Abstracts & Presentations

Michael Baldwin

Faculty Mentor: Dr. Sue Herring, Dr. Zi-Jun Liu
Department of Orthodontics

Objectives: Midfacial hypoplasia (MFH), or underdevelopment of the upper jaw, nose, and cheek bones, leads to Class III malocclusion with impairment of feeding and breathing. Many domestic pigs have MFH making them a novel model for this condition. In early observations the cranial sutures and synchondroses in MFH pigs were not fused; however, the caudal nasal septal cartilage appeared more ossified, forming the hypothesis that increased ossification diminishes an area of cartilage important for normal growth. The objectives of this study were to determine if ossification increases and cell replication decreases in the caudal septum of MFH pigs.

Methods: The midsagittal section of the nasal septum from newborn pigs with (n=5) and without MFH (n=6) was imaged with CT. The area and percentage of ossified septum were measured using ImageJ. The caudal nasal septum was harvested from another sample of newborn pigs with (n=8) and without MFH (n=8) and prepared for histology with H&E. Images (20x) were captured from the dorsal, middle, and ventral thirds. The percentage of replicated cells in each region was estimated by counting cells in isogenous groups. Medians with interquartile ranges were calculated and analyzed with appropriate nonparametric tests.

Results: Area and percentage of ossified septum increased in MFH compared to non-MFH pigs [9.5 (8.9-10.0) vs. 8.7 (8.3-9.0), mm2 divided by body weight, p<0.02 and 17.2% (14.4%-18.9%) vs. 12.8% (11.7%-13.4%), p<0.04]. Cell replication increased from dorsal to ventral in non-MFH pigs [dorsal 40.9% (36.5%-48.1%), middle 44.4% (39.9%-58.5%), ventral 56.7% (50.3%-65.1%), p<0.01]. MFH pigs did not differ from non-MFH pigs in the dorsal two-thirds, but replication decreased ventrally [42.8% (36.7%-46.8%) vs. 56.7% (50.3%-65.1%), p<0.001].

Conclusions: Pigs with MFH have increased ossification and decreased cell replication in the caudoventral nasal septum, suggesting that this region is important for normal growth. Insufficient growth in this region may be a cause of MFH.

Supported By: F30DE028183, T90DE021984, R21DE024814 and R21DE023988.

Jing Li

Faculty Mentor: Dr. Mark Drangsholt,  Dr. Milda Chmieliauskaite, Dr. Nicholas Sotak
Department of Oral Medicine

Objectives: The DC-TMD pain drawing was first introduced to assist the Diagnostic Criteria of Temporomandibular Disorders (DC-TMD) by Schiffman et al. in 2014. It is a unique but simple diagnostic instrument that has five body charts of the human body. This pain drawing depicts the anatomical representation of a patient’s pain experience and has become an indispensable clinical assessment tool for most pain conditions that are “invisible.” It can show: 1) the full bodily extent and experience of pain; 2) patterns of pain to aid in diagnosis; and 3) the efficacy of therapies, e.g., trigger point muscle injections. Observations in the clinic have shown that although the pain drawings are highly valuable, inaccuracies in pain drawing likely occur. To qualitatively describe discrepancies between the pain drawing and assessment in the UW Oral Medicine Clinic as a quality improvement project. To qualitatively describe the challenges of interpreting pain location from a variety of pain drawings completed by patients.

Methods: Observations of patients presenting to the UW Oral Medicine Clinic between December 2022 – January 2023 with completed DC-TMD pain drawings. Patients were assessed by experienced Oral Medicine specialists, and observations were made by faculty and residents. The inclusion criteria were as follows: male or female aged 18 years or older with a diagnosis according to international DC-TMD, including myalgia, myofascial pain, arthralgia, or headache attributed to TMD. As part of the routine clinical protocol, patients completed a pain drawing in the waiting room of OMCS with the standard DC-TMD pain drawing instructions. During their clinical appointment, the attending gave them more detailed instructions on how to draw their present pain, showed a reference standard drawing, and let patients point out their chief complaint of pain with their fingers on their body and face. A second pain drawing was then completed with the assistance of the patient, residents, and attending faculty and served as the reference standard. Patients treated with trigger point injections or dry needling completed an additional pain drawing 10 minutes after therapy. The resident observed the interactions between providers and patients and the pain drawings during clinical sessions and took notes of the discrepancies observed in the pain locations pointed out by the patient and those drawn on the pain drawings.

Results: A total of 50 patients were interviewed and examined for this pilot quality improvement project. The differences noted between the initial pain drawing and the reference standard drawing showed a subgroup that had clear differences. These differences fell into several groups: 1. Pen color influences the clinical diagnosis since if they use a black pen, it is difficult to discern between the patient’s marks and the background drawing. 2. Pain marked with circles and large “X” s are imprecise and can be off target. 3. The time interval of pain experience is not indicated on the pain drawing and needs to be explicit – e.g., pain now, pain last day, or last week. 4. The mouth chart in the pain drawing does not mark the right and left, increasing errors in shading the pain on the wrong side. 5. Confusion with sidedness – i.e., Left or Right, was a relatively common discrepancy. 6. Patients presenting for an orofacial pain concern may omit drawing existing pain in other parts of the body. With the instruction and standard reference pain drawing, the subsequent pain drawings appeared to more accurately depict their state of the pain experience.

Conclusions: This quality improvement project showed clear and common discrepancies, which could be decreased with an enhanced and revised protocol for administering the DC TMD pain drawing. Additional studies with a larger sample size are needed to confirm our study findings and design a 2nd version for DC-TMD pain drawing.

Supported By: UW Department of Oral Medicine

Jocelyn Vivas Castillo

Faculty Mentor: Dr. Mark Drangsholt, Dr. Milda Chmieliauskaite, Dr. Andrea Burke
Department of Oral Medicine and Oral Surgery

Objectives: The aims of this study are to
1) Estimate the prevalence of CP-PTP that presents to the Oral Medicine Clinical Service (OMCS) over a 1-year period.
2) Identify challenges applying ICD-11 CP-PTP diagnostic criteria in a retrospective chart review.
3) Describe the demographic, clinical and psychosocial factors, and the co-occurring comorbidities in patients identified with CP-PTP.

Methods:A 1-year retrospective chart review of UW-OMCS electronic AXIUM records was completed in all patients that had a pain diagnosis based on ICD-9 and ICD-10 codes that had a clinical encounter between December 2021-November 2022. To categorize the fitting cases with the ICD-11 code the following Inclusion criteria were; applied I) >18 years, ii) history of trauma or surgery in the craniofacial region, iii) pain >3 months duration that began or worsened after the surgery or trauma, and iv) the pain distribution is localized to the area of the event or projected to the innervation territory affected. The exclusion criteria were patients < 18 years old, or those with other evident causes of pain.

Data on the inclusion criteria, age, gender, and orofacial diagnoses were collected in REDCap by the PI of the study and 30% of the data was cross checked by a 2nd investigator. Orofacial diagnoses were categorized into broad categories of muscular pain (e.g., myalgia, myofascial pain in the craniofacial region), trigeminal neuropathic pain, burning mouth disorder/oral dysesthesia, oral dystonia, and others available codes in the UW (University of Washington) system. Notes were taken while reading patient charts on challenges encountered when applying ICD-11 CP-PTP criteria to clinical notes. Additional data including Demographic (DOB, ethnicity, marital status, employment, tobacco use), clinical characteristics (specific traumatic or surgical event, the qualitative and quantitative pain characteristics, other comorbidities in the craniofacial area, pain distribution and extra-craniofacial chronic diseases, as well as the psychosocial characteristics (Peg-scale, SCL-90, GAD-7, PHQ-9) will also be collected for each CP-PTP case in RedCap.

Descriptive statistics were used to summarize the findings for objective 1 including: total numbers, percentages, and median age. Qualitative descriptions of challenges are summarized for objective 2. Data for objective 3 is currently being collected and is not presented in this abstract. The study was approved by the University of Washington Institutional Review Board for Human Subjects Research (IRB (Institutional Review Board) #: STUDY00016197).

Results: A total of 1,003 visits in the OMCS occurred between December 2021-November 2022, and 529 (52,7%) cases had a pain diagnosis, 60 (11.34%) cases were omitted due to insufficient documentation. 89 cases (16.82%) fit the CP-PTP criteria. The median age was 57.63 years old (age range 32- 88). Seventy cases occurred in females (76.9%) and 21 (23%) in males. Fifty-eight cases (65.1%) of CP-PTP occurred after craniofacial surgeries, 15 (16.8 %) after trauma, and 16 (17.9 %) cases had both trauma and surgery involved. Most of the diagnoses of patients with CP-PTP were reported as muscular pains (myalgia or myofascial) and/or trigeminal neuropathic pain. Fifty (56.1%) patients had more than 1 diagnosis, and the most common combination (32 cases, representing a 35.1%) was having both trigeminal neuropathic pain and musculoskeletal pain, even if the terminology does not describe completely the clinical characteristics of the disease.

Challenges applying the ICD-11 diagnostic criteria stemmed from incomplete or unclear clinical documentation, insufficient details, and guidance for using the diagnostic criteria from ICD-11, and patient recall or late presentation to the clinic. Limitations included: 1) insufficient documentation about intensity changes in pre-existing pain following surgery/trauma, or confirmation about pain occurring just after the event, 2) details about the trauma in the craniofacial region (e.g. location, extent) if the patient offer information which usually is difficult to describe, 3) time between event and onset of pain (e.g. often described as “after surgery” rather than exact timing after surgery or trauma). Challenges with interpreting the ICD-11 diagnostic criteria were that there is no specified timeline for how soon after an event pain should begin to be considered CP-PTP, and the minimal work up needed to identify other causes of pain (e.g., plain film radiographs v. computed tomography). Additional challenges were recognized when patients were present to the clinic years after the event occurred, with a long-standing chronic pain that had multiple interventions trying to manage the pain, developing then multiple or worsening pain conditions.

Conclusions: 8.87% of individuals seen in the oral medicine clinic over 1 year fit the ICD-11 criteria for CP-PTP. Our sample was most likely to develop neuropathic/nociplastic or muscular pains as the result of possibly associated psychosocial risk factors at the moment of trauma and/or surgery occurs in the craniofacial region. The muscular pain accompanying neuropathic/nociplastic pain (coded as trigeminal neuropathic pain) after surgery was found to be more frequent. Additional work needs to be done to understand how to classify CP-PTP after oral surgical procedures in which prolonged mouth opening may also result in trauma to the musculoskeletal system and to understand the prevalence of these conditions in other settings.

In dentistry, multiple dental procedures (Root Canal Therapy, extractions, dental implants, etc.) involve repeated injuries to the tissues which can sensitize the nerve pathways exacerbating the chronic pain. Then, providing dental surgical treatment in patients with risk factors has been associated with risk of developing CP-PTP. In those patients in which the pain is the reason for intervention, the procedure could worsen the preexistent pain, making future treatments less effective.

In conclusion CP-PTP is an over-dimensioned response to an already-healed injury, often sub-recognized by the dental team reason from which a precise event and pain sequence data collection in conjunction with validated pain and psychosocial questionnaires is recommended to accurately understand the level of risk for developing or confirm a diagnosis of CP-PTP. The risk factor identification can prevent it, or its early diagnosis can avoid the worsening of the condition, especially if the risk factors are modifiable, but also by implementing when needed a treatment based on central and peripheral target therapies.

Sieu Yien (Ashley) Chiam

Faculty Mentor: Kwok-Hung (Albert) Chung
Department of Restorative Dentistry

Objectives: This in vitro study was to assess the effects of using different types of cement on the retention forces between different of titanium copings and implant-supported fixed dental prostheses (IFDPs) using pull-out test.

Methods: Fifty zirconia (ZirCAD, Ivoclar Vivadent) and 20 prepolymerized PMMA resin (AvaDent; Global Dental Science) rectangular (36mm×12mm×8mm) specimens were milled to mimic the lower left segmental portion of the All-on-4 IFDPs. Sixty cylindrical titanium copings (Variobase; Straumann USA) (V) were used and divided into 6 groups (n=10) and 10 conical titanium copings (C) were used as controls. Before cementation, outer surfaces of all titanium copings and intaglio bonding surface of prosthetic specimens were airborne-particle abraded (50-μm Al2O3, 20sec, 0.25MPa at a 10-mm distance). All specimens were cemented following the manufacturer recommendations and instructions according to the experimental design (Figure). After artificial aging (5000 cycles of 5°C-55°C, dwelling time 20 sec; 150 N, 1.5 Hz in a 37°C water bath), all specimens were subjected to retention force testing using a pull-out test using a universal testing machine and a custom fixture with a crosshead speed 5 mm/min. Mode of failure were classified (Type 1, 2, or 3). Retention force values were analyzed by the t-test for the prepolymerized denture acrylic resin specimen groups, and 1-way ANOVA and the Tukey test for the zirconia groups at α = 0.05.

Results: Mean and standard deviation retention force values varied from 101.1±67.1 to 509.0 ±65.2 N for the prepolymerized denture acrylic resin specimen groups. The zirconia groups ranged from 572.8 ±274.7 to 1416.1 ±258.0 N. There is no statistically significant difference in retention force values between V and C specimens cementing with Panavia SA cement (Kuraray Noritake) (p = 0.587). The retention forces and failure modes were influenced by the cement used (p < 0.05). Modes of failure were predominantly Type 2 (Mixed failure) and Type 1 (Adhesive fracture from prosthetic materials) except for the quick-set resin group (Type 3, Adhesive failure from coping).

Conclusions: When bonding IFDPs onto titanium copings, quick-set resin provided significantly higher retention force for prepolymerized denture acrylic resin prostheses. Conical and cylindrical titanium copings performed similarly when cemented with Panavia SA cement under the same protocol. The stability of the bonded interface and retention forces between zirconia prostheses and titanium copings varied from the cement used.

Supported By: American College of Prosthodontics Research Fellowship Grant, Spencer Grant

Courtney M. Hill

Faculty Mentor: Dr. Christy McKinney
Department of Oral Health Sciences

Objectives: Children in food-insecure households have a higher burden of tooth decay than children in food-secure households. Identifying mechanisms underlying the relationship could inform interventions. We examined how sugar-sweetened beverage (SSB) intake and frequent convenience store shopping, a marker of healthful food access, mediated the food insecurity–tooth decay relationship among children in lower-income households.

Methods: Cross-sectional study data included a household survey, beverage questionnaire, and dental examination. The sample included 452 lower-income, racially/ethnically-diverse, child-caregiver dyads in urban Washington State in 2018. The exposure was household food insecurity, the outcome was untreated decayed tooth surfaces, and proposed mediators were log-transformed SSB intake (fluid-ounces/day) and frequent convenience store shopping (≥2 times/week). We used linear regression and Poisson regression to conduct a causal mediation analyses via the potential outcomes framework to estimate proportion mediated.

Results: Fifty-five percent of children were from food-insecure households. Mean number of decayed tooth surfaces among children was 0.87 (standard deviation [SD]=1.99). Mean SSB intake was 17 fluid-ounces/day (SD=35), and 18% of households frequently shopped at a convenience store. After adjusting for confounders, household food insecurity and SSB intake were positively associated with decayed tooth surfaces, but not statistically significantly (mean ratio [MR] 1.60; 95% confidence interval [CI] 0.89, 2.88; p=0.12 and MR 1.16; 95% CI 0.93, 1.46; p=0.19, respectively). Frequent convenience store shopping was associated with more decayed tooth surfaces (prevalence ratio 2.75; 95% CI 1.61, 4.67; p<0.001). SSB intake mediated 10% of the food insecurity–tooth decay relationship (p=0.35) and frequent convenience store shopping mediated 22% (p=0.33).

Conclusions: Interventions aimed at addressing oral health disparities among lower-income children could focus on reducing SSB intake and improving access to healthful foods. Mediation findings suggest that, besides SSB intake and frequent convenience store shopping, there may be other important mechanisms behind the food insecurity–tooth decay relationship.

Supported By: This work was support by funds from Seattle Children’s Research Institute and ARCORA – The Foundation of Delta Dental of Washington. Funding for the Sugary Beverage Tax evaluation in Seattle was provided through ordinance by the City of Seattle. Elected representatives and city staff did not influence the evaluation findings or interpretation of findings.

Philip Walczak

Faculty Mentor: Dr. Cecilia Giachellia
Bioengineering

Objectives: Inorganic phosphate (Pi) is an important factor in hard tissue mineralization. Pi reacts with calcium to form hydroxyapatite (HA), the mineral crystal giving bone its compressive strength. Slc20a2 encodes type III sodium-dependent phosphate transporter PiT-2, the predominant phosphate transporter in hard tissues. Our lab was the first to report that Slc20a2 knockout (KO) mice exhibit stunted bone growth and decreased bone mineral density compared to WT. Here we extend these studies to further investigate the role of Slc20a2 in regulating hard tissue differentiation and mineralization to increase understanding of Pi transport and sensing.

Methods: C57Bl/6NTac-Slc20a2<tm1a (EUCOMM)Wtsi>/Ieg (Slc20a2) mice were used to generate Slc20a2 WT and KO littermates (0.98% Pi diet). Calcein was administered for histomorphometry. Histomorphometry: Bone histomorphometric analysis was performed in the lumbar vertebra. Slc20a2 immunofluorescence was completed on tibia and maxilla over multiple developmental time points and compared with consecutive hematoxylin and eosin staining. MicroCT: MicroCT scans were obtained at 34.42um resolution and 60kV (SkyScan 1076, reconstruction: Bruker NRecon V1.6.9).

Results: Histomorphometric analysis of Slc20a2 KO mice showed significantly lower bone mass, mineral apposition, and osteoblast number without statistically significant changes in osteoclast parameters compared to background matched WT. Slc20a2 KO mice show blunted incisors compared to WT.

Conclusions: Slc20a2 KO mice exhibit impaired osteoblast differentiation and osteogenesis, suggesting that Slc20a2 must play an important role in osteogenesis, supported by our data and other’s findings. In vitro data on osteoblasts suggests that mineralization may not be directly impaired by Slc20a2 KO, but that Slc20a2 plays an important role in osteoblast differentiation. Our mice also exhibit blunted incisors. This observation is supported by others showing Slc20a2 KO mice have impaired dentin formation. Future studies are required to better understand the mechanisms of Slc20a2 signaling in bone and tooth development. A mechanistic understanding of Slc20a2 effects on hard tissue formation may lead to development of therapeutic targets; modulating the function of Slc20a2 could improve osteoinduction in grafts, osseointegration of implants, and regeneration of bone and teeth after injuries.

Supported By: NIH R35 HL139602 to CG; NIH NIDCR NRSA F30DE029094, Morton-Awamura Pediatric Dentistry/Oral Biology Leadership in Children’s Oral Health Research Endowed Fellowship, Magnuson Scholars Award to PW

Juman Al-Haddad

Faculty Mentor: Dr. Alireza Sadr
Department of Restorative Dentistry

Objectives: Polymerization shrinkage may cause gap formation between the cavity floor and bulkfill composites, depending on the polymerization mechanism. This study investigated the adaptation and bond strength of a newly developed dualcure composite with addition of 80 nm spherical zirconia fillers in 4-mm deep preparations compared to commercially available products using optical coherence tomography (OCT) and microtensile bond strength (MTBS).

Methods: Six bulkfill composite products were tested; Bulk EZ Plus prototype (BEZP, Zest), Surefil SDR Flow+ (SDRF, Dentsply), Surefil One self-adhesive capsule (SONE, Dentsply), Filtek One (FTON, 3M), Hyperfil (HYPF, Parkell), and Sonicfill 3 (SNCF, Kerr). Standard composite molds 4-mm in depth were used to observe and score separation of the bonded composite during the polymerization process under OCT (Octina, Yoshida Dental). Preparation 3x3x4 mm in dimensions were prepared in extracted posterior human teeth to conduct the MTBS test with each of the composites placed using ScotchBond Universal (3M) adhesive. OCT data was analyzed by Mann-Whitney U non-parametric tests while MTBS data was analyzed by one-way ANOVA with Bonferroni correction at 0.05 significance level.

Results: OCT data showed that the dualcure composites BEZP followed by HYPF had lowest gap formation during polymerization compared to all other groups (p<0.05). SONE self-adhesive demonstrated the highest debonding from the cavity floor (p<0.05). There was no difference between SDRF and FTON (p>0.05) which had 10% to 50% debonding from the cavity floor. For MTBS analysis excluding the pretest failures due to debonding, SONE showed the lowest bond strength, followed by SNCF both of which were significantly different from SDRF (p<0.05). There was no statistically significant difference among other groups (p>0.05).

Conclusions: The dual-cure bulk fill composites tested exhibited significantly better adaptation and less gap formation than the light cured ones. The difference was not reflected in the bond strength values with the universal adhesive used in the current study. The self-adhesive composite de-bonded frequently in both experiments.

Supported By:  UW Dental Alumni Association

Kevin Huang

Faculty Mentor: Dr. Fritzie Arce-McShane
Department of Oral Health Sciences

Objectives: The purpose of this study was to quantify the impact LOS has on tongue tip kinematics compared to healthy aging.

Methods: 3D tongue and jaw kinematics were collected using XROMM (X-Ray Reconstruction of Moving Morphology) during feeding in two rhesus macaques (RMs R & Y) (Macaca mulatta) before and after a sensory nerve block of specific trigeminal nerve branches and one old RM (E) without sensory nerve block administered. Variation of tongue tip movement, duration, and tongue velocity during the swallowing of half grapes were analyzed to compare feeding performance. Variation of tongue movement and velocity was analyzed +/- 0.5 seconds relative to swallows while duration was measured as the time between maximum gapes in a swallow cycle.

Results: In the absence of sensation following administration of nerve blocks, the variability of tongue movements and its velocity significantly increased (Variance test, p<0.05). Both swallow duration and duration variance increased in the nerve block condition compared to control in subject Y, while it decreased in subject R (Sign rank & Variance test, p<0.05). However, the effects of aging on these movement features were inconsistent. Tongue movement variances in control and nerve block conditions in Y were significantly less in all axes of movement when compared to E except in the superioinferior(y) axis under nerve block. This was not observed in subject R who exhibited less variance in the mediolateral(z) axis in the control condition and greater variance in all axis when compared to E (Ranksum, p<0.05). In the nerve block condition, the variability of tongue movements in all axes was significantly higher in R compared to E (Ranksum, p<0.05). The variability of swallow durations was significantly greater in E compared to young RMs under control conditions (Ranksum, p<0.05).

Conclusions: Our results show that variability in the duration of swallows (compared to both young animals) and in the tongue movements (only in one young subject) increases with aging. There were no strong indications that aging affects the duration of swallows nor does it approximate the effects of LOS. Additional subjects are required to determine the aging effects on swallowing.

Supported By: Funding provided by UWSOD Dental Alumni Association, NIH/NDCR R01DE027236

Bishoy Galil

Faculty Mentor: Dr. Zi-Jun Liu, Dr. Daniel Leotta
Department of Orthodontics/Applied Physics Lab

Objectives: Obesity is a common finding and a major pathogenetic factor in adult obstructive sleep apnea (OSA). Increased visceral and/or upper airway adipose composition and metabolic abnormality have been found in obese/OSA patients. To understand the mechanisms of obesity in relation to OSA, this study was to investigate the tissue stiffness of the tongue in obese OSA minipig model.

Methods: Four validated obese/OSA (two 8-11-month-old Yucatan, two 6.5-year-old Panepinto) and three non-obese/non-OSA (8-11-month-old Yucatan) minipigs were used for studying in vivo tissue stiffness between the middle and posterior tongue as compared with those of their neck muscles. The EUS data were collected in cine-loop format. This produces movie clips that span around 10 seconds for each EUS measurement. The video clips were processed using MATLAB (The MathWorks, Natick, MA).Each elastography frame contains a colorized region of interest (ROI) in which the EUS data were obtained. Custom MATLAB software also divided this color ROI into four quadrants of the color region in the tongue or neck muscle images The color of each ROI image pixel was related to the stiffness of tissues. The obtained stiffness measurements were then averaged according to their respective groups, regions, and quadrants, and bar graphs were produced and compared between the regions and groups. By using a curvilinear scan head with a frequency bandwidth of 1-6 MHz (SSI transducer XC6-1), EUS imaging was taken via the submandibular region with sagittal orientation (for the tongue) and skin surface of the left-side neck.

Results: The results of the EUS give us the mean stiffness, its standard deviation, and the percentage of coverage across all specimens. To narrow the results to be analyzed, we looked at the percent filled and the standard deviation for all the quadrants. From the results, we found that quadrant #1 (Fig. 1) has the highest percent filled and lowest standard deviation. Therefore, quadrant #1 was used for initial analysis due to higher confidence in the elastography measurements. The preliminary results show that there is no significant difference in tissue stiffness between the middle and posterior tongue regions between obese/OSA and non-obese/non-OSA pigs. However, the neck muscle stiffness in obese/OSA was significantly higher than those of non-obese/non-OSA minipigs.

Conclusions: From these results, we might conclude that although there is no significant difference of tongue tissue stiffness between normal control and obese/OSA minipigs, these obese/OSA minipigs presented higher stiffness in their neck muscles. This finding may help clinicians to pay more attention to the fat-infiltrated neck region in OSA patients with obesity.

Supported By: Morell Research Fund from School of Dentistry, University of Washington.

Eli Cosovan

Faculty Mentor: Dr. Fritzie Arce-McShane
Department of Oral Health Sciences

Objectives: The aim of this study is to understand the encoding of 3D tongue direction and the underlying neuronal organization for the control of tongue movements which can aid in developing evaluation tools and therapies for the treatment of sensorimotor dysfunctions.

Methods: Data was collected from young adult rhesus macaques (n=2) while engaged in a feeding task; Experimental conditions: Control feeding and Nerve Block session (following bilateral injections of nerve block to specific sensory branches of the trigeminal nerve) Kinematics: To record 3D tongue and jaw movements, radiopaque beads were surgically implanted into the tongue, mandible, and cranium . The 3D position of jaw and tongue markers was recorded using high-resolution biplanar video radiography (200 Hz, 0.1 mm) and reconstructed using XROMM workflow. Neural Data: Activity from large populations of neurons was recorded using a microelectrode array (100- electrode Utah array or 36-electrode Floating Microelectrode Array) that were chronically implanted in four regions of the orofacial cortex. Data processing: For the kinematic data (3D position of tongue and jaw markers), markers are identified, and their positions tracked using a novel integration of the existing marker tracking workflow (XMALab) with machine learning through DeepLabCut. For the neural data, spike waveforms are sorted offline using Offline Sorter. Data Analysis: The data was processed through MATLAB to analyze single-unit and population activity of action potential firing rates.

Results: Figure 1 displays the directional angle of the tongue during feeding and loss of sensation (nerve block) in the marker at the anterior portion of the tongue (AnteriorM). Figure 2 displays the directional tuning of the tongue during feeding when the NHP had sensation in the tongue (control trial). The directional angle range was largest when the NHP was chewing without nerve block (control trial) ranging between -17 and 12 degrees (figure 2), and smallest during manipulation during trigeminal nerve block, ranging between -7 and 3 degrees (figure 1).

Figure 3 displays the preferred direction of the tongue during chewing under nerve block. The Kruskal-Wallis one way ANOVA test showed that there was directional tuning significance (p<0.05) during chewing with lack of sensation

Conclusions: The Kruskal-Wallis one way ANOVA test showed that there was directional tuning significance (p<0.05) during chewing with lack of sensation. As hypothesized, directional tuning of the tongue was limited during nerve block, yet directional tuning of neurons was still significant.

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

Chao Dong

Faculty Mentor: Dr. Andrea Burke
Department of Oral and Maxillofacial Surgery

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 288 letters of recommendation for 54 applicants selected for interview (79.6% male) analyzed. Of 288 letter writers, 71% 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 = 431, Male= 384, p= 0.715). 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. While there was no significant difference in quantitative text mining and topic modeling, letter writers were more likely to comment community service in letters for male applicants (p=0.038).

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

Katie Gustafson

Faculty Mentor: Dr. Peggy Lee
Department of Oral Radiology

Objectives: The objective of this study was to optimize CBCT scanning protocols for measuring thin alveolar bone height and width.

Methods: 8 pig mandibles (32 teeth) underwent initial scanning to determine reference hole locations. Two reference holes were placed on each selected tooth, followed by CBCT scans at different field of view (FOV), voxel size, and acquisition time (FOV 4×4, 6×6 or 10×10 cm; voxel size 80um or 200 um; scan times 17s or 30.8 s). After protocol scanning, specimens were sectioned into small blocks, then underwent fixation, decalcification, paraffin embedding, sectioning and staining. Bone width and height of each site was measured from the CBCT and histomorphometric measurements. Two observers performed CBCT measurements independently. Histomorphometric measurements were determined by consensus among three observers. Histomorphometric measurements were used as the gold standard. Agreement between measurements were assessed by Bland Altman plots.

Results: The mean measurement of bone height from histological slides was 7.07mm and the mean measurement of bone width was 1.66mm. The overall mean differences between all CBCT measurements and gold standard measurements ranged between 0.04mm and 0.21mm. The mean difference between CBCT protocols ranged between 0.005 mm and 0.10mm. Differences between CBCT protocols were smaller than the differences between CBCT measurements and histomorphometric measurements. For CBCT measurements, the inter-observer agreement was excellent (the intraclass correlation coefficient was 0.95).

Conclusions: Because the mean differences between the gold standard and CBCT measurements for varying protocols were not clinically significant, altering CBCT variables does not change the accuracy of CBCT scans. There is a minimum measurement difference between different CBCT scan protocols. A 200um voxel size is sufficient for accurate CBCT measurements, and a high resolution setting is not necessary for optimization.

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

Fionna Lee

Faculty Mentor: Dr. Philippe Hujoel
Department of Oral Health Sciences

Objectives: Editors of clinical specialty journals have a potential conflict of interest to accept manuscripts which promote their specialty. This study assessed whether this bias impacted dental editors in accepting manuscripts that periodontal treatments improve systemic diseases.

Methods: Dental editors who urged policy makers to dismiss pivotal evidence that a periodontal treatment failed to improve a systemic disease were identified. The manuscripts these dental editors accepted for publication were compared to acceptance decisions made by non-dental editors and characterized with respect to scientific rigor. Favoritism was assessed by the Percentage of Papers by the Most Prolific author and Gini-coefficients (inequality in the distribution of authorship among published authors)

Results: Dental editors, when compared to non-dental editors, were more likely to accept non-registered clinical trials (chi-square test: p < 0.05) and were more likely to accept observational studies with poor control for confounders (two-sample t test: p <0.05). Gini-coefficients and the percentage of papers with most prolific authors showed that dental editors showed favoritism towards authors promoting periodontal-systemic disease associations. Dental editors, when compared to non-dental editors, were 2.6 times as likely to accept statistically significant results and 3.8 times as likely to accept causal terminology (Relative Risk (RR)= 2.6, 95% Confidence Interval (CI): 1.2-5.8 and RR= 3.8, 95% CI: 1.1-13.0, respectively).

Conclusions: Editors of clinical specialty journals who dismissed high-quality evidence accepted for publication the low-quality evidence which supports their clinical specialty.

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

Min Lin

Faculty Mentor(s): Dr. Peggy Lee,  Dr. Daniel Leotta
Department of Oral Medicine, Applied Physics Laboratory

Objectives: Temporomandibular disorders (TMD) are frequently treated using botulinum neurotoxin, which may relieve pain by limiting masseter muscle function and reducing its size. Masseter size changes are generally measured by direct visualization/palpation in follow up clinic visits. Muscle size and/or structural alterations can be objectively and non-invasively imaged using 2-D ultrasound, but protocols have not been standardized. For clinicians to compare muscle thickness between patient visits, the scans need to be repeatable. All studies to date have used single point measurements for muscle thickness and the repeatability of the scans/measurements has not been investigated. This study tested the hypothesis that average muscle thickness measured from the entire muscle would be different from single point measurements. Additionally, the study tested measurement repeatability at different scan locations, muscle contraction states, and operator’s positions. Lastly, the study used the 3-D volume data as the gold standard and compared measurements acquired using traditional 2-D ultrasound imaging. This study aims to identify the most repeatable and accurate imaging protocols to assess masseter muscle.

Methods: Ten adult volunteers (>18 year-old) were recruited. Each volunteer was scanned (right and left masseters) in relaxed and clenched positions. The scanning protocol included 6 scan positions: 3 short-axis views (superior/middle/inferior regions) and 3 long-axis views (anterior/middle/posterior regions). Each subject was scanned twice, roughly 15 minutes apart. Shallow and deep borders of the masseter muscle were manually traced on all 2-D and 3-D images. Center measurements were calculated as the distance between shallow and deep borders of the masseter muscle at the center of each image. Average muscle thickness were calculated as the average thickness of the entire traced muscle using custom software. Agreement between scans was assessed using the Bland-Altman plot.

Results: (1) High intra-rater reliability (ICC 0.99) and inter-rater reliability (ICC 0.98). (2) Muscle thickness measured at the center of the image is similar to the average muscle thickness. (3) Most repeatable scan location is at longitudinal middle in both relaxed and clenched states. (4) Scanning muscle at both relaxed and clenched state are both repeatable with relaxed scans yield slightly higher repeatability. (5) Difference in repeatability when operator stood at the same and opposite site of the muscle scanned is negligible. (6) Location of the maximum thickness increase in the clenched state varied between subjects. (7) Measurements from 2-D scans are correlated to 3-D volume measurements. Clenched: R² = 0.86. Relaxed: R² = 0.95.

Conclusions: Our results demonstrate that our 2-D ultrasound protocol is reproducible regardless of muscle contraction state or the operator position. While thickness at the image center approximates the average muscle thickness, the ability to trace and calculate the entire muscle shape provides more detailed information about regional changes in muscle size after treatment. Most repeatable scan location is at longitudinal middle in both relaxed and clenched states, suggesting that it will be the best location for clinicians to assess the treatment progression. 3D measurement produces a thickness map of the entire muscle. These 3D thickness maps may be useful for clinical exams of TMD patients after neurotoxin treatments, depending on the amount and distribution of muscle size changes in these patients. Our 2-D and 3-D measurements are correlated. However, comparisons for specific regions still need to be done. This is an area for future study. Result of this study provides a reliable protocol of ultrasonography in assessing the parameters of masticatory muscle size. Our protocol can be followed by any clinicians with proper training. With our protocols, clinicians can better see how the muscle change before after the treatment for treatment progression.

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

Kimia Masbough

Faculty Mentor: Dr. Katherine Rafferty
Department of Orthodontics

Objectives: Midfacial hypoplasia (MFH) is the underdevelopment of the upper jaw, nose, and cheek bones. While some research suggests MFH narrows the pharyngeal airway, little is known about the effects of MFH on the structure and function of the nasal cavity. Heating and humidification of the respiratory air are the main functions of the nasal airway. Yucatan pigs are a breed with MFH and as such serve as a naturally occurring large animal model of this condition. The purpose of this project is to compare nasal airway dimensions in pigs with MFH those of normally growing domestic pigs. I hypothesize that shortening of the snout in pigs with MFH reduces the surface area relative to volume (SA/Vol), hampering the ability of the nasal passages to regulate air temperature and humidity.

Methods: MFH pigs (n=14) and normal pigs (n=5) were CT scanned with NSI X5000. (n=5) additional normal pigs were added for SURF. The nasal airway SA/vol for normal and MFH pigs were estimated from 9 coronal slices of the snout using Photoshop, MetaMorph and ImageJ. The surface area to volume ratio was calculated, averaged, and compared using a two-sample t-test.

Results: The average MFH SA/Vol=0.41±0.035 and the average normal SA/vol=0.32±0.089. P-value=0.005 indicates the SA/Vol of the two groups are significantly different.

Conclusions: The results were the opposite of those hypothesized. TheSA/vol was higher in MFH pigs. This may be due to the increase in the actual size of the turbinates or the relative size of the turbinates compared to a smaller air chamber volume in MFH. It is significant to know the compensation is possible in pigs, and it may also occur in humans. This research will provide new insights about the consequences of MFH that will be relevant for better treatment of this condition.

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

Wolfgang McLelland

Faculty Mentor: Dr. Fritzie Arce-McShane
Department of Oral Health Sciences

Objectives: Functional connectivity is when cortico-cortical interactions within and across different regions of the brain show synchronized or coherent activity during neurophysiological events. Functional connectivity of the somatosensory and motor cortex for orofacial behavior (feeding and breathing) is mostly unexplored and how that relates to normal, or loss of function has yet to be determined. This project will focus on how neurons in the orofacial sensorimotor cortex interact to create effective feeding and how loss of oral sensation impacts functional connectivity.

Methods: Data were collected from two 8-10y/o adult male rhesus macaques (Macaca mulatta). Macaques were fed foods presented by the experimenter via a long stylus. On some days, animals engaged in the feeding task following bilateral injections of nerve block to specific sensory branches of the trigeminal nerve. Neurons were tracked with microelectrode arrays that were chronically implanted in four regions of the orofacial cortex. Spike waveforms are sorted offline using Offline Sorter. Spectral coherence analysis will be performed.

Results: The Theta band has the largest and greatest magnitude of coherent neurons between both macaques and conditions. Nerve blocks caused a change in coherence. One subject had a compensatory increase in coherent activity, while the other had an overall decrease in all areas. Neuronal pairs in the control condition either ceased to exhibit significant coherence or had a reduced coherence in the nerve block. The time of peak coherence shifted from the control to the nerve block.

Conclusions: Functional connectivity in the orofacial sensorimotor cortex is affected by loss of sensation in oral structures during feeding. Somatosensory research involving touch with the hands find similar conclusions in that nerve blocks impact somatosensation. Future studies could inform treatment strategies of sensory impairments associated with dental implants, trigeminal neuralgia, temporomandibular disorders, orofacial pain, and for the restoration of sensory feedback.

Supported By: University of Washington Dr. Douglass L. Morell Dentistry Research Fund and NIDCR Grant R01DE027236.

Yikang Dong

Faculty Mentor: Dr. Zi-Jun Liu
Department of Orthodontics

Objectives: The internal kinematics of the tongue base (TB) is crucial for oropharyngeal function but remains largely unknown. This study aimed to analyze the 3D deformations of the TB during natural chewing and swallowing.

Methods: Eight 7-8-months-old Yucatan minipigs were used. Under anesthesia, eight 2mm ultrasonic crystals were implanted into the TB to circumscribe a cubic region. The distances between crystal pairs were recorded simultaneously with EMG during natural chewing and swallowing. The dorsal/ventral lengths and anterior/posterior thicknesses were measured based on the chewing side defined by EMG, but no chewing side was defined for widths measurement. For swallowing, distance changes and durations were measured. All deformational measurements were converted to percentage of initial distance at rest.

Results: On both ipsilateral and contralateral sides, the dorsal length increased 20-40% with larger elongation on ipsilateral side. In contrast, changes in ventral lengths presented both elongation and shortening with larger changes on contralateral side. Anterior and posterior thickenings were seen on both sides. The anterior thickening was about 20% larger than the posterior thickening. The anterior dorsal/ventral and posterior dorsal widths increased up to 27% whereas posterior ventral width narrowed about 3.2 to 10.2%. During swallowing, the ventral/dorsal lengths increased by 10.6-11.4% and 30.0-35.7% respectively, and anterior/posterior thickness increased by 5.8-41.0%. The anterior/posterior dorsal and anterior ventral widths increased by 14.5-16.4%, and 8.6-23.6% respectively with slightly thinning (0.8-1.0%) in posterior ventral width. Overall, swallow showed similar elongation and shortening pattern compared to chewing but exhibited faster and sharper deformations. The average swallowing duration was 0.5439s.

Conclusions: The TB elongates, thickens, and widens with a narrowing in posterior width, and presents inconsistent changes of ventral length. Swallowing exhibits a similar pattern to chewing but the deformational shifts of each dimension are more abrupt and faster than those seen in chewing.

Supported By: NIH

Kimia Imani

Faculty Mentor: Dr. Donald Chi
Department of Oral Health Sciences

Objectives: Dental care provided in emergency departments (EDs) is almost entirely non-definitive and palliative, with treatment consisting mostly of short-term analgesics and antibiotics This study evaluated whether children with special healthcare needs (SHCN) were more likely to use EDs for nontraumatic dental conditions (NTDC) compared to children without SHCN. Among children that used the ED for NTDC, we also examined whether the likelihood of an opioid prescription after an ED visit for NTDC differed between children with and without SHCN.

Methods: This analysis was based on 2017 Oregon Medicaid enrollment, claims, and pharmacy data for children ages 0-17 years (n=225,614) To assess associations between SHCN, NTDC-related ED use, and receipt of opioid prescriptions, confounder-adjusted odds ratios (OR) and 95% confidence intervals (CI) were generated using logistic regression models.

Results: About 14% (n=31,867) of the children had SHCN and the prevalence of NTDC-related ED use was 0.36% for all children. In the adjusted model, the odds of NTDC-related ED use were 2.67 times greater among children with SHCN compared to children without SHCN (95% CI=1.33, 4.75; p=0.002). Among children that used the ED for NTDC (n=908), 8.30% received an opioid prescription. In the adjusted model, children with SHCN were at higher odds of receiving an opioid prescription following NTDC-related ED admission compared to children without SHCN, but this difference was not statistically significant (pOR= 1.43, 95% CI= 0.33, 6.60, p= 0.63).

Conclusions: Medicaid-enrolled children with SHCN have higher odds of NTDC-related ED admissions, however, they do not have higher odds of receiving an opioid prescription following the ED visit compared to children without SHCN. These findings support the need to understand the underlying mechanisms to prevent ED admissions for NTDC for Medicaid enrollees and low-income SHCN pediatric patients.

Supported By: U.S. National Institute of Dental and Craniofacial Research (grants T90DE021984 and K08DE020856)

Lucas Honnlee

Faculty Mentor: Dr. Zi-Jun Liu
Department of Orthodontics

Objectives: Oropharyngeal structures are highly active during mastication and swallowing, but their spatial relationships are less understood. This study was to analyze the dynamics of soft palate(SP), tongue base(TB), and epiglottis(EG) during chewing and swallowing.

Methods: Voluntary chewing and swallowing of barium-mixed diet were video-taped(30 frames/s) for 5-10 minutes using x-ray fluoroscopy in five 7-8-month-old Yucatan minipigs. Frames of 2-5 masticatory cycles and bolus swallowing episodes from each animal were digitized using the video-analysis tool. The digitized points were the tip of SP and EG, and the midpoint of dorsal surface of TB. The reference point was the most gingival point on the distal surface of the upper last molar. An X-Y coordinate was set up in each frame to standardize the excursion ranges and direction of each structure and bolus. Previously implanted 2mm ultrasonic crystals in TB were used for calibration. All distance changes were converted to % of the given distances at rest.

Results: Distances between tongue base to soft palate, tongue base to epiglottis, and epiglottis to soft palate shortened at 49.21-75.01±9.69%, 4.06-25.30%±8.77%, and 5.82-26.25%±8.02 in opening, and elongated at 33.10-117.79%±35.81%, 5.41-30.18±10.68%, and 5.64-33.51±10.52% respectively in closing and power stroke during chewing.

The distance between the TB to SP increased immediately before swallowing, followed by a 20.14% to 60.5% decrease ±14.39% from the average oropharyngeal space during swallowing.

Decreased distance of 5.65% to 38.02% and 1.57% to 42.73% were observed between EG to TB and EG to SP respectively, but phasic changes of EG to TP and EG to SP were not as definitive as the change seen between SP and TB.

Conclusions: The spatial relationships of three oropharyngeal structures decrease and increase in distance and the largest changes in chewing is between TB and SP due to jaw movements. The closer spatial relationship of these three structures during swallowing propels the bolus through the oropharynx.

Supported By: R01 DE028864 from NIH/NIDCR

Drew Marchant

Faculty Mentor: Dr. Jasjit Dillon
Department of Oral and Maxillofacial Surgery

Objectives: The purpose of the study was to understand the continued impact of social distancing policies and its long-term effects on the epidemiology of facial fractures.

Methods: “The authors designed a retrospective cohort study and enrolled a sample of subjects who presented to Harborview Medical Center (HMC) an urban Level 1 trauma center for the evaluation and management of oral and maxillofacial fractures (OMF) between January 1 and December 31 in the years 2018 through 2021. The primary predictor variable was evaluation of OMF during a period during or after social distancing policies (2020, 2021 – investigational group) or without social distancing policies in place (2018, 2019 – control group). The primary outcome variables were the mechanism of injury, abbreviated injury scale (AIS) and the injury severity score (ISS). Univariate and bivariate analyses were performed with a statistical significance at p<0.05.

Results: 4064 subjects presented with OMF. The number of subjects in each year remained the same (n=1018 in 2018, n=963 in 2019, n=1020 in 2020, n=1063 in 2021). Most subjects were male (73.3%). The incidence of males presenting for OMF increased significantly from 2018 (69.9%) and 2019 (71.34%) to 2020 (75.20%) and 2021 (76.4%) (p<0.001). There was a significant increase in the number of Hispanic individuals presenting with OMF (8.84% in 2018, 7.89% in 2019, 10.20% in 2020 and 11.57% in 2021; p<0.01).

The most common mechanism of injury was falls (35.2%), which decreased slightly but not significantly (36.15% in 2018, 36.86% in 2019, 35.49% in 2020, 32.64% in 2021; p=.10). Firearm (gun) injuries accounted for 5.2% of OMF and increased significantly from 2018/2019 to 2020/2021 (4.13% in 2018, 4.98% in 2019, 4.70% in 2020, 7.14% in 2021; p<0.05).

Injuries secondary to interpersonal violence slightly increased from 2018/2019 compared to 2020/2021 but not significantly (15.42% in 2018, 15.68% in 2019, 16.27% in 2020, 18.34% in 2021; p=0.12). Additionally, patients presenting with facial fractures secondary to domestic violence also increased slightly but not significantly (1.18% in 2018, 1.56% in 2019, 1.27% in 2020, 2.45% in 2021; p=0.19).

Subjects in 2020/2021 presented with a lower injury severity score overall (mean ISS = 18.45 ± 12.77) compared to 2018/2019 (mean ISS = 19.25 ± 12.89) (p<0.05). Subjects in 2020/2021 had a lower severity of craniofacial trauma (mean head/face AIS = 2.96 ± 1.16) compared to 2018/2019 (mean head/face AIS = 3.04 ± 1.14) (p<0.05).

Conclusions: The investigators found that during and after the period of social distancing through the COVID-19 pandemic, the number of OMF cases overall remained the same and the severity of oral and maxillofacial and overall injuries was lower. There was an increase in firearm (gun) related OMF and the incidence of both males and Hispanics presenting for OMF increased.

Supported By: N/A

Priyanka Kumari

Department of 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. 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: MPRA identified SNPs from multiple loci with allele-specific effects on enhancer activity. We pursued ten such SNPs at the 1q32/IRF6 locus with luciferase reporter assays. For two of them, at 10 kilobases and 21 kilobases upstream of the IRF6 transcription start site, we separately engineered cell clones to be homozygous for the risk-associated allele or the non-risk associated allele. Interestingly, for both SNPs, the expression level of IRF6 was relatively lower in the former. The IRF6 -10kb and IRF6 -21kb SNP had allele-specific effects on binding of transcription factor FOXA1 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: NIH DE027362, USA

Yan Ting (Blair) Zhao

Biochemistry

Objectives: The angiopoietin-Tie2 pathway regulates blood vessel stability, remodeling, and permeability. Understanding Tie2 signaling can improve vascular functions to treat oral diseases such as dental sepsis, oral lesions, and periodontitis. Previously, we demonstrated that angiopoietins’ activity falls into two broad phenotypic classes distinguished by the number of presented F-domains (Zhao et al. 2021 EMBO Rep). Ang1-like ligands with high F-domain valency activate pAKT, while Ang2-like trimeric F-domain scaffolds inhibit pAKT. Interestingly, Ang1 at high oligomeric states accelerates cell migration and promotes vascular stability, but how Tie2 performs these seemingly opposing cellular functions is unclear.

Methods: We utilized de novo designed 2-dimensional protein sheets conjugated with Ang1 F-domain to cluster Tie2 receptors and analyzed the potential associating components in endothelial cells. We also used computationally designed Tie2 super agonists to evaluate the role of Tie2 signaling in tight junction formation in mature and developing endothelial cells: iPSC-derived endothelial cells and 3D vascular organoids.

Results: In the present study, we show that Tie2 forms two classes of complexes: Tie2-α5β1 integrin and Tie2-tight junction complex. We show the  Tie2-α5β1 complex promotes cell migration by forming focal adhesions and recruiting VE-cadherin out of the cell-cell junction. We also demonstrate that the Tie2-α5β1 complex is critical to activating the pAKT/FOXO1 signaling axis to promote cell survival. In contrast, the Tie2-tight junction complex consisted of ZO1, claudin-5, and occludin that facilitate tight junction assembly for vascular stability. We further demonstrated that computationally designed Tie2 super agonists up-regulate tight junction expression in mature and developing endothelial cells. Tie2 super agonists accelerate junctional ZO1 and claudin-5 re-assembly after chemical disruptions in HUVECs, indicating that the Tie2 signaling directly modulates the assembly of tight junctions at the endothelial paracellular space.  Tie2 super agonist also ameliorated diabetic-induced vascular defects through up-regulating tight junction formation in vascular organoids.

Conclusions: In conclusion, we illustrated the mechanism of Tie2-mediated cell migration and tight junction formation via two classes of Tie2 complexes to regulate angiogenesis.

Supported By: T90 post-doc fellowship

Cameron Randall

Department of Oral Health Sciences

Objectives: Use of evidence-based behavior guidance helps facilitate dental treatment completion while minimizing distress and negative experiences with dentistry. The American Academy of Pediatric Dentistry (AAPD) provides best practice recommendations for behavior guidance, outlining numerous basic and advanced techniques. This study aimed to determine: (1) which of these nonpharmacological techniques pediatric dentists routinely use, and (2) their perception of caregiver acceptance.

Methods: All active AAPD dentist members were invited to participate in this cross-sectional study. Participants (N=517) completed an online questionnaire that queried which behavior guidance techniques listed in the AAPD recommendation statement they routinely use, the frequency with which they encounter caregiver hesitancy or refusal when using the techniques, and practice characteristics and demographics. Data were analyzed using descriptive statistics.

Results: All participants had completed or were completing a pediatric dentistry residency, and 78% were board certified; 79% worked in solo or group private practice, and 13% were affiliated with an academic institution. Nearly all reported using basic behavior guidance techniques such as tell-show-do (98.3%) and positive reinforcement or descriptive praise (95.1%), with fewer endorsing use of audio-visual distraction (77.0%), desensitization (74.9%), voice control (59.2%), and parental absence (56.3%). Even fewer endorsed use of physical restraints (30.6%) and cognitive behavioral therapy (4.3%). Of the 26 behavior guidance techniques participants endorsed using, caregiver hesitancy or refusal was encountered most frequently for voice control, parental absence, and physical restraints (mean scores between “rarely” and “occasionally”).

Conclusions: This survey of current practice patterns suggests that pediatric dentists routinely use basic behavior guidance techniques recommended by the AAPD, but use more resource-intensive (e.g., audio-visual or virtual reality distraction) and advanced (e.g., memory restructuring, cognitive behavioral therapy) techniques less frequently. With a few exceptions, these techniques are accepted by caregivers. Future research should identify ways to promote the implementation of more advanced techniques, which may offer greater benefit.

Supported By: NIH/NIDCR K23DE028906

Kristopher Kerns

Department of Periodontics

Objectives: The objective of this study was to characterize the effects of localized oral inflammation associated with experimental gingivitis on distant contralaterally located healthy tooth sites that had maintained oral hygiene for a period of 21 days.

Methods: An experimental gingivitis study enrolled and consented 21 young healthy individuals ages 18-35 and carried them through experimental gingivitis with a split mouth design, essentially providing intra-oral controls for each study subject. Each subject refrained from brushing a randomly assigned test set of 3 teeth which allowed for the natural accumulation and maturation of dental plaque within these sites while oral hygiene was maintained in the rest of the mouth for the duration of the study. Using sub gingival plaque and gingival crevicular fluid we performed both 16S DNA sequencing and host mediator analysis respectively. Furthermore we account for the variation in observed in human gingival inflammation associated with experimental gingivitis, and account for this variation by stratifying our analysis based on responder phenotypes previously observed (High, Low, and Slow).

Results: Our analysis reveals localized inflammation resulting from experimental gingivitis effects distant healthy tooth sites and results in temporally resolved shifts in key pro-inflammatory host mediators, including IL-1B, IL-6, IL-8, and TNF-a, that then precedes a shift within the sub gingival microbiome community towards a dysbiotic composition observed in distant test sites (Shift from commensal associated Firmictutes to periodontal disease associated Bacteroidetes). Furthermore, we observe this non-localized subclinical effect is responder dependent, being most represented in the High and Slow responder groups and less evident in the Low responder group.

Conclusions: This study provides highly resolved temporal evidence of how healthy tooth sites in the human mouth are impacted by distant localized sites of microbially-induced inflammation associated with experimental gingivitis and provides mechanistic insight into how otherwise healthy tissues may be impacted or become affected by localized periodontal inflammation.

Supported By: NIH TR002317, T90 DE021984, Colgate-Palmolive clinical research grant

Sunil Kumar Singh

Department of Oral Health Sciences

Objectives: Most of the heritable risk for orofacial cleft (OFC) has not been assigned to any gene or locus. Genes accounting for the missing heritability are likely to encode members of the same transcriptional regulatory networks (TRNs) as known OFC-associated genes. A subset of the OFC-associated genes encodes members of a TRN governing differentiation of periderm, the most superficial cell layer of embryonic structures including of the palate shelves. We hypothesized that this subset would be enriched among the relatively highly-connected members of the periderm TRN. If this is true, then other highly-connected members of the TRN are candidates to harbor the missing heritability for OFC. Here we test this hypothesis by generating a computational model of the periderm TRN, comparing the ability of several algorithms to predict a set of experimentally-confirmed network edges.  We use the zebrafish gastrula-stage periderm, or enveloping layer (EVL), as a tractable model of human oral periderm.

Methods: We dissociated mid-gastrula zebrafish embryos into single cells and performed single-cell assay-for-transposon-accessible-chromatin (ATAC), revealing open chromatin regions, and single-cell RNA sequencing. We used hundreds of individual EVL cell expression profiles for co-expression-based TRN inference using three algorithms (weighted gene co-expression network analysis, WGCNA; GEne Network Inference with Ensemble of trees, GENIE3; and graphical least absolute shrinkage and selection operator, gLASSO). In addition, we used single cell ATAC seq data in combination with predicted transcription factor (TF) DNA-binding motifs to guide the estimation of TF interactions with gene-associated enhancers.  We compared the network predictions against gene expression (RNA-seq) and chromatin (ChIP-seq and CUT&RUN) datasets. We used top-performing network models to identify a) hub TFs based on their centrality and the targets of the TFs encoding other categories of proteins (effector proteins). Finally, we performed enrichment analysis for known OFC risk genes and loss-of-function de novo variants detected in OFC patients in the list of TFs, ranked by connectivity, and the effector protein list, ranked by the number of TFs targeting them.

Results: Precision-recall curves using gold standard datasets revealed that incorporating the prior information based on TF-binding motifs in open chromatin regions significantly boosted the accuracy of the network recovery. Some of the highly connected TFs in the TRN model like, Grhl3, Tfap2a, Klf17, Cebpb, are known for their role in periderm differentiation. Interestingly one of three modules in the network contained all the TFs previously implicated in periderm development. Consistent with our hypothesis, genes encoding highly connected (hub) TFs and highly connected effector proteins are enriched for known OFC risk genes relative to poorly connected ones.

Conclusions: These results indicate that other highly connected genes in the EVL TRN are candidates to be OFC risk genes and may guide efforts to evaluate pathogenicity of DNA variants detected in patients with OFC.

Supported By: NIH R01 DE023575 and NIH R01 DE027983

Milda Chmieliauskaite

Department of Oral Medicine

Objectives: The aims of this study were to contribute to the existing literature on TMD in low-income older adults by: (1) assessing the extent of TMD in low-income community-dwelling older adults (62-years and older) using the validated TMD pain-screener; (2) investigating the association between TMD and sociodemographic and psychosocial factors, oral health related quality of life, and coping skills.

Methods: The secondary data utilized in this study was part of a cross-sectional investigation assessing the psychometric properties of an Illness Perception Questionnaire Revised for Dental Use in Older/Elder Adults (IPQ-RDE).28 Participants were recruited from sixteen facilities that provide low-income, older (62 and older) tenants with subsidized housing funded by the U.S. Housing and Urban Development (HUD, Section 202), and other low-income housing tax credits. Eight of the sixteen facilities also included private pay (PPT) market rate rental apartments for tenants above HUD’s income eligibility guidelines. Eligibility criteria included 1) older adults aged ≥ 62 years who were either HUD or PPT tenants; 2) presence of caries or periodontal disease assessed through a screening dental exam 3) Mini-Mental State Examination score > 23 (MMSE) and 4) comprehension and speaking English language. Stratified sampling was used to obtain equal samples of HUD and PPT tenants. The data collection occurred between January 2016 and April 2017. The study was conducted in accordance with ethical principles, including the World Medical Association Declaration of Helsinki and was approved by the University Hospitals Cleveland Medical Center Institutional Review Board. Informed consent was obtained from the participants.

Trained study staff administered paper based surveys to the participants and collected data on; i) socio-demographic variables self-reported by patients, ii) six-item temporomandibular disorder pain-screener (a reliable and validated measure used to detect the presence of painful-TMD in the last 30 days, iii) oral symptoms in the last six months, iv) The Geriatric Oral Health Assessment Index (GOHAI) (an oral health-related quality of life measure for older adults), v)The Patient-Reported Outcomes Measurement Information System (PROMIS®) short forms v1.0 (for anxiety and depression), vi) The abbreviated medical outcomes study (MOS) social support survey, and the vii) thirty two-item Tobin Coping-Strategies Inventory-Short Form (CSI-SF). Descriptive statistics along with bi-variate analysis and logistic regression were used to investigate associations.

Results: 198 older adults (mean age 73.54 ± 7.89) participated in this study. Thirteen percent of the sample screened positive for painful-TMD and nine percent of those reported jaw joint pain specifically (p<0.0001). No significant relationships were detected between a positive TMD screen and age, gender, race, marital status, level of education, or time since the last visit to the dentist. After adjusting for confounding variables, the odds of screening positive for TMD was positively associated with higher scores of anxiety with an odds ratio of 1.25. While the odds of low oral health related quality of life was 1.21 times significantly higher in individuals with painful-TMD than those without TMD.

Conclusions: Low-income older adults in this urban setting had painful-TMD rates higher than previously reported in US older adults. This is among the first studies to our knowledge that investigated associated psychosocial factors in low-income older adults who screened positive for TMD.

Supported By: National Institute of Dental and Craniofacial Research of the National Institutes of Health under award numbers R21 DE025057 and UL1 TR000439 the Clinical and Translational Sciences Collaborative of Cleveland from the National Center for Advancing Translational Sciences (NCATS).

Mark Drangsholt

Department of Oral Medicine

Objectives: N-of-1 trials provide a mechanism for making evidence based treatment decisions for an individual patient. They use key methodological elements of group clinical trials to evaluate treatment effectiveness in a single patient, for situations that cannot always accommodate large scale trials: rare diseases, comorbid conditions, or in patients using concurrent therapies (Voyra S, et al, 2015). However, the trials have not been feasible in clinical practice since randomization and blinding is not easily achievable. N-of-1 trials without randomization and blinding may have sufficient rigor to elevate clinical practice. Our objectives are to:

1. Describe several examples of feasible N-of-1 clinical trials conducted in craniofacial pain practice.
2. Within the same patients, show how multiple validated measures can deeply phenotype patients to aid in a more personalized approach of clinical subtypes.

Methods: Five patients with chronic continuous craniofacial pain for greater than one year who had seen multiple practitioners are described. Each was deeply phenotyped at baseline via measures such as PainTracker, a web-based validated questionnaire of key aspects of the condition such as all pain locations, pain intensity, pain interference, psychological distress, which is completed by the patient at each visit. Next, multiple sequential trials of therapeutic agents were administered to each person and after one to four weeks each outcome measure was reassessed. This allowed for measurement of changes over time in relation to each therapy within each patient. Blinding and randomization of therapies was not done but key covariates were also collected. The Nof1 or within patient trial would usually be – ABCDEF- whereby A is the baseline and B-F are separate therapeutic agents sequentially introduced. Other trials were for example, ABBBBC, with the repeating of a successful agent. The effect of each therapy on the key patient-reported outcome measures was measured.

Results: The five patients all had craniofacial pain, including temporomandibular disorders, trigeminal neuropathic pain and burning mouth disorders, alone and in combination. Common therapies included muscle relaxant medication, trigger point injections into muscles, topical clonazepam and neurosensory vinyl stents. Each showed graded reductions in the pain intensity and pain interference scores after introduction of successive interventions.

Conclusions: The five cases of deeply phenotyped craniofacial pain treated via the feasible Nof1 trials (within-patient trials) showed substantial improvement in the patient reported outcomes of pain intensity, pain impact and lower psychological distress. The methodological rigor of this approach would likely be higher than routine clinical practice although lower than fully randomized and blinded Nof1 trials. More work is needed to ascertain the utility of such an approach.

Supported By: Oral Medicine Research Fund