Chang AM, Liu Q, Hajjar AM, Greer A, McLean JS, Darveau RP
Oral Health Sciences, Periodontics
Oral gingival tissue, especially the junctional epithelium (JE), is constantly exposed to sub-gingival plaque. A key component of gingival health is the regulation of the number of neutrophils that migrate into the gingival crevice to counteract its harmful effects. This report investigates the contribution of innate defense receptors, Toll-like-receptor-2 (TLR), TLR4 and both TLR2/4 to the maintenance of neutrophil homeostasis in the JE.
Bacterial composition was analyzed from whole oral swabs collected from 12-14 week-old wild-type (WT), TLR2, TLR4, TLR2/4 knock-out (KO) mice (n=5-6/group) using a MiSeq platform targeting the V3-V4 region of the 16S ribosomal RNA gene. Mandibles were histologically examined for quantification of neutrophils present in the JE and for bone loss (n=30 JE/group, with 5 mice/group) . Lastly, total bacterial load was quantified using quantitative real-time PCR (n=5-6/group).
Compared to WT, all TLR KO mice displayed significantly increased recruitment of neutrophils (p=0.0079) into the JE that did not correlate with bacterial load. Rather, TLR4 and TLR2/4 KO mice demonstrated a significant increase in the number of bacteria (p=0.022 and p=0.0152, respectively). Furthermore, comparative compositional analyses of the oral microbiome revealed that each KO strain harbored unique microbial communities that are distinct from each other but maintained similar levels of alveolar bone.
Neutrophil migration into healthy mouse JE does not require TLR2 or TLR4. However, a significant increase in the number of neutrophils as well as a significant change in the oral microbial composition in both TLR2 and TLR4 KO mice demonstrate that these TLRs contribute to the homeostatic relationship between bacteria and the host in healthy periodontal tissue.
Supported by: National Institutes of Health Grants R01DE023453 and TL1TR002318.
Slichter S, Woodward A, Gharpure AS, Hsu J, Chiu Y, Ko A, Lwo Y, McLean J, Yajima M, Deng J, Gottardo R, Prlic M, Dixon DR
MAIT or human mucosal invariant T-cells are a recently identified lymphocyte population. Activated MAIT cells produce pro-inflammatory cytokines and cytotoxic effector molecules. Recently, our group showed the presence of MAIT cells in both healthy and inflamed oral mucosal tissue. However, we questioned whether MAIT cells were present in diseased, inflamed tissue around dental implants, and if so, what were their role or function.
In the first aim, we tested for the presence and quantification of MAIT cells in both normal tissue as well from mucosa surrounding dental implants diagnosed with peri-implant disease. Second, we are determining the functional properties of MAIT cells within peri-implant tissue biopsies.
In Aim 1, we obtained human oral mucosa from diseased implant sites as well as matched patient blood samples. Flow cell cytometry was used to test ex vivo samples of both tissue and matched blood samples. Lymphocyte populations were isolated and quantified. In Aim 2, utilizing next-gen RNA sequencing, we are testing these ex vivo cells for functional properties and activation levels as compared to naive cells extrapolated from collected matched patient samples allowing for comparison of functional states linked to cell transcriptome.
Flow cell cytometry experiments have determined the ability to accurately identify MAIT cells in inflamed tissue samples from peri-implant sites and quantify amounts compared to matched blood samples. Initial results indicate that MAIT cells are proportionally elevated in inflamed peri-implant tissue. Next, we are determining if MAIT cells are hyper or hypo-functional compared to naïve cells.
The experiments performed thus far have identified not only the presence of MAIT cells within peri-implant tissues and suggest a proportional increase that may be different than what is observed in other oral mucosa. These findings are both immunologically and clinically relevant and indicate potential for future therapeutic intervention strategies.
Supported by: National Institutes of Health Grant R21DE026565.
Baldwin M, Liu Z, and Herring S
Oral Health Sciences, Orthodontics
Many domestic pigs have midfacial hypoplasia (MFH), or underdevelopment of the upper jaw, nose, and cheek bones, making them a natural and novel model for this condition in humans. However, the frequency and severity of MFH has not been measured and compared across breeds, and the cause of MFH in this animal is unknown. The objectives of this study were to 1) compare the frequency and severity of MFH in various pig breeds and 2) determine if the cranial base cartilages, which are believed by many to be the primary drivers of facial growth, are defective in MFH pigs.
Objective 1: 125 dry skulls from various pig breeds were examined for the presence of MFH. The severity of MFH was determined by measuring the amount of anterior crossbite. Objective 2: Heads from 8 MFH pigs and 7 normal pigs, ages newborn to 4 months, were harvested, parasagittally sectioned, and compared for structural differences in the cranial base.
Objective 1: 100% (16/16) of Yucatan minipigs had MFH, whereas only 15% (12/79) of non-Yucatan minipigs, and 27% (8/30) of farm pigs had MFH. On average, the anterior crossbite was much larger in Yucatans (11.1mm), compared with other minipigs (3.4mm) and farm pigs (1.9mm) (ANOVA, p<0.0001). Objective 2: The cartilage tail of the nasal septum was ossified in all MFH pigs, but present in all normal pigs. In addition, the perpendicular plate of the ethmoid was more advanced in length and height in MFH pigs compared to normal pigs. The rest of the cranial base appeared normal.
All Yucatans have some degree of MFH, which is much less common and severe in other pigs with MFH. Early mineralization of the ethmoid may be one possible cause of MFH in pigs, and may relate to MFH in humans.
Supported by: National Institutes of Health Grants T90DE021984, R21DE024814 and R21DE023988; Magnuson Scholarship and ARCS Scholarship.
Finkleman SA, Todoki LS, Choi KW, Greenlee GM, Huang GJ, National Dental PBRN Collaborative Group
A variety of treatment modalities are employed to treat anterior openbite (AOB) in adults. This publication describes a large, prospective cohort study examining recommendations and treatment for adult AOB.
Participants consisted of practitioners, recruited from the National Dental Practice-based Research Network, and their adult AOB patients in treatment. Demographic and treatment data was acquired through questionnaires.
91 practitioners and 347 patients were recruited. Practitioners reported using fixed appliances and elastics frequently for adult AOB patients. Only 33% of practitioners reported using aligners frequently; 10-13% reported frequently recommending TADs or orthognathic surgery. The mean pre-treatment overbite was -2.4mm. The mean mandibular plane angle was 38.8°. Almost 40% of patients had previous orthodontic treatment.
This publication presents the demographic data and the practitioners’ self-reported treatment preferences.
Supported by: National Institutes of Health Grant U19DE22516.
Kuriakose P, Khosravi R, Bollen AM
Dental Monitoring®(DM) is a software system that allows orthodontists to remotely monitor a patient’s treatment. This study evaluated the use of DM during rapid palatal expansion treatment (RPE) and patient acceptance of DM.
Consecutively treated RPE patients were invited to participate. Participants were trained to capture intraoral photos/videos using the DM app. Intermolar width (in mm) and posterior crossbite correction measured by DM were compared to digital scan and intraoral measurements.
So far, out of 30 patients enrolled, 5 withdrew (1 from technical difficulty with phone) and 8 were unsuccessful in using DM app. There was no significant difference in intermolar width measurements obtained with DM, digital scan or intraoral. DM was not a viable option for 40% of the patients. One third of the patients preferred DM over a clinical visit.
DM software can be used to remotely evaluate RPE treatment but is not an option for all patients.
Supported by: University of Washington Orthodontic Alumni Association.
SURF and 4th Year Dental Students
Freed A, Johnson SC, Pan A, Sun GX, Witkowski M, Li L, Ford JM, Howard C, Sedensky MM, Morgan PG
Anesthesiology and Pain Medicine
General anesthesia has become a key component of patient care during invasive dental procedures and pediatric care and is considered safe in healthy individuals. However, pre-clinical studies in mice, rats, and monkeys have repeatedly demonstrated that exposure to anesthetic agents during early postnatal periods can lead to acute neurotoxicity and later-life defects in cognition, typically assessed using behavioral assays for learning and memory. Although the potential for anesthetics to cause damage to the neonatal brain is well-documented in laboratory-based studies, the clinical significance of these animal models remains unclear. Here, we provide a systematic evaluation of various physiological parameters in post-natal day 7 neonatal mice exposed to 1.5% isoflurane for 2-4 hours, representing the most common anesthesia-induced neurotoxicity paradigm in current literature. We find that 2 or more hours of anesthesia exposure results in dramatic respiratory and metabolic changes, unlike those seen during clinical exposures. Our data indicate that pre-clinical anesthesia-induced neurotoxicity models in mice are inappropriate models of human exposures.
Supported by: National Institutes of Health Grant R01GM118514 and University of Washington Dr. Douglass L. Morell Dentistry Research Fund.
Ly D, Leotta DF, Balu N, Lu Y, Eftekhar M, Liu ZJ
Obesity is a major risk factor for obstructive sleep apnea (OSA) because the increased fat composition and decreased tissue stiffness in oropharyngeal structures could result in airway collapse during sleep. The purpose of this study is to investigate tissue properties of the tongue base (BT), soft palate (SP), and pharyngeal wall (PW) in non-obese/non-OSA and obese/OSA minipigs.
After sleep monitoring to verify naturally-occurring OSA on 7 minipigs: 5 Yucatans (8.5-11 months; 3 non-obese and 2 obese) and 2 Panepinto (6.5 years, both obese), fat-weighted MRI (Dixon), elastography ultrasound (EUS), and sleep video-fluoroscopy (SVF) were performed under sedation. The quantifications of fat composition (%) in MRI and tissue stiffness (kPa) of the TB, SP and PW on EUS, and respiratory excursion of the SP (mm) on SVF were performed by software analysis.
All 4 obese and 3 non-obese minipigs were confirmed to be with and without OSA, respectively. The fat composition gradually increased from 20% to 50-70% from anterior to posterior TB regardless of presence of obesity/OSA. However, this trend was not seen in the SP and PW. The PW presented the highest fat composition (50-80%) as compared with the TB and SP (20-65%). Young obese/OSA Yucatans showed stiffer TB (20-30kPa) than their controls(15-20kPa). However, the TB of older obese/OSA Panepintos with severer OSA presented lower stiffness (10-18kPa) as compared with young controls. The range of SP excursion was greater in both anterior-posterior and dorsal-ventral directions in OSA/obese (4.5-9.5mm) than controls (3.0-3.5mm), and the largest excursion ranges were seen in older obese/OSA Panepintos.
More fat composition in the posterior TB could be a predisposing factor for oropharyngeal airway collapse, and less stiff TB might be potential factors for more severe OSA in older obese Panepintos. More extended excursion of the SP may result from heavy snoring seen in all obese/OSA minipigs.
Supported by: National Institutes of Health Grant R21DE023988 and University of Washington Dr. Douglass L. Morell Dentistry Research Fund.
Smith KK, Herring SW, Rafferty K
Midfacial hypoplasia, the underdevelopment of the upper jaw, can be caused by slow growth or premature fusion of craniofacial sutures. This research’s goal was to (a) assess whether a cyclic loading procedure can improve the growth rate of sutures in Yucatan minipigs, which have midfacial hypoplasia, and (b) begin the search for mechanisms involved through bromodeoxyuridine (BrdU) staining of proliferating cells.
Suture specimens from pigs labeled with fluorescent bone markers were embedded in plastic and sectioned (n=4 loaded and n=4 sham pigs). Images of the nasofrontal and coronal sutures were merged to perform a histomorphometric analysis of the average widths of sutural space and newly deposited bone. The analysis for each was done by two different observers blinded to pig treatment. Other suture samples, including some from farm pigs with normal jaws, were decalcified, embedded in paraffin, and sectioned. Animals had been injected with BrdU. Farm pig and Yucatan sections were reacted with anti-BrdU to identify dividing cells within the sutures (n=12).
Neither the sutural space width nor the fluorescent marker width of the Yucatan minipigs showed differences between experimental and sham in any of the sutures examined. However, the coronal suture widths were significantly narrower than those of the nasofrontal sutures. Similarly, there were no significant differences between BrdU+ cell counts of experimental and sham animals for either the osteogenic or the middle zone of the sutures. However, the osteogenic zones always had higher labeled cell density than middle zones and Yucatans were more heavily labeled than farm pigs.
The hypotheses cannot be accepted because there was no treatment effect. Although this treatment has been reported to be enhance suture width and growth in normal animals, it did not work on the poorly growing sutures of the midfacially hypoplastic Yucatan pigs.
Supported by: National Institutes of Health Grant R21DE024814 and University of Washington Dental Alumni Association.
Mills C, Beussman K, Popowics T, Sniadecki N
Oral Health Sciences
This study sought to understand how periodontal ligament (PDL) cells (frozen and freshly isolated) respond to changes of the extracellular matrix (ECM). Hyaluronan (HA), a major component of the ECM, is broken down from its high molecular weight (HMW-HA) form in health to low molecular weight (LMW-HA) form in disease. We tracked cell migration distance and speed and traction forces exerted on their surroundings. Our hypothesis was that PDL cells will migrate further, faster, and exert less force on their surroundings in a disease (LMW-HA) state in order to find sites of tissue damage.
Cellular migration was studied by treating cells with LMW-HA or HMW-HA against controls and capturing image data every 5 min for 10 hours, which was processed using cell tracking in ImageJ. Traction forces were studied by seeding cells on silicone microposts, treating cells for 1 hour, and fixing staining to study micropost deflection, which was visualized using phase-contrast, fluorescent microscopy. Data were analyzed using custom MATLAB code.
Our migration hypothesis was confirmed using ANOVA with Tukey post hoc, and statistically significant differences in migration velocities [µm/min] were seen between the LMW-HA and HMW-HA (P=3.3295e-7;P=.0054) and control (P=.0047;P=.0034) frozen and fresh cells. Our traction force hypothesis was rejected. LMW-HA average force per post (nN) was higher than HMW-HA and control groups (P=.00231;P=.073). Both LMW-HA and HMW-HA groups had a lower degree of spreading (µm2) than the controls (P=.0061;P=.0089).
Differing forms of HA have significant effects on PDL cell motility. Cells treated with LMW-HA migrate faster and exert more force on their surroundings in doing so. This could be to pull themselves along the ECM whereas cells treated with HMW-HA may be entering a more quiescent state. The LMW-HA treated cells may compact themselves to be more mobile resulting in less cellular spreading.
Supported by: National Institutes of Health Grant T90DE021984 and University of Washington Dr. Douglass L. Morell Dentistry Research Fund.
Hanson-Drury S, Alghadeer A, Zhao YT, Mathieu J, Ruohola-Baker H
Biochemistry, Oral Health Sciences
Destruction of tooth enamel is irreversible in nature due to the loss of ameloblasts, or enamel secretory cells, following tooth development. The goal of this project is to, for the first time, develop a differentiation protocol for human induced pluripotent stem cells (hiPSC) derived human oral epithelium and furthermore, ameloblasts for therapeutic purposes. To reach this goal, we will first develop a novel differentiation protocol of hiPSC to oral epithelium in vitro by identifying key differentiation signaling molecules involved and specific markers for the mature oral epithelium. Successful differentiation of hiPSCs into oral epithelium will be confirmed by the increased expression of transcription factor Pitx2 using qRT-PCR analysis.
hiPSC were cultured for 10-days under varying differentiation conditions to test the effect of multiple factors including base culturing media (RPMI or DMEM/F-12), and supplementation with BMP4, SAG, FGF10, GSK3-i and EGF (N=3 per differentiation condition). Signaling molecules analyzed include OCT4 to indicate successful differentiation from a pluripotent state, epithelial cell marker KRT14, and the specific marker of dental epithelium expressed during initiation stage Pitx2. Upon harvest, qRT-PCR was performed and analyzed via Student’s t test. Positive controls include fetal human canine tooth bud, tongue, lip and dental lamina (N=1). All results were compared and normalized to a control group of undifferentiated hiPSC (N=3).
Pluripotency stem cell marker OCT4 expression is significantly downregulated in hiPSCs differentiated with only BMP4 as well as those with BMP4 and SAG compared with wild type control cells. OCT4 expression is very significantly (P=0.0029) downregulated 102-fold in cells supplemented with only BMP4. Epithelial stem cell marker KRT14 expression is significantly upregulated 14-fold in cells differentiated in RPMI media compared to DMEM/F-12 media (P=0.0247) and 8-fold in cells supplemented with GSK-3i (P=0.0357). KRT14 expression is not significantly changed in cells differentiated with only BMP4; both BMP4 and SAG; FGF10; or EGF. Pitx2 expression is upregulated in cells differentiated with only BMP4 as well as those with BMP4 and SAG compared with wild type control cells, with extremely significant (P=0.0001) 155-fold upregulation in cells supplemented with both BMP4 and SAG. Pitx2 expression is extremely significantly upregulated 5-fold in cells supplemented with FGF10 (P=0.0001). Pitx2 is very significantly downregulated 24-fold in cells supplemented with GSK-3i (P=0.0042) and significantly downregulated 3-fold in those supplemented with EGF (P=0.0157). Pitx2 expression is not significantly changed in cells differentiated in RPMI media compared to DMEM/F-12.
The extremely significantly increased Pitx2 expression observed in cells supplemented with BMP4, SAG, FGF10 indicate that these cells have successfully differentiated into oral epithelium. Significantly upregulated KRT14 in cells differentiated in RPMI media indicate successful differentiation into epithelium.
Supported by: National Institutes of Health Grants T90DE021984 and P01GM081619, University of Washington Dr. Douglass L. Morell Dentistry Research Fund and STEMFD.
Nguyen K, Kotsakis G, Chrepa V
Human multipotent mesenchymal stem cells (MSCs) isolated from extracted dental tissues have been evaluated in cell-mediated therapeutic strategies. Various dental sources of MSCs has been characterized in order to facilitate the development of regenerative dental procedures, tissue engineering applications, and the future current good tissue practices (cGTP) protocols for clinical banking of MSCs. Standardized criteria for MSCs include specific surface antigen expressions and multipotent differentiation potentials. Our goal was to characterize human alveolar bone mesenchymal stem cells (ABSCs) and dental pulp mesenchymal stem cells (DPSCs) using these criteria.
Human alveolar bone and dental pulp samples were collected from periodontal surgeries and freshly extracted teeth, respectively. Cells were isolated and processed for stem cell surface marker staining (CD73, CD90 and CD105) and the pan-leukocyte marker CD45 using multicolor flow cytometry. Osteogenic and adipogenic expression was evaluated using Alizarin red stain and Oil red O stain, respectively, under light microscopy.
Alveolar bone tissue contained about 24.61% CD45-negative cells, 99.17% of which showed positive co-expression of the MSC markers CD73, CD90 and CD105. Human dental pulp tissue had 68.76% CD45-negative cells; 94.58% of those expressed all 3 MSC markers. Under osteogenic conditions, both cell populations showed positive Alizarin Red staining, with DPSCs producing more robust mineral deposits. Stained lipid droplets were more evident in ABSC cultures compared to DPSCs.
Cells isolated from human alveolar bone and dental pulp tissue samples expressed MSC markers while being negative for the pan-leukocyte marker CD45 and showed differentiation potentials into osteogenic and adipogenic lineages. Greater osteogenic differentiation was observed in DPSC cultures compared to ABSCs under light microscopy, while opposite findings were observed during adipogenesis.
Supported by: University of Washington Dental Alumni Association.
Lutgen PJ, An SW, Chan DC, Sadr A
We evaluated the performance of full coverage restorations on structurally compromised teeth with different core buildups cycled under incrementally increasing loads using a dynamic chewing simulator. Internal stress patterns contributing to failure were analyzed with optical coherence tomography (OCT; Yoshida Dental MFG). 16 extracted human premolars were root canal treated and randomly divided into 4 groups to receive core buildups. G1: 2mm ferrule, 2mm composite core (Clearfil PhotoCore, Kuraray Noritake Dental). G2: no ferrule, 4mm composite core with peripheral reinforcement by continuous fiber (Ribbond Ultra, Ribbond). G3: no ferrule, 4mm composite core. G4: no ferrule, fiber post (ParaPost Fiber, Coltene) with composite core. Specimens were prepared with 1mm margins and received full coverage lithium disilicate crowns (IPS e.max CAD, Ivoclar Vivadent). Specimens mounted in a dynamic chewing simulator (CS-4.4, SD Mechatronik) were initially loaded with 10kg with 5kg of additional weight added every 250,000 cycles until continuous loading with 25kg produced catastrophic failure. OCT imaging was conducted at the conclusion of each set of 250,000 cycles from proximal, buccal, lingual, and occlusal views. There was a significant difference among groups. Specimens without ferrule receiving either composite core or composite core with fiber post perform similarly, with some specimens failing during the incremental loading stage. Continuous fiber-reinforcement within core buildups provides significantly increased performance compared to fiber posts or composite alone. The ferrule group showed highest survival. Catastrophic failures were primarily initiated in the lithium disilicate crown itself or the lingual restorative margin. Ferrule remains an important factor in the structural integrity of full coverage crowns. Continuous fiber reinforcement of core composite distributes stresses peripherally and may provide an alternative restorative option for structurally compromised teeth and no ferrule. Failures observed were caused by microcracks within the crown that led to complete fracture or cement debonding due to lingual tensile forces.
Supported by: University of Washington Dr. Douglas L. Morell Dentistry Research Fund.
Chiu AC, Chen YW, Hayashi J, Sadr A
The advancement of intraoral scanners has allowed for more efficient workflow in the dental clinical setting. The use of intraoral scanners has been widely adapted in recent years with the 3Shape TRIOS scanner proven to be one of the most accurate systems. However, limited data exist regarding the accuracy of digital impressions produced using various manufacturer settings. The purpose of this in-vitro study was to evaluate and compare the accuracy of digital impression at the preparation margin using different scanning resolutions.
An all-ceramic crown preparation of #30 was constructed in a typodont and a control scan (n=3) was created with a laboratory scanner. Digital impressions using three scan settings – high-resolution (HR), standard resolution (SR), and combined resolution (SHR) were compared (n=20). 3D inspection was performed to measure the discrepancy between TRIOS and control scans along the preparation finish line. Scanning time and the number of images captured per scan were recorded. Statistical analysis was performed with one-way ANOVA, two-way repeated measures ANOVA, Pearson’s correlation, and Dunnett’s T3 test (a = 0.05).
For discrepancy on the finish line, no significant differences were observed between group HR (31.5 ± 5.5 μm), group SHR (33.2 ± 3.7 μm), and group SR (33.6 ± 3.1 μm). Scanning time in group SR was on average 34.2 seconds less than group SHR and 75.1 seconds less than group HR. Although no significant differences in discrepancy were observed between scanning resolutions, significant differences in discrepancy were observed among tooth surfaces, with the distal surface showing the highest discrepancies.
The data showed significant differences in scan time and numbers of images captured between different scan resolution settings. While the scanning resolution setting was not a significant factor.
Supported by: University of Washington Dental Alumni Association.
Walczak PA, Sadr A, Seibel EJ
Restorative Dentistry, Mechanical Engineering
Early characterization and acidogenicity assessment of oral biofilms may facilitate management of cariogenic oral biofilms. The time interval at which biofilm is active below the individual’s critical enamel pH is important in maintaining tooth integrity. This pilot study’s primary objective is to show efficacy of a prototype intra-oral device for measurement of oral biofilm pH in a clinical setting.
Six subjects (3 F, 3 M, mean age=22.7, SD=1.34) were recruited for the study. One tooth in each quadrant with high plaque load was selected by a calibrated operator. The labial surface of an upper central incisor served as control due to expected lower plaque loads. A spectrometer was used to measure pH-dependent fluorescence spectra of FDA-approved fluorescein (500uM) at 420 nm excitation, compared with pH strips and salivary pH measurement (4 subjects). After baseline measurements (N=3 per surface), the subjects were administered a sucrose challenge (10%, 10 mL for 15s). Occlusal plaque pH was monitored for 15 minutes. Measurements were also conducted on freshly extracted teeth with rampant decay.
The optical device exhibited pH 0.2 uncertainty in repeated baseline measurements (resting pH) throughout the study. Biofilm pH was found to be higher and closer to salivary pH in control than most posterior measurements, showing the readings were not merely measuring salivary pH. Sucrose pH response curves were observed, with some biofilms displaying classical responses and others being less consistent. Repeated measurements (N=2) on extracted teeth showed at least moderate relationship between readings (>0.630).
This is the first clinical testing of an optical pH measurement device of oral biofilms. Results indicate the device may have diagnostic potential in the future with further development, but larger, longitudinal studies are required. A future study with high caries risk patients is planned with a second-generation device.
Supported by: National Science Foundation Grant 1631146 and University of Washington Dr. Douglass L. Morell Dentistry Research Fund.
Panahpour Eslami N, Chan DC, Sadr A
Silver diammine fluoride (SDF) is an increasingly popular anti-cariogenic agent for minimally-invasive dentistry. This study addressed a common question regarding an optimal application frequency of SDF when used with glass-ionomer cement (GI) for remineralizing dentin in advanced carious lesions.
25 freshly extracted human posterior permanent teeth with advanced carious lesions were used in this study. After superficial removal of infected dentin, single (G3), double(G4), and triple(G5) applications of SDF (Advantage Arrest) followed by a layer of GI (GC Fuji IX GP) were compared to no treatment (negative control, G2), and GI only (G1). All teeth were stored in artificial saliva (AS) solution for 2 weeks after final treatment. Micro-computed X-ray tomography(mCT) scans were obtained at each stage and analyzed using Amira (FEI VG) and ImageJ (NIH) to plot mineral density vs. depth and calculate lesion depth (LD) and mineral loss (dZ) for 30 lesions (n=30) per treatment group. Comparison within and between groups with appropriate parametric and non-parametric statistical tests were carried out(α=0.05).
Mean LD and dZ at baseline ranged from 638 to 1008 µm and 2327 to 10013 µm.vol%. Paired t-test showed that LD and dZ changed significantly within all groups from baseline to treatment weeks following AS storage (p<0.05) except for LD in the control (p>0.05). Mean reductions in groups G1 to G5 were 6.8%, 0.4%, 19.5%, 13.2% and 10.6% for LD, and 49.1%, 11.3%, 54.3%, 66.1% and 48.1% for dZ, respectively. One-way ANOVA showed significant difference among groups for both variables (p<0.05). Collectively, G3 and G4 showed the highest mineral gains.
The remineralization effect of SDF when used in conjunction with AS solution and GI is enhanced. One or two applications of SDF are effective in healing advanced dentin lesions with additional application not demonstrating additional benefit. These results set the stage for future studies on a strategy to remineralize advanced lesions using SDF, potentially in vivo.
Supported by: University of Washington Dental Alumni Association.
Bashizada E, Wallace E, Cunha-Cruz J, Stein C, McKinney CM
Pediatrics, Seattle Children’s Research Institute, Oral Health Sciences
Greater intake of water may decrease caries prevalence; however, the risk may differ based on whether consumption is from fluoridated tap water or bottled water, which typically has suboptimal fluoride content. Our objective was to examine associations between water intake by type (tap and bottled water) and dental caries.
We conducted a cross-sectional study in King County, Washington. We enrolled 452, majority low-income children between 1st and 9th grade. We collected sociodemographic data, frequency and amount of beverage intake, including tap and bottled water separately, non-sugar sweetened beverages [SSBs], SSBs, 100% fruit juice, and milk via survey. We conducted oral health examinations to estimate number of decayed, missing due to caries and filled teeth in primary dentition (dmft). We estimated descriptive statistics using means and percentages. We employed a multivariable model using Poisson regression and robust standard errors to estimate prevalence ratios (PR) for associations between water intake and dmft, adjusting for potential confounders (sociodemographic, SSBs and 100% juice intake).
We enrolled a racial diverse, low-income sample from the Seattle and King County area with 88.2% reporting a race/ethnicity other than non-Hispanic White. In addition, 63.7% were below the 130% federal poverty line (FPL). In adjusted analysis, we found an association between increased overall water consumption and decreased dmft (prevalence ratio [PR] ranging from 0.67-0.71). Fluoridated tap water has a statistically significant association with decreased dmft (PR ranging from 0.55-0.77) with the largest decrease seen for >1-3 servings (PR=0.55). There is no association between increased bottled water consumption and decreased dmft prevalence with PR>1 for all servings/day >3 (p>0.05).
Fluoridated tap water is protective against caries. Bottled water is not associated with reduced caries. Efforts to ensure fluoridated water is the primary source of water intake may be needed to prevent caries in population health.
Supported by: University of Washington Dr. Douglass L. Morell Dentistry Research Fund, Seattle Children’s Center for Child Health and Behavior Stimulus Fund and ARCORA, the foundation of Delta Dental of Washington.
Finkleman S, Ramsay D, Popowics T, Arola D, Mancl L
Oral Health Sciences, Orthodontics
Orthodontic patients are at risk for experiencing dental injury during participation in sports-related activities. However, little is known about the impact behavior of orthodontic mouthguards. This study developed a protocol for benchtop testing of orthodontic mouthguards and compared the protective capacities of four commercial orthodontic mouthguards during simulated blunt trauma.
Four commercially available orthodontic mouthguards were studied: OPRO Gold Level Mouthguard for Braces, Under Armour Braces Strapless Mouthguard, Shock Doctor Ultra Mouthguard for Braces, SISU Aero. The orthodontic mouthguards were thermomolded to a custom-fabricated orthodontic model and 0.5 J of energy were delivered at the central incisor with a drop-tower. Sensor films were analyzed to quantify the pixel area corresponding to different magnitudes of pressure with an open-source image-processing software.
Overlay images demonstrated that pressure was concentrated on brackets and archwire. SISU Aero broke during each first run and was removed from the study thereafter. The average pixel area in the impact incisor region for the highest pressure magnitude was lowest for Under Armour (p-value = 0.024; F1,4 =12.7), but was not significant at a level of 0.05 significance after controlling for multiple tests. Restricting the data to first impact only revealed the average pressure away from the impact site was significantly lower for SISU (p<0.05).
A definitive conclusion about the relative protective capacities of the four orthodontic mouthguards could not be reached. Future research should increase sample sizes, improve the protocol to minimize non-impact marks, and improve mouthguard designs to better distribute impact forces.
Supported by: University of Washington Dr. Douglass L. Morell Dentistry Research Fund.
Other Dental & Engineering Students
Hwang MJ, Dodson TB
Oral and Maxillofacial Surgery
Unlike football helmets, standard bicycle helmets do not include a face mask to protect the face. The purpose of this study is to measure the effects of bicycle helmets on the frequency and severity of maxillofacial injuries. The specific aims were to measure and compare the frequencies and severity of maxillofacial injuries between injured bicyclists who did or did not wear helmets.
We implemented a retrospective cohort study and enrolled a sample of bicyclists injured between January 4, 2012 and March 31, 2018 who presented to Harborview Medical Center for evaluation and treatment. The primary predictor variable was helmet use (yes/no). The primary outcome variables were 1) presence of a maxillofacial injury (yes/no) and 2) injury severity measured using the facial abbreviated injury scale (FAIS). Other study variables were grouped into demographic or injury-related variables. Appropriate uni-, bi-, and multiple logistic regressions statistics were computed and statistical significance was set at p < 0.05.
The sample was composed of 1379 subjects with a mean age of 39.6 ± 18.9 (1 – 91) years and 78.4% were male. The frequencies of facial injuries between helmeted and non-helmeted bicyclists were 17.3% and 30.6%, respectively (relative risk = 0.6, 95% CI = 0.5 – 0.7, p < 0.0001). The protective effect held after adjusting for age, sex, and alcohol exposure. The FAIS between helmeted and nonhelmeted riders was 1.8 ± 0.5 versus 1.9 ± 0.6 (p = 0.8), respectively.
Bicyclists who wear helmets are 40% less likely to have a facial injury from bicycle accidents. There was no difference in facial injury severity among bicyclists who wear helmets when compared to those who do not wear helmets. We conclude that standard bicycle helmets protect against facial injury, but may not decrease maxillofacial injury severity.
Supported by: University of Washington Department of Oral and Maxillofacial Surgery Laboratory for Applied Clinical Investigation, University of Washington Department of Oral and Maxillofacial Surgery Research and Training Fund.
Hurst, P, Hwang MJ, Dillon JK
Oral and Maxillofacial Surgery
Dog bite injuries to pediatric patients (minors) are not uncommon. Current literature shows that minors are at a higher risk of injuries to the head, neck and face (HNF) from dog bites. Less is known about the specific facial regions that are injured. The purpose of this study is to: 1) measure the frequency of injuries to the HNF region among pediatric and adult patients and 2) assess the patterns of maxillofacial injuries.
A retrospective chart review on patients presenting with dog bite injuries to a Harborview Medical Center from January 2000 to December 2015 was implemented. The primary predictor variable was age group (pediatric versus adult). The primary outcome variable was type of injury (HNF injury versus non-HNF injury). Facial injuries were categorized as upper, middle, and lower thirds.
The sample comprised of 465 subjects. Mean age 26.3 ± 21.5 (0.7 – 94.4). Minors and adults comprised 43.7% and 56.3%, respectively. The frequencies of HNF injuries between minors and adults were 90.1%% and 49.6%, respectively (relative risk = 1.8, 95% CI = 1.8 – 2.1, p < 0.0001). Among the HNF injuries, facial injuries were most common (93.6%). Upper facial third injuries were more common in the minors than adults, 29.3% and 15.1% (p = 0.005), respectively. A larger proportion of minors suffered a middle third injury, 82.2% versus 60.5%, for adults, respectively (p <0.0001). There was no significant difference in the lower third.
Minors are 1.8 times more likely to have a HNF injury from dog bites than adults. Both groups were more likely to have an injury to the middle and lower facial thirds. Minors are 1.9 and 1.4 times more likely to suffer an injury to the upper or middle facial thirds, respectively. There was no difference in risk for the lower third.
Latimer J, Eftekhar M, Roll K, Daubert D, Kotsakis G
The Alveolar Bone Changes in Diabetes (ABCD) Study is a prospective cohort study that aims to assess pathophysiologic alterations of mandibular alveolar bone structure as well as safety and efficacy of implant placement in patients with well-controlled (WC) versus poorly-controlled (PC) Type II Diabetes Mellitus (T2DM). This study consists of an initial screening, implant placement, and a series of follow up appointments over a 3-year period. HbA1c values were taken as a measure of glycemic control at baseline and 3 months following implant surgery. Bone cores were collected intra-operatively; histological, histomorphometric, and immunohistochemical assessments were used to evaluate structural quality, marrow adiposity, vascularity, PPAR-y activity, and RUNX2 activity. Clinical measures used to assess implants included maximum insertion torque (MIT), implant stability quotient (ISQ), intraoral examination, and periapical radiographs. Patient perceived success of implant surgery was assessed with oral health impact (OHIP) questionnaires quantifying patient perceived pain, disability, and social handicap at each appointment.
10 WC and 10 PC patients received the allocated intervention. Data analysis revealed a positive correlation between HbA1c and PPAR-γ activity (r = 0.16) as well as bimodal patterns of bone and fat fraction in relation to Hba1c. Increasing HbA1c in the PC group was associated with higher bone fraction (ρ = 0.38) and reduced vascular density (r=-0.13). Baseline and 3-month post-surgery ISQ measurements were clinically acceptable (p<0.05). No adverse events occurred and overall OHIP scores decreased for both groups, indicating improvements in patient perceived oral health related quality of life. These findings suggest that chronic hyperglycemia in T2DM may lead to dysregulation of PPAR-γ activity and increase in alveolar bone fraction without conservation of bone quality. Clinically, these changes did not affect implant stability, integration, and early survival, thus supporting implant placement to improve oral function in patients with T2DM, even without ideal glycemic control.
Supported by: University of Washington Elam M. and Georgina E. Hack Memorial Research Fund. Partial material support provided by Straumann USA.
Baioumy YM, Yucesoy DT, Fong H, Sarikaya M, Dogan S
Chemical Engineering, Restorative Dentistry
Incorporating biomimetic mineral layer to the molecular structure of enamel has been a long-standing challenge. Aim of this in-vitro study was to evaluate ADP5-guided remineralization of enamel and ability to control the biomimetic mineral layer properties.
Extracted healthy human molars were used to create white spot lesions on enamel with acetic acid. Samples were incubated in a 15-AA long ADP5 amelogenin-derived peptide solution for 10 mins at 37°C. Samples were treated with Ca/PO4 for 1h at 37°C (Group 1). Samples treated with Ca/PO4 without ADP5 served as positive controls (Group 2). Enamel without any treatment were used as negative controls. Scanning electron microscope (SEM) was used to evaluate the morphology and thickness of new mineral layer. Energy dispersive X-ray spectroscopy used to measure chemical composition of new layer. Structural characterization was done by transmission electron microscopy (TEM). Nanoindentation measurements and microhardness testing were performed to assess mechanical properties. The average values and standard deviations were calculated as the mean ± standard error.
SEM images showed continuous layer of plate-like crystals growing from the surface of the underlying enamel lesion in Group 1 with a 10μm-thick continuous remineralized layer. Ca/PO4 clusters without substantial remineralization were observed in Group 2. TEM showed hydroxyapatite (HAp) crystals of 30−50 nm were encountered in healthy enamel tissue which correspond to plate-shape mineral found in Group 1. Elemental analysis of the samples from Group 1 revealed Ca/P ratio of 1.54±0.12. The microhardness and nanomechanical revealed the values between that of untreated enamel and dentin in Group 1.
This study demonstrated crystalline mineral layer is formed on an artificially created lesion on human enamel in the presence of Ca/PO4 ions under physiologically conditions by using ADP5. The approach offers future clinical implementations as a novel dental health care for biomimetic remineralization.
Supported by: Washington State Life Sciences Discovery Fund and University of Washington Dean and Margaret Spencer Endowed Clinical Research Fund.
Lepe X, Streiff K, Johnson G
Determine if current automixed cements provide clinically acceptable retention of high-strength zirconia crowns following long-term aging.
Mounted in epoxy, extracted human molars prepared with flat occlusal plane, 20-degree taper, 4mm axial length and axio-occlusal slightly rounded. Teeth equally distributed into three cementation groups (n=12) by preparation surface area.
Occlusal bar added to facilitate removal of cemented crown of high-strength zirconia (IPS e.max ZirCAD LT). Cement space set at 45µm axially and 55µm occlusally. After sintering and before delivery, fit of crowns confirmed and intaglios air-abraded with 50µm alumina at 1-bar pressure for 3-seconds, then steam cleaned.
Cements: original powder-liquid RelyX Luting (RMGI; RXL) as control, and paste-paste, automix systems RelyX Luting Plus Automix (RMGI; RXLA) and RelyX Unicem 2 Automix (self-adhesive, modified resin; RXUA). Crowns cemented under 20-kg force, placed in 37°C oven at 100% humidity for setting, then thermocycled (5°C-55°C) for 5000 cycles monthly for 6 months. Crowns removed axially with Instron at 0.5 mm/min. Removal force recorded and dislodgment stress calculated using surface area.
ANOVA revealed significant differences (P<0.001). Levene’s test significant therefore Games-Howell used for pair-wise tests (≠). Χ2 analysis significant. RXLA demonstrated significantly lower crown retention compared to RXUA and RXL. Modes of failure displayed cement only in crown intaglio for RXLA, while others showed cement residing on dentin and intaglio or mostly in crown intaglio.
RXL RXUA RXLA P
Stress (MPa) 3.1(1.1) = 3.1(0.4) ≠ 1.3(0.3) P<0.001
Force (Kg) 42.6(16.8) = 41.6(6.0) ≠ 17.8(3.7) P=0.005
Cement (%N) 58%-mixed;= 50%-mixed; ≠ 100%-in crown P<0.001
42%-on dentin 50%-in crown
With long-term aging, high-strength zirconia crowns were strongly retained by two of three cements. However, crown retention for RelyX Luting Plus Automix was less than half in comparison. These results serve as basis for clinical cement selection for high-strength zirconia crowns.
Supported by: University of Washington Dean and Margaret Spencer Endowed Clinical Research Fund.