behavior. To compare the effects of sensory loss and healthy aging, we administered a nerve block to temporarily eliminate oral tactile sensations while preserving motor and proprioceptive signals in young animals.

Results: We found an overall increase in stereotypy of tongue trajectories from the anterior to posterior region of the tongue under all conditions, as shown by antero-posterior decrease in the variance of trajectory length and trajectory end points. The posterior region of the tongue exhibited distinct stereotypy, although the effect is reduced in older animals. To evaluate whether the regional variations in tongue kinematics were reflected in the informational content of the neuronal responses, we computed the time-evolved mutual information (MuI) between the tongue kinematic data and the spiking activity. We found differential patterns of MuI as a function of time relative to swallow and tongue regions; the anterior region of the tongue exhibited peak MuI at the start of the swallow cycle, while the posterior region had peaks at the minimum gape in the young animal. In the absence of sensation, the peak MuI associated with movements of the posterior tongue occurred after the minimum gape. These patterns were observed for both MIo and SIo.

Conclusions: Our results suggest that regional variation in tongue stereotypy and in the dynamics of information in the spiking activity of MIo and SIo neurons during swallows may underlie differing contributions of lingual action for swallowing. This knowledge provides valuable insights into the neuromechanical changes associated with healthy aging.

Supported By: NIH

Cameron Renteria

Departments: Materials Science and Engineering, Oral Health Sciences

Faculty Mentors: Dr. Dwayne D. Arola, Dr. Douglas Ramsay

Objectives: To characterize the effects of aging on the durability of tooth enamel in terms of the apparent fracture toughness (KApp) and brittleness (B) through an unsupervised machine learning approach.

Methods: Enamel samples were prepared for characterization from donor teeth collected at dental practices in Seattle, WA with IRB approval and divided into Primary (age≤12 years, n=6), Young Adult (20≤age≤50 years, n=6) and Senior Adult (age≥ 55, n=6) groups. The hardness and reduced modulus distributions of the enamel were evaluated using nanoindentation with a Berkovich indenter and 50 nm tip radius as a function of distance from the Dentin Enamel Junction. The chemical composition of the enamel was evaluated using Raman microspectroscopy. All scans were performed over the extended spectral range: 300 – 1200 cm-1, using a 1200 gr/mm diffraction grating with a spectral resolution of 2 cm-1. A co-located analysis of apparent fracture toughness (KApp) and brittleness (B) was performed using microindentation techniques. Microindentations and cracking were induced using a Vickers semi-automated microscope. Finally, the interdependencies between aging and the enamel properties were assessed using a combination of traditional Pearson’s correlation coefficient matrices, K-means clustering, and self-organizing maps (SOMs) via unsupervised machine learning.

Results: Results showed that KApp was negatively correlated to the E, H, degree of crystallinity, and fluoridation, but positively correlated with carbonate content; the opposite trends were observed in B. Interestingly, the SOMs showed that the outer enamel of the old group underwent a degradation in durability (decrease in KApp and increase in B) that was related to multiple contributions, whereas the inner enamel did not undergo this change. The degradation in durability, which was exhibited by enamel of the senior adult group, was not apparent in the primary or young adult groups. However, a spatial stratification of properties with age was observed via K-means clustering across the age groups. Clinically, these findings could stimulate the design of age-targeted oral health care products that are aimed at reducing the brittleness of senior adult enamel.

Conclusions: The application of K-means clustering on the trained SOMs provided new insights into the contributions to changes in enamel durability with aging. They also provided a unique way for visualizing high-dimensional data onto 2D plots and identified new research directions that would not have otherwise been discovered. Overall, the findings demonstrate how machine learning techniques can provide new understanding on aging of enamel and their promise in pursuits aimed at developing age-targeted oral health care.

Supported By: Colgate-Palmolive Company, NIH-NIDCR T90 DE021984

Brian Bhaskar

Department: Oral and Maxillofacial Surgery

Faculty Mentor: Dr. Andrea Burke

Objectives: Given the increasing use of electric scooters (e-scooter, ES) for transportation and recreation, the purpose of this study is to compare patterns and severity of maxillofacial and head injuries related to e-scooter and bicycle accidents and to identify factors contributing to injury severity.

Methods: The authors implemented a multi-center retrospective cohort study and enrolled a sample of ES riders and bicyclists who sustained maxillofacial injuries between September 2020 and September 2022. The subjects were evaluated at Harborview Medical Center, the University of Washington Medical Center Montlake and Northwest campuses. The primary predictive variable was the type of vehicle involved in the accident (bicycle / electric scooter). The primary outcome variables included the presence of maxillofacial and head injuries, the pattern of injuries, and the severity of these injuries using both the Injury Severity Score (ISS) and Face and Head Abbreviated Injury Scale (AIS).

Results: The final sample was composed of 205 total subjects, of which 52 (25.4%) were in the ES group and 153 (74.6%) in the bicycle group. Isolated midface injuries were the most common hard tissue pattern in both groups. The most common soft tissue injury patterns included the upper face and midface in e-scooters (19.2%) and the midface in bicycles (22.9%). There was no difference in maxillofacial injury severity between groups, but head injuries were more severe in the electric scooter group (p < 0.0002), with a greater likelihood of intracranial injury (p < 0.0001). Subjects involved in ES accidents were more likely to have positive blood alcohol level (p < 0.05) and less likely to wear a helmet (p < 0.0001) when compared to bicycles. Using regression analysis, drug use was found to have a significant impact on mean Injury Severity Score (p < 0.002) and alcohol use had the greatest effect on mean head AIS (p=0.08).

Conclusions: These findings suggest maxillofacial injury patterns and severity are comparable among e-scooters and bicycles. However, e-scooters riders are at greater risk of severe head injuries compared to bicycles and riding while intoxicated has the greatest effect on injury severity.

Supported By: Supported in part by the Laboratory of Applied Clinical Research and Research and Training Fund of the Department of Oral and Maxillofacial Surgery

Charlie Smith

Department: Oral and Maxillofacial Surgery

Faculty Mentor: Dr. Andrea Burke

Objectives: The purpose of this study was to measure and compare the total milligrams (mg) of propofol administered to subjects with and without self-reported cannabis use (CU vs. NCU) undergoing third molar (M3) removal under IVGA in a university based, outpatient oral and maxillofacial surgery (OMS) clinic. The specific aims of the study were to: 1) compare how self-reported CU vs. non-cannabis use (NCU) affects the total mg of propofol delivered during IVGA; 2) compare how CU vs. NCU affects other IVGA anesthetic agents during third molar extraction; and 3) identify questions about CU that may affect the amount of anesthetic administered.

Methods: This prospective cohort study was conducted at the University of Washington’s Oral and Maxillofacial Surgery clinic between May 1, 2022 and February 28, 2023. Subjects completed a modified “Daily sessions, Frequency, Age of Onset, and Quantity of Cannabis Use Inventory (DFAQ-CU)” questionnaire, developed at Washington State University, to quantify cannabis use pre-operatively. Subjects who were eligible were at least 21 years of age, and scheduled for M3 removal (involving at least one upper and lower M3) with IVGA. Subjects under 21, pregnant patients, not eligible for IVGA, with self-reported substance use disorder, developmental delay, or ASA classification of 4 were deemed ineligible for the study. This study was approved by the UW Institutional Review Board (IRB) (no. 00014619).

Results: In comparison to non-cannabis users (NCUs, n=11), cannabis users (CUs, n=38) were administered 55.5% more total propofol (225.5mg vs 145.0mg; p≤ 0.038) and 60.7% more bolus propofol (107.4 mg vs. 66.8 mg; p ≤ 0.016). In contrast, CUs were administered 20% less fentanyl compared to NCUs (58.7mg vs. 70.6mg; p≤0.046). A regression tree analysis indicated that CUs with self-reported consumption of cannabis “multiple times per day” for “more than 2-3 years” had a higher mean total propofol requirement of 262mg, compared to 169mg for those with less frequent and shorter duration of use.

Conclusions: This study found CUs required a statistically significant increase in propofol (both total and bolus), and decreased fentanyl during IVGA for M3 removal compared to NCUs. Further studies will be needed to reliably quantify anesthetic requirements in this population. Such studies would ideally utilize electronic vital sign monitoring, comparing vital sign responses over a set duration with a standardized anesthetic regimen.

Supported By: NA

Priyanka Kumari

Department: Oral Health Sciences

Objectives: Genome wide association studies (GWAS) have detected single nucleotide variants (SNPs) at multiple genetic loci that are associated with risk for isolated (non-syndromic) orofacial cleft (OFC). For instance, variation at the 1q32/IRF6 locus accounts for about 12% of genetic contribution to isolated OFC, the most at any single locus. We aim to identify the subset of SNPs, among those identified by GWAS, that directly contribute to OFC pathogenesis.

Methods: To identify the functional subset of SNPs at 1q32 (IRF6) and 7 other loci we conducted a massively parallel reporter assay (MPRA) in a human fetal oral cell line (GMSM-K). SNPs of interest from the MPRA were pursued with traditional reporter assays, and ultimately two were investigated using genome editing followed by RT-qPCR, to assess the SNP’s effects on expression levels of IRF6, and chromatin immuno-precipitation to assess its effects on transcription factor binding.

Results: From each locus we identified multiple SNPs with allele-specific effects in the MPRA. Of 11 such SNPs tested in traditional luciferase assays in primary keratinocytes, 9 showed allele-specific effects, including two promising SNPs (the IRF6 -10kb and IRF6 -21kb SNP) at the IRF6 locus. We engineered both SNPs (separately) to homozygosity for the risk or non-risk associated allele in human induced pluripotent stem cells, then differentiated the cells into embryonic oral epithelium. Intriguingly, cells homozygous for the risk allele of either SNP expressed lower levels of IRF6 than cells homozygous for the non-risk allele. The IRF6 -10kb and IRF6 -21kb SNP had allele-specific effects on binding of transcription factor FOXE1 and ETS2, nominating genes encoding these transcription factors as OFC risk genes. Conditional analyses suggest that the IRF6-10kb and IRF6-21kb SNPs together account for most or all of the association signal at 1q32/IRF6 for cleft lip only and for cleft lip with or without cleft palate.

Conclusions: These experiments identify non-coding variants that directly affect risk for orofacial cleft; such knowledge has the potential to underpin more accurate genetic counseling and the design of next-generation therapies for this disorder.

Supported By: NIDCR R01DE027362

Sunil Kumar Singh

Department: Oral Health Sciences

Objectives: Orofacial cleft (OFC) is one of the most common structural birth defects which affects about 1 in every 700 babies worldwide. Most of the heritable risk for non-syndromic OFC (nsOFC) has not been assigned to any gene or locus. The common variants are thought to contribute less than half of the overall heritable risk for nsOFC, with much of the remainder presumed to reside in rare variants. Rare variants can be identified by whole genome or exome sequencing approaches, but they identify thousands of variants. Thus, there is a need to prioritize the candidate genes to harbor the heritable risk variants. Two known OFC risk genes, IRF6 and GRHL3, encode members of the transcriptional regulatory network (TRN) governing differentiation of oral periderm in murine embryos and of the enveloping layer (EVL) in zebrafish embryos. We reasoned that highly-ranked members of the periderm or EVL TRN are candidates to harbor the missing heritability for OFC.

Methods: We performed single-cell multiome sequencing on zebrafish shield-stage embryos and recovered paired ATAC-seq and RNA-seq profiles from 394 EVL cells. We fed these profiles to four TRN inference algorithms which use two types of evidence to infer TF-to-target gene edges: first, TF-binding sites present in chromatin regions open in EVL cells, and second, correlated gene expression levels within EVL cells. We tested the performance of the resulting TRN models against gold standard datasets, including RNA-seq from embryos depleted of seven TFs (separately) and ChIP-seq or CUT&RUN for three TFs in embryo lysates. We ranked the TFs and effector genes based on their connectivity in the best performing model. We tested a few highly ranked TFs for their effect on periderm integrity by using morpholinos and CRISPR-Cas9 genome editing. Finally, we investigated the average connectivity of known OFC genes in our network model.

Results: Pair-wise correlation-based network inference algorithm performed better in predicting true edges than the more complex regression methods. TRN models integrating both types of evidence performed better than those using just correlated gene expression. A sub-network module of the top-performing model contained Irf6, Grhl3, and other regulators of periderm integrity. Embryos depleted of newly identified top-ranked TFs within this module recapitulated the phenotypes of irf6 or grhl3 loss-of-function mutants. In addition, orthologs of genes associated with OFC had higher centrality scores than genes not associated with OFC.

Conclusions: These results suggest that TRN models of relevant cell types can prioritize candidate genes to harbor the variants with missing heritable risk for OFC.

Supported By: NIDCR R01 DE023575 and R01 DE027983

Riana Grewal

Department: Orthodontics

Faculty Mentor: Dr. Zi-Jun Liu
R. Grewal*, A. Vimawala, D.H. Rosero Salazar, Z. J. Liu Dept. Orthodontics, University of Washington, Seattle, USA, *Presenter

Objectives: The aim of the present study is to characterize the morphology of the pharyngeal airway spaces by quantifying the volumetric features and the fat compositions of the different pharyngeal structures by MRI images.

Methods: Eight 7-8 months old Yucatan minipigs of both genders were sedated and placed in prone to have magnetic resonance imaging (MRI) using Vista and fat-weighted sequences with synchronized physiological monitoring and respiration gating. The volumes of nasal/velar and oral airway and the retroglossal space from MRI Vista images were acquired through anatomical segmentations and 3D reconstructions using Radiant-DICOM Viewer and ITK-SNAP software through a coronal view. The fat compositions in fat-weighted images were first processed with the script digitized using DICOM software and then parsed giving a relative fat composition for each individual pixel in 3D format. Then they were measured for the tongue body and base, soft palate, and pharyngeal wall above the soft palate using add-on MATLAB software. The fat composition of the left masseter muscle was measured as a reference.

Results: The total volumes per region were: 15.35 ± 2.84 cm3 naso/velar pharynx, 3.80 ± 1.67 cm3 oropharynx; and 2.76 ± 1.28 cm3 retroglossal space. The areas in the cross-sectional views were 105.34 ± 12.75 mm2 nasopharynx, 62.02 ± 5.35 mm2 oropharynx, 39.47 ± 3.49 mm2 retroglossal. The nasopharyngeal volume and area were significantly larger than those in other airspaces (p < 0.05).  Total fat composition was 69.91 ± 3.21 % tongue body and 79.29 ± 2.85 % tongue base both significantly larger than 56.30 ± 2.85 % soft palate, 18.80 ± 2.14 % pharyngeal wall, and 25 ± 1.27 % masseter muscle (p < 0.05).

Conclusions: The volume and area of the nasopharynx were the largest out of the three pharyngeal airway spaces. The largest and smallest fat compositions were observed in the tongue base and the pharyngeal wall, respectively. These findings provide a basis for better understanding of the morphological and tissue characteristics of the pharyngeal airway spaces and structures for better understanding of clinical conditions such as obstructive sleep apnea (OSA) due to obesity, and other pharyngeal functional disorders.

Supported By: NIH/NIDCR R01 DE028864