35TH ANNUAL

RESEARCH DAY

Ed 2 Student BridgeUniversity of Colorado Anschutz Medical Campus

Friday, February 22, 2019 | 12 p.m.-1 p.m.

ABSTRACTSPoster Presentations

Undergraduates

Designing Opto-mechanically Responsive Molecules as Coatings on Pit and Fissure Sealants Kasra Roostan1, Krithika Baskaran2, Dixa Gautam3, Gannon M. Kehe3, Dylan I. Mori3, Michael J. Schurr4 and Devatha P. Nair3,5

1Department of Psychology and Neuroscience, University of Colorado, Boulder, Boulder, CO, USA; Departments of 2Restorative Dentistry and 3Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 4Department of Immunology and Microbiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA, 5Materials Science and Engineering Program, University of Colorado, Boulder, Boulder, CO, USA Pits and fissures on the surface of teeth account for 88% of all occlusal caries in children. Although dental sealants applied on enamel surfaces can restrict bacterial ingress by up to 50%, their efficacy can be enhanced by anti-bacterial strategies that target early cariogenic colonizers such as Streptococcus mutans (S. mutans). This translational study evaluates the ability of azopolymer coatings to disrupt biofilms on commercial pit and fissure sealants. Objectives: This study investigates the mechanical properties and biological impact of applying opto-mechanically active azopolymer coatings on commercial pit and fissure sealants. Methods: Extracted human molars were prepared with 35% phosphoric acid etch ( NOVO™ , 30s), followed by the application of sealant and azocoatings (Embrace™ wetbond™).Strenght was assessed via microtensile tests (cross-head speed -1 mm/min , n ≥ 3, Mini Bionix II, MN,USA). Extracted and sealed molars were exposed to S. mutans biofilms grown under sucrose-dependent conditions and quantified via CFU counts.

Results: The mechanical strength of the sealant was not compromised with the introduction of the azopolymer (14.0 ± 2 vs 3.2 ± 4 MPa, p = 0.325). The presence of azocoatings affected biofilm formation on the tooth samples (a) and had an extended impact beyond the substrate as seen via biofilm formation in the wells (b). No live S. mutans were detected on the OH-A AZO well. Conclusion: Preliminary results indicate that azobenzene coatings can be successfully incorporated into commercial pit and fissure sealants as anti-bacterial coatings.

Microtensile bond strength ( µTBS ) assessment

OH - AAZO

AAZO

Azomolecules used in the study

Tooth Samples

( ) a ( b )

Contr

AAZ

OH - AAZO

DDS Students

Protective Effect of Theobromine Against Acid Attack on Tooth Enamel Elizabeth Tremblay and Clifton Carey Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. Objectives: Theobromine is a potential therapeutic agent and fluoride alternative that can help protect tooth enamel from the acid produced by bacteria after consumption of sugars. Our hypothesis is that a 100 ppm solution of Theobromine will protect tooth enamel from an acid attack. We also want to validate the use of non-destructive µCT methods for the measurement of enamel erosion compared to transverse microradiography. Methods: Both interproximal surfaces of 8 human caries-free teeth were polished with 400 grit paper to make flat surfaces and remove the outer ~50 µm of enamel (i.e., two surfaces per tooth). A portion of the surfaces were protected with fingernail polish. Four teeth samples were randomly assigned to the control group of 0 ppm of Theobromine solution and the other 4 were assigned to the 100 ppm of Theobromine solution. The samples were soaked in the test solutions for 2 min, briefly rinsed with dH2O then exposed to a 1% citric acid attack at pH of 3.9 for 10 min. Samples were stored in saliva-like storage solution for 24 hours before measurement of surface loss by µCT. The measurements and statistics were blinded. Results: Enamel loss for the control samples was 13.2±8.0µm; loss for the theobromine treated samples was 1.5±4.0µm the difference is significant at p=0.0405. Conclusions: The hypothesis is supported that theobromine protects tooth enamel from an acid attack at 100ppm concentration. These erosion results are not statistically different (p>0.05) from results of prior experiments under the same conditions as here where enamel erosion was determined by transverse microradiography after theobromine exposure (3.1 ± 1.1 µm, n=16). This confirms the use of the non-destructive µCT for the measurement of erosive losses.

Intraoral findings in newborns: prevalence and associated factors Claudia L. Chandler1, Isabelita D. Azevedo2, Manoelito F. Silva, Jr. 3, Johnnatas M. Lopes2, Antonio Gordon-Nunez4 and Silvana A. Pereira2 1ISP Program, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2Federal University of Rio Grande do Norte – UFRN, Natal-RN, Brazil; 3State University of Ponta Grossa – UEPG, Ponta Grossa-PR, Brazil; 4State University of Paraiba – UEPB, Araruna-PB, Brazil. Objective: To investigate the prevalence of intraoral characteristics and associated factors with neonatal and parent variables in a group of Brazilian newborns. Methods: This cross-sectional study with a descriptive and inferential approach, whose data was obtained through clinical examination, interview and the collection of medical record information. The sample was selected from babies up to three days old, of both sexes, and born between January and December 2013 in the Ana Bezerra University Hospital, in the city of Santa Cruz-RN, Brazil. The exposure variables included neonatal (sex, weight, gestational age, type of delivery and Apgar score) and parent (presence of systemic disease(s), drug use and consanguinity between the parents) variables. Descriptive analysis and Poisson regression were performed to estimate the ratio of gross and adjusted prevalence of intraoral findings (Epstein pearls, Bohn’s nodule, dental lamina cyst and ankyloglossia) with the neonatal and parent variables (p<0.05). Results: Of a total of 168 examined newborns, 56.5% (n=95) were male. The most prevalent intraoral feature was the fibrous cord of Magitot, 62.5% (n=105), and the most frequent alteration was Epstein pearls, 44.6% (n=75). Female gender was a factor for Bohn’s nodule (OR=0.90; 95%CI:0.82-0.99), and no tobacco use by the father was a protective factor for Epstein pearls (OR=0.73;95%CI:0.55-0.97). Additionally, lower mother’s age, between 20-34 years of age (OR=1.58;95%CI:1.07-2.35) and under 19 years of age (OR=1.61; 95%CI:1.03-2.52) increases the chance of having Epstein pearls. Conclusion: There was a high prevalence of alterations in the newborns’ oral cavities and there were associations between neonatal and parent variables.

ISP Students

Detection of White Spot Lesions by PTR-LUM Technology Minh Trinh and Clifton Carey Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. Objectives: Early stage WSL are often difficult to detect without some means to indicate the presence of the demineralization. There is a sensitive caries detection system based on photothermal radiometry-modulated luminescence (PTR-LUM) technology called the Canary System. This system uses an infrared laser that penetrates 5mm into the tooth which creates a fluorescence signal that is related to mineral integrity. The signal is converted into a “Canary Number” (CN) which is interpreted to indicate the presence of caries. We wanted to determine if PTR-LUM technology is effective for detecting early stage WSL. Our hypothesis was that there would be a positive linear relationship between the CN and visual WSL scores. Methods: 36 premolars that were extracted for orthodontic therapy were evaluated for WSL by the Canary System and visually for the presence of WSL under magnification. CN were determined at the labial gingival, incisal surfaces and left and right interproximal surfaces. The CN were averaged for each tooth. Visual determination was with the teeth gently dried and observed under a 10x dissecting microscope for WSL in the same zones as for the Canary System measurements. The number of zones with WSL for each tooth were summed to yield the visual WSL score. Digital microphotographs were taken for reference. Results: The average CN score was 28.1±5.6, range 19-39, n=36; and the median visual WSL was 1, range 0-3, n=36. Because the visual WSL score is not parametric, a Spearman's correlation was run to determine the relationship between the CN and visual WSL scores. There was a strong, positive monotonic correlation between CN and WSL scores (Rs=0.743, n = 36, p<0.0001). Conclusions: The hypothesis is supported, there is a strong positive relationship between CN and visual WSL scores. This shows the Canary system is sufficiently sensitive to detect early WSL, thus giving an early warning of caries activity allowing for early intervention to preserve tooth integrity.

Irradiation of a Dual-Wavelength Photosensitive Molecule to Overcome Oxygen Inhibition

Kimberly K. Childress,1 Kangmin Kim,2 David J. Glugla,3 Charles B. Musgrave,1,2,4,5 Christopher N. Bowman1,4 and Jeffrey W. Stansbury1,6

Departments of 1Chemical and Biological Engineering 2Chemistry and Biochemistry, 3Electrical, Computer, and Energy Engineering and 4Materials Science and Engineering, University of Colorado, Boulder, Boulder, CO, USA; 5National Renewable Energy Laboratory, Golden, CO, USA; 6Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

Objective: Oxygen inhibition is detrimental to free radical polymerizations and can result in reduced polymerization kinetics and final mechanical properties. Photosensitizers have previously been incorporated in monomer formulations to excite molecular oxygen to its inert singlet state prior to irradiation of a photoinitiator, thereby avoiding unwanted consumption of free radicals by oxygen. In this work, a single photoresponsive molecule was shown for the first time to perform the tasks of both oxygen-scavenging photosensitization and photoinitiation.

Methods: Zinc 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (ZnTTP) was intoduced into di(ethylene glycol) ethyl ether acrylate (DEGEEA). Independent irradiation of ZnTTP’s UV and visible peaks with 365 and 635 nm LEDs, respectively, was conducted at varying exposure times and intensities. Polymerization conversion and ZnTTP concentration were measured in real-time via a coupled FT-NIR/UV-Vis analysis.

Results: Irradiation of ZnTTP’s UV band resulted in polymerization of DEGEEA. At 10 mW/cm2 for 0.2 mM ZnTTP, an induction time of ~290±7 s preceded the onset of polymerization. Increasing irradiance to 90 mW/cm2 reduced the induction time to 36±3 s, and increasing [ZnTTP]0 from 0.1 to 10 mM reduced the induction time from 479±15 s to 58±3 s. However, red-light pre-irradiation of the visible band of ZnTTP with a dose of ~6500 mJ/cm2, regardless of the irradiance/expose time combination, resulted in sufficient singlet oxygen generation to completely eliminate any induction time upon UV-activated polymerization. Only minor amounts of ZnTTP photodegradation occurred during the pre-irradiation stage (~4%), leaving sufficient ZnTTP available to promote efficient photoinitiation.

Conclusions: This work highlights the potential of ZnTTP to perform the polymerization functions that multiple molecules were previously required to achieve. The utility of ZnTTP as a photosensitizer and photoinitiator via irradiation of its two absorption bands resulted in immediate polymerization following adequate singlet oxygen generation.

Funding: IUCRC and ALTANA

Graduate Students

Srsf3-mediated alternative RNA splicing downstream of PDGFRa signaling in the palatal mesenchyme Brenna J.C. Dennison1,2, Eric D. Larson3 and Katherine A. Fantauzzo1

1Department of Craniofacial Biology; 2Graduate Program in Cell Biology, Stem Cells and Development; 3Department of Otolaryngology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Craniofacial development is a critical morphological event during embryogenesis, defects in which result in highly prevalent human birth defects. In both humans and mice this process relies on signaling through the platelet-derived growth factor receptor alpha (PDGFRa). Mutations in human PDGFRA are associated with cleft lip/palate and mouse models with mutations in this gene similarly display facial clefting phenotypes. PI3K is the main downstream effector of PDGFRa signaling during mouse development. We previously performed a mass spectrometry-based phosphoproteomic screen to identify targets of PI3K/Akt-mediated PDGFRa signaling in primary mouse embryonic palatal mesenchyme cells (MEPMs), revealing an enrichment for proteins that regulate RNA splicing. Objectives: We hypothesize that one of these Akt phosphorylation targets, Srsf3, mediates tissue-specific alternative RNA splicing downstream of PDGFRa signaling in the palatal mesenchyme. Methods and Results: We have biochemically confirmed using immunoprecipitation assays the PI3K/Akt-mediated phosphorylation of Srsf3 upon PDGF-AA ligand treatment of MEPMs and further demonstrated that this phosphorylation drives Srsf3 translocation into the nucleus. We revealed that expression of Srsf3 is enriched in the maxillary processes and palatal shelves of mid-gestation mouse embryos. Moreover, RNA-sequencing analysis of palatal shelf mesenchyme derived from wild-type versus autophosphorylation mutant knock-in embryos in which PDGFRa is unable to bind PI3K identified differentially alternatively-spliced transcripts containing Srsf3 binding sites that are associated with craniofacial defects. Finally, we showed that ablation of Srsf3 in the neural crest lineage results in embryos with midline facial clefting, facial bone hypoplasia and exencephaly. Conclusions: Taken together, our results point to a novel role for the PDGFRa-PI3K/Akt-Srsf3 signaling axis in regulating RNA processing during craniofacial development.

Tailored Nanogels as Antibacterial Agents

Humberto Escobedo1, Dixa Gautum2, Toan Nguyen 3, Michael J Schurr 4, Devatha P. Nair1,2 1Department of Pharmaceutical Science, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA;2Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 3Biomedical Sciences Program, Regis University, Denver, CO, USA; 4Department of Immunology and Microbiology, School of Medicine University of Colorado Anschutz Medical Campus, Aurora, CO, USA The mechanism by which bacteria have evolved to evade targeted, antibiotics is the formation of biofilms, which renders them 10-1000 times more resistant to common antibiotic drugs. As non-traditional antibiotic strategies that target biofilms have continued to garner interests, multiple studies have shown the potential of nanoparticles as a substitute and/or complement to strategies to combat antimicrobial resistance. We examine the ability of tailored nanogels from 2- 200 nm synthesized to induce bacterial membrane disruption via passive, non-lytic mechanisms as a promising route to inhibit and disrupt biofilm growth. Objective: Study the bacteriostatic effects of tailored nanogels synthesized via two different mechanisms on biofilm formation of gram-positive and gram-negative bacteria Methods: Nanogels were synthesized and characterized via both a solution polymerization (NG1, 2-10 nm) and an emulsion polymerization (NG2, 50-100 nm ) mechanism from monomers 2-(Dimethylamino)ethyl methacrylate (DMEAMA)/Tetraethyleneglycol dimethacrylate (TTEGDMA) for NG1 and DMEAMA /Polyethylene glycol (PEG400DMA) for NG2. The impact of the nanogels on biofilm formation on uropathogenic E. coli (UPEC) and S. mutans was studied to determine its bacteriostatic effect and quantified via CFU counts.

Results: Nanogels were synthesized and characterized (GPC and Zetasizer, NG1 2 nm, NG 2, 100 nm) and the bacteriostatic effects quantified via CFU counts indicate that both there is both a charge and concentration dependence on the outcomes. Conclusion: Tailored Nanogels synthesized via two different mechanisms to control the size and charge on nanoparticles can impart an inhibitory and disruptive growth effect on both gram-negative and gram-positive bacteria. Future work will focus on optimizing the synthetic protocol for nanogels and determining the ideal charge, size and concentration to prevent biofilm formation

Biofilm inhibition

Biofilm disruption

New Approaches to Reactive Oligomers and Prepolymers in Photo-cured Systems

Guangzhe Gao1, Parag K. Shah1, Tao Liu1 and Jeffrey Stansbury1, 2 1Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA; 2Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Objective: The introduction of functional nanogels prepared by a chain-growth free radical polymerization process has demonstrated utility in enhanced performance of dental resins for adhesive and composite applications as well as in hydrogels and controlled-release coatings. Construction of reactive nanogels by a step-growth mechanism offers access to different nanogel network structure options and properties that are examined here. Methods: A series of nanogels were synthesized by diisocyanate addition to a modest stoichiometric excess of multi-thiols with ambient reactions completed in one hour. Residual thiol group concentration within nanogels was assessed by Ellman’s test and this functionality was then used to attach polymerizable methacrylate groups. Nanogel characterization involved gel permeation chromatography, dynamic light scattering, rheology and bulk photopolymerization kinetics. Nanogel formulations as dispersions in TEGDMA over the entire compositional range provided resins that were analyzed for reaction kinetics, polymer thermomechanical properties and polymerization stress. Results: Nanogels with sizes less than 10 nm were designed as room temperature liquids with narrow glass transition temperatures (Tg’s; tandmax = -21 to -10°C). Bulk nanogel viscosities varied widely (<50 to >10,000Pa×s). Residual thiol concentrations, which were subsequently converted to methacrylate linkages, gave 18-24 functional groups/nanogel on average. All four bulk nanogels underwent rapid, near-quantitative conversion during ambient photocure. Depending on nanogel structure, polymeric Tg’s obtained upon bulk nanogel polymerization varied (23-44°C). Photopolymerization of nanogel+TEGDMA mixtures showed dramatically enhanced reaction rate and conversion compared with TEGDMA homopolymer. Flexural strength and modulus were maintained (P<0.05) up to 35wt% nanogel addition while shrinkage stress was reduced by at least 25%. Conclusions: Functional nanogels can be synthesized efficiently through a “clickable” step-growth approach where internal network densities and reactive group concentrations can be well controlled. This study shows potential for beneficial changes in resin reactivity and stress. Funded by IUCRC

Oncogene Regulation of the HNSCC Immune Microenvironment Sean Korpela1, Trista Hinz2, Jacob Calhoun2, Raphael Nemenoff3, Sana Karam4, Ayman Oweida4, Lynn Heasley2,5

Departments of 1Pharmacology, 2Craniofacial Biology, 3Medicine, and 4Radiology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 5Veterans Administration Eastern Colorado Health Care System (VHAECH), Aurora, CO, USA Objectives: The Epidermal Growth Factor Receptor (EGFR) is a key component of a receptor tyrosine kinase (RTK) network that functions as a non-mutated “driver” in head and neck squamous cell carcinoma (HNSCC) and is the target for the FDA-approved agent, cetuximab. Individually, HNSCC patients exhibit wide-ranging extent of response to EGFR inhibitors, even in combination with chemo- or radiotherapy. Herein, we explored the idea that the EGFR-MEK-ERK pathway acts as a repressor of innate immune signaling in HNSCC such that targeted drugs de-repress this response to yield increased paracrine communication with the immune microenvironment which may directly influence the variability observed in therapeutic responses. Methods: Human and murine HNSCC cell lines were treated with gefitinib (an EGFR TKI), AZD8931 (a pan-ERBB inhibitor) or trametinib (a MEK inhibitor) to block oncogenic signaling through the EGFR-MEK-ERK pathway. The murine HNSCC cell line, B4B8 was propagated orthotopically in immunocompetent Balb/c mice and treated for 1 week with AZD8931 (50mg/kg). Results: Transcriptomic analysis with Affymetrix GeneChips of gefitinib treated human HNSCC cells revealed marked induction of innate immune response genes. These findings were validated by qRT-PCR. Induction of CXCL10 protein, was variably induced by gefitinib, AZD8931 and trametinib, and was IKK-NFkB pathway dependent. A 1-week treatment of B4B8 tumors in syngeneic mice with AZD8931 elicited tumor shrinkage and significant increases in immune cell populations. Conclusions: The data support the overall hypothesis that targeted EGFR-MEK-MAPK axis inhibitors de-repress an innate immune signaling cascade that induces a variable spectrum of anti- and pro-tumorigenic chemokines and cytokines that lead to direct participation of recruited immune cells in the therapeutic response. Identifying mechanism-based combinations of agents that induce anti-tumorigenic signals or block pro-tumorigenic signals may ultimately influence the residual disease state and thus extend progression-free and overall survival in HNSSC patients.

Examining PDGFR dimer-specific dynamics using bimolecular fluorescence complementation Madison A. Rogers1,2 and Katherine A. Fantauzzo1 1Department of Craniofacial Biology; 2Graduate Program in Cell Biology, Stem Cells and Development, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in this process in humans and mice. Pdgfra and Pdgfrb mutant mouse models display varying degrees of midline clefting as well as subepidermal blebbing. In addition to homodimeric receptor complexes, we have previously demonstrated that PDGFRa and PDGFRb genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers. Objectives: We hypothesize that the various PDGFR dimers have different patterns of expression and ligand affinities and, further, that each dimer binds to a unique complement of intracellular signaling molecules to generate distinct cellular outputs during midface development. Methods: Here, we highlight ongoing experiments to visualize and purify PDGFR dimers using bimolecular fluorescence complementation (BiFC), which circumvents several limitations with antibody-based approaches. We cloned plasmids expressing C-terminal fusions of each PDGFR with BiFC fragments corresponding to the N-terminal (V1) or C-terminal (V2) regions of the Venus fluorescent protein. Results: By transiently transfecting cells with combinations of PDGFR-V1 and -V2 plasmid pairs and stimulating with various PDGF ligands, we detected PDGFRa homodimer, PDGFRb homodimer and PDGFRa/b heterodimer formation at the cell membrane and in numerous endosomal compartments, to differing extents. Further, we employed BiFC coupled with affinity purification following ligand stimulation to selectively purify and biochemically analyze the various PDGFR dimers, revealing differences in the timing of receptor autophosphorylation, signal molecule binding and the amplitude and duration of downstream intracellular signaling. Conclusions: This approach will likely serve as a powerful tool to quantify the dimer-specific dynamics of PDGFR activation, signal molecule binding and internalization.

Understanding the role of Prdm1a in zebrafish melanocytes in development and disease Brittany Truong1,3, Ritsuko Iwanaga1, David Orlicky2, Craig Ceol4 and Kristin B. Artinger1

1Department of Craniofacial Biology; 2Department of Pathology; 3Human Molecular Genetics and Genomics Graduate Program; University of Colorado Anschutz Medical Campus, Aurora, CO, USA. 4Program in Molecular Medicine, University of Massachusetts, USA. Neural crest cells are highly regulated, transient, multipotent population of cells with the potential to differentiate into various cell types, including melanocytes among other derivatives. Mutations in neural crest development genes can lead to misregulation of neural crest-derived cells, thus promoting defects in development leading to disease. Prdm1a is a master regulator of neural crest specification during embryonic development, and loss of prdm1a results in a partial loss of neural crest cell derivatives, including pigment cells. Objective: Here, we investigate the role of Prdm1a in melanocyte development and show that Prdm1a regulates the melanocyte stem cell population through sox10. Methods/Results: prdm1a zebrafish mutants have increased sox10 expression, but a decrease in expression of differentiated melanocyte markers, dct, mc1r, mitfa, and tyr. Pigment production measured by tyrosinase activity is significantly reduced as well. Conclusion: Our data suggest that Prdm1a is necessary for differentiation of melanocyte stem cells into mature melanocytes. Understanding the development of melanocytes and regulatory genes involved will elucidate the mechanisms driving neural crest derived pigmentation diseases, such as melanoma.

Post-doctoral Fellows

The intersection of biochemistry and developmental biology: Investigating the disease mechanism of a novel EDNRA mutation linked to severe human craniofacial birth defects Stanley M. Kanai1, Amanda Barone Pritchard2, Andre L.P. Tavares1, Nevin A. Lambert3, Elaine H. Zackai2, and David E. Clouthier1

1Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. 2The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 3Department of Pharmacology and Toxicology, Medical College of Georgia-Augusta University, Augusta, GA, USA. Facial morphogenesis requires the establishment of cranial neural crest cell (NCC) identity in the mandibular and maxillary portions of the first pharyngeal arch. This process is dependent on G protein-coupled endothelin receptor type A (EDNRA, ETA) and is highly conserved among gnathostomes, as demonstrated in mouse and zebrafish gain- and loss-of-function (LOF) models and in the human conditions Auriculocondylar Syndrome and Mandibulofacial Dysostosis with Alopecia, in which ETA signaling is altered. Although many ETA dependent gene expression networks have been identified in mutant models, understanding changes in intracellular signaling that precede gene expression has proven more difficult. Objective: To determine whether we can efficiently elucidate how EDNRA disease alleles impact receptor function and intracellular signaling, we adopted experimental approaches used in biochemistry and pharmacology. Method: We used a range of assays based on bioluminescence resonance energy transfer (BRET) that monitor ETA receptor biology in real-time. We specifically tested subcellular localization, G protein activation, and G protein recruitment. Results: To prove the utility of this approach, we examined the consequences of a novel mutation in EDNRA. An individual with a homozygous missense mutation in exon seven of EDNRA, p.Glu381Pro, was identified by whole exome sequencing. The individual died shortly after birth with notable micrognathia/microstomia, an aortic arch anomaly and a ventricular septal defect. Our BRET assays showed the mutation prevents G protein-coupling by ETA. Our findings suggest that p.Glu381Pro is a LOF mutation that renders ETA incapable of recruiting and activating Gaq/Ga11, thus resulting in severe craniofacial and cardiovascular defects. Conclusion: These results illustrate that our approach can quickly define the molecular basis of developmental disorders and thus may impact treatment decisions and options in the future.

A zebrafish model of frontonasal dysplasia

Jennyfer M. Mitchell, Juliana Sucharov, Elliott Brooks, Kuval Ray and James T. Nichols

Department of Craniofacial Biology, School of Dental Medicine University of Colorado Anschutz Medical Campus, Aurora, CO, USA

Mutations in the human transcription factor encoding gene ALX3 are associated with abnormal facial development. ALX3 deficiency is linked to the autosomal recessive disorder frontonasal dysplasia (FND). FND phenotypes include midline skeletal defects like palatal clefting and nasal defects. Yet, knock-out of the mouse Alx3 ortholog does not cause FND phenotypes. The zebrafish palatal skeleton is under similar genetic control to the amniote palatal skeleton, and therefore is a useful model for understanding FND.

Objectives: With this study we want to examine the role that alx3 has in palate development of zebrafish.

Methods and Results: We performed single-cell RNA-sequencing to reveal that the alx gene family is strongly, and specifically, expressed in zebrafish frontonasal Neural Crest Cells (NCCs). NCCs give rise to the anterior neurocranium, the zebrafish palate. CRISPR/Cas9 mutagenesis demonstrates that alx3 function is required for zebrafish palate formation. We generated two independent alx3 mutant alleles that both cause a consistent set of robust craniofacial phenotypes. Zebrafish homozygous alx3 mutants display clefting and shape changes in palatal bones and cartilage. Interestingly, heterozygous individuals also show palatal defects, contrary to the human inheritance for this disorder. Both alx3 homozygous mutants and heterozygotes exhibit discrete patches of reduced chondrogenesis within the palatal skeleton. Because cells are present in these patches, but do not secrete matrix, we hypothesize that alx3 is required for skeletal cell differentiation and not NCC migration or proliferation. Since the alx3 paralogs alx1 and alx4a are also expressed in NCC, we mutagenized the entire gene family. Surprisingly, our current alx1, alx4a and alx4b mutant alleles do not show overt phenotypes in isolation.

Conclusions: That alx3 is the only gene in the alx family required for craniofacial development conflicts with previous reports using morpholino knockdown in zebrafish. Our studies leverage the strengths of the zebrafish system to understand the developmental, genetic and cellular mechanisms underpinning human FND.

Kill-and-release: Using phenolic azobenzenes to remove Streptoccocus mutans biofilms from restorative materials Dylan I. Mori1, Gannon M. Kehe1, Michael J. Schurr2, and Devatha P. Nair1

1Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2Department of Microbiology and Immunology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Secondary caries account for 60-75% of all oral restorative work, as restorations are an ideal location for bacterial colonization. The Nair lab has demonstrated that while the photofluidization of azopolymers is effective at removing several types of biofilms, Streptococcus mutans (S. mutans) biofilms grown under sucrose-rich conditions are exceptionally adhesive and do not respond to induced photofluidization. This study is aimed at designing novel antibacterial azopolymers that successfully disrupt S. mutans biofilms (‘kill’) while maintaining opto-mechanical biofilm removal abilities (‘release’). Objective: To develop a photoresponsive polymeric coating that can inhibit the growth of S. mutans biofilms and detach the biofilm from the coating surface. Methods: Phenolic azobenzene monomer (OH-AAZO) was synthesized, characterized (via NMR), and polymerized onto a resin to mimic a glassy coating on a dental restoration. The OH-AAZO’s cytocompatibility was confirmed via ISO9993 using L929 mouse fibroblast cells. The detachment of an established S. mutans biofilm from the coating surface was initiated via intermittent light exposures (FlashMaxTM dental lamp, 3 sec flashes) and washes in PBS. Biofilms were imaged (Zeiss digital microscope) and quantified via serial dilutions and CFU counts. Results: In the absence of any external stimuli, the presence of a polymerized OH-AAZO coating successfully inhibits S. mutans biofilm formation. The effectiveness of OH-AAZO is greater with higher concentration coatings compared to lower concentration coatings. Interestingly, a combination of phenolic and azobenzene groups results in the optimal inhibitory effect. OH-AAZO has also demonstrated long-term S. mutans inhibition in both sucrose-dependent and -independent conditions.

Digital microscope images of an S. mutans biofilm grown on an

uncoated sample (left) and on a substrate coated with OH-AAZO (right). No live S. mutans was detected on the OH-AAZO

substrate.

Conclusions: Preliminary results indicate that OH-AAZO has potential to prevent S. mutans biofilm formation on the surface of oral restorations (patent pending) while maintaining the ability to detach biofilms from the surface. Future work will involve synthesizing new azobenzene compounds to maximize the detachment forces of dead S. mutans from the coating surface.

Role of chromatin modifiers in craniofacial and cardiac development Rwik Sen1, Sofia A. Pezoa2, Lomeli Carpio Shull1, Laura Hernandez-Lagunas1, Lee A. Niswander2, Tamim H. Shaikh3, and Kristin Bruk Artinger1 1Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, CO, USA; 3Division of Pediatrics, Children’s Hospital Colorado, Aurora, CO, USA Objectives: Cranial and cardiac neural crest cells (NCC) undergo cellular growth, patterning, and differentiation in the branchial arches to form cartilage and bone of the face, and the great arteries, cardiac ganglia, and outflow tract myocardium of the heart respectively. Defects in chromatin modifiers result abnormalities in both NCC populations, including in Kabuki syndrome patients, suggesting a role in regulating normal NCC development. Here, we investigated the role of two histone acetyltransferases Kat2a and Kat2b that modify Histone 3 lysine 9 (H3K9) in craniofacial development, a histone H3K4 methyltransferase Kmt2d and two de-methylases Kdm6a/al which target methylated histone H3K27 in cardiac development. Methods: We knocked out these genes in zebrafish using gene editing tools like CRISPR and TALEN, and employed assays to analyze RNA and protein levels, and cartilage formation. These genes are conserved among zebrafish and humans. Results: Single and double kat2a and kat2b zebrafish mutants have an overall shortening and hypoplasia of cartilage elements and disruption of posterior ceratobranchial cartilages, likely due to reduced domains of expression of cartilage and bone-specific markers, sox9a and col2a1, and runx2a and runx2b, respectively. This reduction is likely a result of an overall reduction of H3K9 acetylation in kat2a mutants. Craniofacial patterning genes including tbx15, tgfb3, tgfbrap1 and nf2b are differentially expressed upon RNA sequencing analysis of sox10:GFP positive neural crest cells in kat2a and kat2b mutants. The analysis suggests altered cellular energetics in mutants. Disruption of kmt2d and kdm6a/al result in both cardiac and craniofacial defects, with reduction of heart rates. Ongoing studies will examine the expression of cardiac progenitor markers such as hand2 and mef2ca as well as examination of transcriptome of human Kabuki patient lymphoblastoid cells where kmt2d and kdm6a/al protein levels are reduced. Conclusions: Together, these studies suggest important roles of epigenetic modifiers in craniofacial and cardiac development.

PRDM histone methyltransferases regulate cranial neural crest development Lomeli C. Shull1, Joseph Gerlach1, Lee Niswander2, Kristin B. Artinger1

1Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Denver | Anschutz Medical Campus, Aurora, CO, USA; 2Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA

Defects in cranial neural crest (cNCC) development are associated with birth defects including cleft lip/palate and chromatin remodelers have been linked to these craniofacial disorders. The mechanisms of how chromatin regulators function in normal cNCC development and their association with congenital diseases remains unknown. Objective. We investigated the role of lysine methyltransferases, prdm3 and prdm16, during craniofacial development. prdm3 and prdm16 methylate lysine residue 9 on histone 3 to repress gene expression. Methods. CRISPR/Cas9 was used to generate prdm3 and prdm16 zebrafish mutants. Phenotypes were analyzed by Alcian blue and Alizarin red staining of the craniofacial skeletons. Transcriptomic (RNA-seq) studies in zebrafish were used to identify mechanisms of prdm3 and prdm16 in cNCCs during craniofacial development. Results. Loss of prdm3 or prdm16 in zebrafish causes subtle craniofacial defects including reduced development of the posterior ceratobrancial cartilages. prdm3-/-;prdm16-/- double mutants do not survive longer than 3 dpf, however, different combinatorial loss of alleles causes more severe craniofacial phenotypes at 4 dpf. RNA-seq revealed global changes in gene expression with loss of prdm3 or prdm16 specifically in pathways associated with cell junctions, tubulin dynamics and neurogenesis. RNA-seq also revealed an upregulation of prdm1a in each mutant suggesting genetic compensation between prdm1a, prdm3 and prdm16. prdm1a knockdown in combinatorial prdm3/prdm16 mutants causes very severe craniofacial defects at 4 dpf. Characterization of prdm1a-/-;prdm3-/-;prdm16-/- triple zebrafish mutants is ongoing, as well as defining the mechanism of PRDM orthologs in mediating proper chondrocyte condensation/adhesion versus activation of cell fate during craniofacial development. Conclusion. These studies reveal important roles for Prdm3 and Prdm16 in cNCCs and formation of the craniofacial skeleton.

ROS modulates ERK signaling after DNA damage Jordon T. Speidel, Angela M. Ohm, Trisiani Affandi and Mary E. Reyland Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Treatment of head and neck cancers typically involves chemotherapy and/or radiotherapy. Because of the non-selective nature of these treatments the adjacent normal, healthy tissue is often also affected. If the salivary gland is injured the secretory acinar cells can undergo apoptosis, resulting in decreased saliva production leading to poor health outcomes. Therefore, the modulation of apoptosis in the healthy non-targeted tissues provides a potential therapeutic target. To this end, our lab has demonstrated that nuclear localization of PKCδ after DNA damage is necessary for apoptosis. Additionally, it is well established that DNA damage induces reactive oxygen species (ROS) generation. However, the interplay between DNA damage induced PKCδ activation and ROS generation has yet to be elucidated. We hypothesize that induction of ROS by DNA Damage is regulated by PKCδ and mediates many of the downstream effects of PKCδ in apoptosis. Objective: 1) Determine the role of PKCδ in the activation of ERK after DNA damage; 2) Determine the role of ROS in ERK activation after DNA damage; 3) Determine if PKCδ and ROS work antagonistically or synergistically. Methods: In vitro studies were performed in rat parotid salivary gland acinar cells (ParC5) between passages 10-35. Immunoblot analyses were performed using 25ug total protein lysates. Cellular ROS was detected using CellROX Green (Life Sciences) by a Gallios flow cytometer (Beckman Coulter). Reverse Phase Protein Array (RPPA) analysis was performed at MD Anderson’s RPPA Core Facility. Results: Etoposide induced apoptosis and DNA damage are pERK dependent. PKCδ overexpression induces ROS dependent DNA damage. We identified the first phase of ERK activation to be ROS dependent while the second phase is PKCδ dependent and may be ROS independent. Conclusions: We have identified that the DNA damage induced biphasic activation of ERK is both ROS and PKCδ dependent.

PKCδ regulates chromatin conformation and DNA double stranded break repair in response to irradiation

Trisiani Affandi, Angela M. Ohm, Annie Pham, and Mary E. Reyland

Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

Treatment for head and neck cancer typically includes irradiation (IR) alone or in combination with chemotherapy. Although IR kills cancer cells, it also damages normal tissues in the oral cavity. IR-induced damage to the salivary gland is mediated by apoptosis, and causes loss of salivary gland function. Our lab has previously shown that protein kinase C delta (PKCδ) regulates apoptosis in response to DNA-damaging agents, and that loss of PKCδ function results in radioprotection of normal, but not tumor cells.

Objective: Understanding the mechanism by which nuclear PKCδ regulates DNA damage-induced apoptosis may allow us to identify new targets for radioprotection of the salivary gland and oral tissues of HNC patients. We hypothesize that PKCδ regulates double stranded break repair, and that inhibition of PKCδ activation increases repair of IR-induced DNA damage through an effect on chromatin accessibility.

Methods: DNA damage was analyzed by γH2AX foci quantification and Comet assay. In vivo fluorescent reporter assay was used to directly quantify homologous recombination (HR) and non-homologous end joining (NHEJ). Chromatin remodeling was investigated by looking at specific histone modifications involved in DNA damage response and by performing micrococcal nuclease (MNase) assay.

Results: Our data demonstrates that IR-induced γH2AX foci are resolved more rapidly in PKCδ-depleted cells, which correlates with a more rapid decrease in DNA damage assayed by Comet. We show that depletion of PKCδ increases DNA repair through both NHEJ and HR. Furthermore, depletion of PKCδ results in increased activation of DNA-PK, and the addition of a DNA-PK inhibitor reverses this increase. We show that in PKCδ-depleted cells, H4K16ac is increased, while expression of nuclear targeted PKCδ dramatically decreases H4K16ac. Furthermore, depletion of PKCδ results in an increase in MNase sensitivity, suggesting a more open DNA conformation.

Conclusions: Our data suggest a novel mechanism for control of apoptosis by PKCδ mediated through regulation of chromatin conformation and DNA repair.

Professional Research Assistants

TKI induced innate immune signaling and response to therapy in EGFR mutant lung cancer Ahmed Al Salman1, Natalia J Gurule1, Caroline McCoach3, Trista K Hinz1, Karen Ryall3, Aik-Choon Tan3, Robert C Doebele3, and Lynn E Heasley1, 3 1Department of Craniofacial Biology, School of Dental Medicine, Anschutz Medical Campus, Aurora, CO, USA; 2MDivision of Medical Oncology, School of Medicine, Anschutz Medical Campus, Aurora, CO, USA; 3Veterans Administration Eastern Colorado Health Care System (VHAECH), Aurora, CO, USA

Oncogenic mutations that occur in the receptor tyrosine kinases (RTKs) EGFR, ALK and ROS1 are present in a significant subset of lung cancer patients, most which present with advanced stage disease. Tyrosine kinase inhibitors (TKIs) that target tumor cells dependent on these oncogenic RTKs yield tumor shrinkage but fail to eliminate 100% of the tumor. Instead, drug tolerant persisters yield residual disease that eventually contributes to the wide range of therapeutic responses observed in patients. Objectives: We seek to understand molecular mechanisms that generate residual disease in response to oncogene specific TKIs. We hypothesize that oncogene targeted TKIs induce rapid transcriptional reprogramming in the lung cancer cell compartment that contributes to a varying degree of immune surveillance by the TME through release of multiple pro- and anti- tumorigenic chemokines and cytokines. Methods/Results: Preliminary data from RNAseq performed on matched pairs (baseline and 2 weeks of TKI treatment) of biopsies from two EGFR mutant lung tumors reveals evidence of marked and variable induction of interferon (IFN) a and g pathways and effector immune cell signatures. Studies with EGFR mutant cell lines treated with a 3rd generation EGFR TKI unveils rapid and dynamic transcriptional reprogramming. Bioinformatic analysis demonstrates induction of the IFNα and IFNγ pathways in response to TKI, representing increases in pro- and anti-tumorigenic chemokines and cytokines that instruct adaptive and innate immune cell types. Pro- tumorigenic chemokines such as IL6 and TGFB2 as well as anti-tumorigenic chemokine CXCL10 are induced to variable magnitudes and with distinct kinetics across our panel of EGFR mutant cell lines. Their expression is sensitive to IKK inhibition, suggesting that the NFkB pathway may be an important transcriptional regulator of EGFR TKI mediated chemokine and cytokine induction. Conclusions: Our data demonstrate that inhibitors of oncogenic EGFR induce an innate immune signaling response in cell line models as well as matched patient biopsies. Studies are ongoing to test the function of TKI mediated changes to the innate immune system on response to therapy.

CU Faces of Hope – A craniofacial and dental biorepository Cristan Carter, David E. Clouthier, Trevor Williams and Kristin B. Artinger Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO USA The craniofacial complex is the most complex region of the human body and thus prone to genetic insult. As a result, there are wide ranges of syndromes that include craniofacial abnormalities. The range of these syndromes encompass over one third of all congenital malformations. Although there have been numerous advances in research focusing on the causes and treatment of these conditions, there is a critical need to determine the genetic and developmental underpinnings of these abnormalities. This lack of knowledge makes it a challenge to translate potential therapeutic and preventative approaches into the clinic. Objective: We aim to develop a large biorepository to support a transformational research program, named CU Faces of Hope, which involves a multidisciplinary team of biomedical researchers, clinicians and data scientists located across multiple departments, divisions, institutes and centers at the University of Colorado who will work together toward a single goal: to compare genetic information from patients with defects in craniofacial formation and unaffected siblings/parents to identify mutations that may be causative for the syndromic phenotype. Methods/Results: The Craniofacial and Dental Biorepository has a goal to enroll up to 2500 individuals with various syndromes associated with craniofacial conditions between the ages of 6 months (inclusive) and 99 years (inclusive), and 1000 controls. Clinical data will be obtained from medical health records, radiographs, family history, and cognitive evaluations that are part of standard clinical encounters. We will collect both DNA or RNA from blood (or cheek swab) and primary teeth, and whole genome sequence will be generated for each participant in our project. Conclusion: The Craniofacial and Dental Biorepository will provide the samples necessary to significantly advance our understanding craniofacial birth defects, enabling the development of novel diagnostics and therapeutic tools for patients affected by craniofacial conditions.

Smart Material Strategies to Combat Contact Lens Microbial Keratitis

Gannon M. Kehe1, Christopher J. Carpenter2, Rooban Nahomi3, Ram Nagaraj3, Michael J. Schurr4, Devatha P. Nair1,5

1Department of Craniofacial Biology, University of Colorado-School of Dental Medicine, Anschutz Medical Campus, Aurora, CO, USA; 2Department of Biology, Metropolitan State University of Denver, Denver, CO, USA; Departments of 3Ophthalmology and 4Immunology and Microbiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA; 5Materials Science and Engineering Program, University of Colorado, Boulder, Boulder, CO, USA Abstract: Microbial keratitis (bacterial infection of the cornea) is a leading cause of global blindness, with over two million new cases annually reported. As contact lens (CL) wear significantly increases the risk of MK via Pseudomonas bacterial infections, non –traditional strategies such as ‘smart’ are of significant importance. Herein, the athermal photofluidization of azobenzenes in response to transient light exposure as a novel approach to disrupt and detach biofilms is studied. Objectives: The aim of this research is to formulate and characterize smart azopolymer coatings that can disrupt and remove biofilms from the surface of contact lenses in response to transient light exposure. Methods: Acrylated azomonomers were synthesized and characterized (NMR). Azomonomers were polymerized (double-bond conversion - FTIR) on the surface of a CL as a coating (concentrations 100, 50. 0.5 mgs/ml coatings). Corneal epithelial cell cytocompatibility of the coated CLs were evaluated and P. aeruginosa biofilms were grown on CLs and exposed to LED Light source (3M EliparTM / FlashmaxTM) initiating the photofluidization of the azo-coatings. Detachment of biofilms was imaged and quantified (CFU counts). Results: The acrylated azomonomers were characterized as CL coatings and concentration dependent ability to detach biofilms characterized. The initial (before light exposure) and final biofilms were quantified. Corneal Epithelial cell compatibility indicates that the coatings are non-toxic.

Different concentrations of the azocoating on CLs are studied and their ability to detach biofilms on exposure to light (FlashmaxTM, 1 W, 1 min exposure) quantified.

Conclussion: Our results indicate the azocoatings on CLs can inhibit the growth and attachment of Pseudomonas aeruginosa biofilms. Future work will focus on optimizing the efficacy of the azocoatings by tailoring the concentration and structure of the azomolecules

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Tuning genetic networks allows development to overcome genetic perturbation

Kuval Ray, Juliana Sucharov, Elliott P. Brooks and James T. Nichols Department of Craniofacial Biology, School of Dental Medicine, University Colorado Anschutz Medical Campus, Aurora, CO, USA In humans, loss-of-function mutations in the transcription factor encoding gene MEF2C are associated with many craniofacial dysmorphologies. But, at least one human has been found that is resilient to the mutation, displaying no craniofacial abnormalities. To understand how the same mutation only sometimes causes a phenotype, known as incomplete penetrance, we compare two zebrafish strains that we selectively bred to have either low- or high-penetrance of mef2ca-associated phenotypes. We pushed penetrance to extreme ends of the spectrum by selective breeding. Objectives: We hypothesized that differences in Notch signaling might underlie differences in dlx5a expression and thus differences in penetrance between our low- and high-penetrance strains. Methods and Results: We created a low-penetrance strain in which homozygous mutants survive and develop into fertile adults. We also created a high-penetrance strain in which the mutation is fully lethal and craniofacial phenotypes even manifest in heterozygotes. These strains allow us to probe the mechanisms of genetic resilience. To understand how genetic networks might differ between strains, we examined the mef2ca transcriptional target dlx5a in low- and high-penetrance lines. We find that low-penetrance fish have relatively high dlx5a expression compared to the high-penetrance strain. To test if dlx5a function affects penetrance, we introduced a dlx5a loss-of-function allele into the low-penetrance strain. We find that removing functional copies of dlx5a dose-dependently increases penetrance of mef2ca-associated phenotypes. Notch signaling is known to act oppositely to mef2ca on dlx5a and repress its expression. To test this, we pharmacologically disabled Notch signaling in a high-penetrance family. This treatment reduces the penetrance of mef2ca-associated phenotypes, rescuing mef2ca mutants and phenocopying our low-penetrance strain. Conclusions: It is possible that human MEF2C mutants might use a similar mechanism to overcome deleterious mutations. Thus, we propose that tuning opposing pathways might be a widespread, natural mechanism leading to genetic resilience to many mutations.

Faculty

Remineralization of White Spot Lesions with Apatite-forming Salt Clifton Carey Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. Category: Faculty Objectives: White spot lesions (WSL) are the earliest signs if enamel demineralization which can develop into cavitations and tooth loss. Currently, no commercial product has been identified that can reliably reverse WSL. One new product for that contains apatite-forming calcium-phosphate salts (Ca/P) may induce WSL remineralization. This study evaluated the remineralization of artificial WSL by use of the SalivaMax™ product that contains apatite-forming calcium-phosphate salts. Methods: Fifteen caries-free human premolars were sliced to remove the cusps and the root to yield tooth discs 5 mm thick. Three groups of 5 discs were joined together with x-ray transparent epoxy to form columns of teeth samples. One side of the column was sanded with 600 grit paper to expose flat fresh enamel surfaces. Each set of samples was attached to a plastic rod for handling and soaked in 1% lactic acid at pH 3.6 for 48 h to form WSL. Two of the sets were suspended in stirred solutions of SalivaMax™ (1 packet in 35 mL dH2O), and one set was suspended in dH2O (control). SalilvaMax™ and H2O were replaced daily. All samples were photographed and scanned with microCT, voxel size 4.4µm, before demineralization, after WSL formation and at 2.5d, 21d and 35d. Mineral density was determined for sound enamel and the lesion body. Results: The table shows that there is a significant increase in remineralization at 21 and 35 days exposure to Ca/P-containing SalivaMax™ compared to controls. Time 0 (%) 2.5 days (%) 21 days (%) 35 days (%) Control (dH2O)

0.0±0.0 9.5±9.8 10.7±11.0 18.2±10.8

Ca/P treated 0.0±0.0 7.2±6.4 56.7±3.7 73.7±5.4 t-test p - 0.5897 0.0000 0.0000 Results are percent remineralization average ± standard deviation, n=10 Ca/P treated and n=5 controls.

Results are percent remineralization average ± standard deviation, n=10 Ca/P treated and n=5 controls. Conclusions: The Ca/P-containing SalivaMax™ is able to cause significant remineralization of artificial WSLs. This method for remineralizing WSL should be tested on naturally occurring WSL.

Photodynamic Nanogels as Hybrid layers in Adhesive Dentistry

Rinku .K. Trivedi1,Gannon M. Kehe1, Dylan.l.Mori1, Dixa Gautam1 and Devatha P. Nair1,2

1Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2Materials Science and Engineering Program, University of Colorado, Boulder, Boulder, CO, USA Achieving optimal resin-dentin bonding is key to long-lasting dental restorations. The 30 ± 11 nm interfibrillar spaces between the type 1-collagen fibrils serve as diffusion channels for resins to infiltrate around the collagen to form the hybrid layer that anchors the resin to the tooth. We hypothesize that by applying a reactive photodynamic azobenzene nanogels (≥ 5 nm) as primer to form the hybrid layer and the composite-adhesive interface, the mechanical properties of the adhesive resin can be tailored for optimal adhesion and function. Objective: The aim of my research is to synthesize photodynamic nanogels and assess the viscosity, mechanical strength and anti-bacterial properties of the nanogel layers in adhesives Methods: Nanogels were synthesized using Solvent Yellow7, 2- Isocyanatoethyl methacrylate, Glycerol and Hexamethylene Disisocyanate(characterized via triple detector GPC). The viscosity of the solvated nanogels was evaluated using (CAP2000+;Brookfield,USA) and while the mechanical strength for two different nanogel concentrations was assessed via microtensile bond tests (n ≥ 3, Mini Bionix II, MN,USA). S. mutans biofilms grown on the nanogel-resin samples imaged (Zeiss digital microscope) and quantified (CFU counts). Results: Nanogels were successfully synthesized (GPC Mw = 12 kDa, Rh =2 nm). The dry strength of the adhesive was enhanced by the nanogels (41 ± 9 MPa and 27 ± 7 MPa for the 5wt% and 40 wt% samples vs 20.4 ± 1MPa for the control) while the presence of the nanogels up to 40 wt% did not influence the anti-bacterial behavior of the BisGMA/HEMA control.

The viscosity of the ethanol solvated photodynamic nanogels can be tailored via exposure to light (3M Elipar TM, 1200 mW/cm2 60 s exposure)

Conclusions Preliminary results indicate that photodynamic nanogels applied as hybrid layers can be tailored for optimal viscosity and mechanical properties at the adhesive-dentin and adhesive-composite interface. Future work will include Microtensile dentin bond strength tests and optimizing the viscosity of the hybrid layer.

Research Associates

Radiation curing technology using nanogels to achieve reduced oxygen inhibition and low photoinitiator migration in coating applications Parag Shah1, Kimberly Childress1 and Jeffrey Stansbury1,2

1Department of Chemical and Biological Engineering, University of Colorado, Boulder, Boulder, CO, USA; 2School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Objectives: Oxygen inhibition has a negative effect on free radical polymerization, resulting in reduced rates of polymerization, especially at the surface in applications that utilize thin polymer films. Commercially, resins are loaded with high amounts of photoinitiators to achieve faster polymerization but this results in high residual initiator content that has the possibility of leaching out and can be hazardous in applications such as coatings for food packaging and in dental materials. Nanogels can be used to create macroinitiators that have a low probability of migration outside the coating as well as reduce oxygen inhibition due to their inherently crosslinked structure. Methods: Nanogels were synthesized from butyl methacrylate and urethane dimethacrylate and post-functionalized with methacrylate groups. A model resin system was made from EOTMPTA (65 wt%), Genomer 3364 (25 wt%) with Irgacure 2100 (1 wt%) as the photoinitiator to which nanogels were added at 10 wt% and 50 wt% to create resin samples with different loading of nanogels. Photopolymerization kinetics were followed using an FTIR under air and nitrogen atmospheres. A photoinitiator, 2-hydroxy-2-methylpropiophenone (HMP) tethered to a nanogel and the efficiency of polymerization using this macroinitiator was studied. Results: All the samples in an atmosphere of air were slower to polymerize than the corresponding samples in an inert nitrogen atmosphere. For the same ambient conditions, an increase in nanogel loading led to a concomitant increase in reaction rate (Figure 1). The HMP initiator was successfully integrated into a nanogel to form a macroinitiator that initiated polymerization upon irradiation by UV light. Increasing amounts of the macroinitiator resulted in faster polymerization. However, the overall rate of polymerization was slower than resins with a comparable amount of free initiator. Conclusions: Nanogels were successfully used to reduce oxygen inhibition during photopolymerization of thin resin layers by essentially forming a barrier to oxygen diffusion from the atmosphere. A macrophotoinitiator made from a nanogel was effective in initiating photopolymerization albeit at slower rates compared to free initiator. Studies are on to evaluate the efficiency of polymerization of the macroinitiator.

Figure 1 – Photopolymerization kinetics of resin with various loading levels of nanogels (0, 10 and 50 wt%) in air and nitrogen.

Irradiation conditions – 405 nm LED @ 10 mW/cm2

Funding: ALTANA