Rena N. D'Souza - US grants

Throughout life, the dental pulp nurtures cells that are responsible for the main tenance, renewal and repair of dentin, a principal component of the tooth. The unique association between pulp and dentin begins with the development of the dental papilla. Odontoblasts maintain this intimacy through normal function, reactions to dental treatment and in repair by synthesizing new dentin. This proposal is based on the fundamental hypothesis that Transforming Growth Factor - Beta (TGF-BETA) influences odontoblast differentiation during tooth formation and in repair. TGF-BETA is implicated as a regulatory factor in the control of cell growth, differentiation and function, and it appears to be of major importance in embryogenesis and in the repair of tissue injury. In addition to promoting angiogenesis, TGF-BETA is a potent chemotactic and mitogenic agent for several types of cells, including platelets, macrophages, lymphocytes and fibroblasts. Many of the regulatory activities of TGF-BETA are a function of its ability to enhance the formation of extracellular matrix. TGF-BETA dramatically influences osteoblasts, cells highly specialized for synthesis of extracellular matrix during bone formation. Cultured osteoblasts have receptors for TGF-BETA, and also produce and secrete the factor. Odontoblasts are cells related in function to osteoblasts; they synthesize dentin extracellular matrix during tooth development and repair. The specific aims of the proposed study are: 1. To determine if TGF-BETA is present in developing rat molars, 2. To describe the relationship between the temporal and spatial patterns of TGF-BETA expression and odontoblast differentiation in developing rat molars, 3. To characterize the presence of TGF-BETA in adult human pulps, and 4. To compare TGF-BETA levels in healthy, adult human pulps with those in inflamed human pulps. Immunolocalization experiments using a polyclonal antibody preparation that recognizes TGF-BETA, will be performed on rat tooth germs in various stages of development. The results will be correlated with morphogenetic and histogenetic events known to occur at each stage of development. Human pulps from healthy and carious teeth will be subjected to immunohistochemistry and Western immunoblot analysis for TBF-BETA. Incident levels of TGF-BETA and its expression within pulp cells will be compared in the two groups. Future experiments will utilize well characterized in vitro approaches to examine the effect of TGF-BETA on the synthesis of extracellular matrix proteins in odontoblasts. The long-range goals of this research are to provide a better understanding of the mechanism of odontoblast differentiation during tooth formation, and to study the origin and nature of precursor odontoblasts in pulp during reparative dentin formation, a basic phenomenon that influences the practice of clinical operative dentistry.

Dentin is produced by specialized matrix producing cells called odontoblasts. Although it is known that the extracellular matrix (ECM) plays an important role in epithelial-mesenchymal interactions during tooth development, little is known about the molecular mechanisms controlling odontoblast terminal differentiation and dentin formation. A long range goal of the proposed research is to study the regulatory autocrine and paracrine factors that influence odontoblast formation during tooth development. Multiple lines of evidence suggest that the transforming growth factor-beta (TGF-beta) gene family controls embryonic processes and terminal differentiation events by modulating ECM. The fundamental hypothesis to be addressed by these studies is that members of the TGF-beta family are intrinsic regulators of tooth development whose coordinated action plays a major role in ECM formation by odontoblasts. The developing tooth provides a powerful, yet unexplored opportunity to study the role of TGF-beta in regulating these events. To test this hypothesis, we will perform correlative and functional studies using in vivo and in vitro developing tooth systems and a combination of immunohistochemical, organ culture, biochemical and molecular techniques. The Specific Aims are: 1. To study the temporal and spatial patterns of expression of TGF-beta during rat molar development in vivo. 2. To determine whether TGF-beta is synthesized and secreted by rat molar organs in culture. 3. To study the effects of TGF-beta on ECM formation by rat odontoblasts in vitro. 4. To determine whether a NF-1 binding site involved in TGF-beta activation of the alpha2(1) collagen promoter, mediates activation of this gene in odontoblasts. Collectively, these studies will provide new data on the biology of the TGFs-beta and will increase our understanding of the mechanisms controlling odontoblast differentiation and function. This information can be applied to studies on reparative dentinogenesis where odontoblasts respond to caries, dental materials and operative procedures by producing a distinct ECM, and to the mechanisms of odontogenic tumorigenesis.

DESCRIPTION: Dentin matrix protein 1 (Dmp1) is a non-collagenous phosphoprotein found in dentin. Dmp1 is synthesized by odontoblasts as well as osteoblasts and cementoblasts, suggesting a function in biomineralization. This R03 application aims to elucidate the function of Dmp1 by generating and characterizing the phenotype of Dmp1 null mice. The hypothesis to be tested is that functional Dmp1 is essential for normal dentin. Aim 1 will introduce a null allele in the Dmp1 gene using homologous recombination in embryonic stem (ES) cells. Mutant cells will then be injected into blastocysts to generate germline chimeras carrying the targeted Dmp1 gene. Aim 2 will characterize neonatal Dmp1-/- mice by comparing them to wildtype and heterozygote animals using morphologic, radiologic, histologic and molecular approaches. The establishment of Dmp1 deficient mice and their basic characterization will provide the basis of future studies addressing the functional role of Dmp1 in dentin formation.

Among the complex network of cytokines that influence odontoblast function during development and repair, TGF-beta1 is unique in its dual abilities to function as a potent immunosuppressant and as an inducer of extracellular matrix (ECM) production. The molecular mechanisms of TGF-beta1 action during dentin formation are poorly understood. To better understand the precise physiologic functions of TGF-beta1 in dentinogenesis we have studied mice with a loss-of-function mutation at the TGF-beta1 locus. TGF-beta 1(-/-) mice die at weaning from widespread inflammation due to the loss of a critical regulator of immune function. Our studies of adult denition in TGF-beta1 (-/-) mice kept alive on dexamethasone show extensive pulp and periapical pathology and a marked attrition of occlusal surfaces. Teeth of TGF-beta1(-/-) mice backcrossed onto immunodeficient backgrounds also showed significant changes in dentin likely caused by defects in mineralization. The impairment in dentinogenesis is thus linked directly to the loss of TGF-beta1 rather than to the secondary effects of inflammation. In the proposed studies, TGF-beta-1(-/-) athymic nude mice will be used to rigorously test the hypothesis that TGF-beta1 is important for odontoblast function during primary and reparative dentin formation. In Aim 1, the structure and integrity of primary dentin will be assessed in TGF-beta1(-/-) nude mice by tetracycline labeling, Fourier transform infrared spectroscopy, ultrastructural and biomechanical approaches. Aim 2 will use cellular and molecular approaches to assess whether the absence of TGF-beta1 affects the expression of alkaline phosphatase and key dentin ECM genes, using in situ hybridization, RT-PCR and immunolocalization analyses. The biosynthetic abilities of odontoblasts will be tested by labeling organ cultures with 3H-proline and 45Ca. In Aim 3, the genetic pathways involving TGF-beta1 will be explored. The expression and functional relationship of genes like Bmp2 and Msx2 that may potentially interact with TGF-beta1 will be assessed in TGF-beta1(-/-) nude and Msx2(-/-) mice whose teeth also appear poorly mineralized. Studies in Aim 4 will assess whether TGF-beta1(-/-) odontoblasts can form reparative dentin in vivo. The effects of exogeneous TGF-beta1 placed at sites of pulp exposure will be compared in TGF-beta1(-/-) vs. TGF-beta1(+/-) nude littermates. The overall goal of these studies is to provide definitive information on TGF-beta1's role in dentin formation. Such insights will contribute to the development of bioactive dental materials to treat and prevent injuries of the dentin-pulp complex.

DESCRIPTION (adapted from the Investigator's abstract): Genetic and molecular studies in humans and mice indicate that Osf2/Cbfal is a critical transcriptional regulator of bone formation. Heterozygous mutants in Osf2/Cbfa1 cause cleidocranial dysplasia (CCD), an inherited disorder in humans and mice characterized by skeletal defects. Mice lacking a functional Cbfa1 gene die at birth and lack bone. CCD also results in supernumerary teeth, defects in tooth form and structure and delayed eruption. Analysis of the expression of Osf2/Cbfal mRNA show that it is restricted to dental mesenchyme during morphogenesis and that epithelial signals regulate Osf2/Cbfa1 expression in mandibular/dental mesenchyme. Cbfal(-/-)_ molar organs show aberrations in size and shape and fail to advance beyond the early bell stage. They lack overt odontoblast differentiation and normal dentin. These data suggest that Osf2/Cbfa! plays a non redundant role in tooth development. The proposed studies will test the central hypothesis that Osf2/Cbfa1 is a key mesenchymal factor with two critical functions in tooth development, one to influence the morphogenetic patterning of dental epithelium, and the other to establish the competence of dental mesenchyme to respond to epithelial signals that subsequently direct the differentiation of odontoblasts. Aim 1 will analyze the expression of Osf2/Cbfa1 mrna and protein and will assess the histologic and molecular changes in Cbfa1(+/-) and Cbfal(-/-) dentition. Aim 2 will study the developmental fate of Cbfa1(-/-) tooth organs when transplanted into wildtype mice and will use epithelial-mesenchymal recombination to assess when and where in tooth morphogenesis and cytodifferentiation, Osf2/Cbfa1 exerts its most critical influence. In Aim 3, the involvement of Osf2/Cbfa1 with signaling molecules that influence tooth morphogenesis and cytodifferentiation will be studied. Aim 4 will directly assess Osf2/Cbfa1's role in the differentiation and function of odontoblasts. DNA transfection and antisense oligonucleotide blocking assays will assess whether Osf2/Cbfa1 can induce/upregulate the expression of dentin ECM genes in established pre-odontoblastic and odontoblastic cell lines. Finally, dominant-negative transgenics that express a transcriptonally inactive form of Osf2/Cbfa1 driven by the osteocalcin promoter will be used to study the role of Osf2/Cbfa1 in maintaining the functional odontoblast phenotype. These data will establish Osf2/Cbfa1's role/s in tooth organogenesis and will provide new insights into the biology of tooth development that are critical to our understanding of the pathogenesis of genetic and acquired diseases that involve dentition.

Among the complex network of cytokines that influence odontoblast function during development and repair, TGF-beta1 is unique in its dual abilities to function as a potent immunosuppressant and as an inducer of extracellular matrix (ECM) production. The molecular mechanisms of TGF-beta1 action during dentin formation are poorly understood. To better understand the precise physiologic functions of TGF-beta1 in dentinogenesis we have studied mice with a loss-of-function mutation at the TGF-beta1 locus. TGF-beta 1(-/-) mice die at weaning from widespread inflammation due to the loss of a critical regulator of immune function. Our studies of adult denition in TGF-beta1 (-/-) mice kept alive on dexamethasone show extensive pulp and periapical pathology and a marked attrition of occlusal surfaces. Teeth of TGF-beta1(-/-) mice backcrossed onto immunodeficient backgrounds also showed significant changes in dentin likely caused by defects in mineralization. The impairment in dentinogenesis is thus linked directly to the loss of TGF-beta1 rather than to the secondary effects of inflammation. In the proposed studies, TGF-beta-1(-/-) athymic nude mice will be used to rigorously test the hypothesis that TGF-beta1 is important for odontoblast function during primary and reparative dentin formation. In Aim 1, the structure and integrity of primary dentin will be assessed in TGF-beta1(-/-) nude mice by tetracycline labeling, Fourier transform infrared spectroscopy, ultrastructural and biomechanical approaches. Aim 2 will use cellular and molecular approaches to assess whether the absence of TGF-beta1 affects the expression of alkaline phosphatase and key dentin ECM genes, using in situ hybridization, RT-PCR and immunolocalization analyses. The biosynthetic abilities of odontoblasts will be tested by labeling organ cultures with 3H-proline and 45Ca. In Aim 3, the genetic pathways involving TGF-beta1 will be explored. The expression and functional relationship of genes like Bmp2 and Msx2 that may potentially interact with TGF-beta1 will be assessed in TGF-beta1(-/-) nude and Msx2(-/-) mice whose teeth also appear poorly mineralized. Studies in Aim 4 will assess whether TGF-beta1(-/-) odontoblasts can form reparative dentin in vivo. The effects of exogeneous TGF-beta1 placed at sites of pulp exposure will be compared in TGF-beta1(-/-) vs. TGF-beta1(+/-) nude littermates. The overall goal of these studies is to provide definitive information on TGF-beta1's role in dentin formation. Such insights will contribute to the development of bioactive dental materials to treat and prevent injuries of the dentin-pulp complex.

DESCRIPTION (provided by applicant): This is a new NRSA Institutional Research Training Grant (T32) application that is directly responsive to the recommendations of the Blue Ribbon Panel and the training initiatives of the NIDCR. The program is named the University of Texas Training Program in CraniofaciaI-Oral Biology Research that is Comprehensive and Integrated with the Health Science Center at Houston (UT-TORCH). UT-TORCH builds on our successful predoctoral dental student research program that has been supported by an NIDCR Short-Term Research Training Grant for Health Professional Students (T35) since 1994. Its primary mission is to identify, recruit, train, and retain dental faculty who can become independent investigators with the life-long learning and collaborative skills needed to address emerging opportunities in craniofacial and oral research. UT-TORCH draws from the enriched resources of a highly diverse and research-intensive academic health science center environment. It is comprehensive, integrative, and interdisciplinary in every respect and brings together a core of 52 outstanding and highly committed faculty research mentors from existing programs within the Dental Branch, Graduate School of Biomedical Sciences, Medical School, School of Public Health, and the School of Health Information Sciences at UT-Houston as well as from MD Anderson Cancer Center, Baylor College of Medicine, and Rice University. Research training is offered through a continuum of five tracks that span various stages of career development. Track I - A short-term program of summer research experiences for predoctoral dental students; Track II - An individual PhD in Biomedical Sciences program; Track III - An integrated DDS/PhD degree program; Track IV- Postdoctoral training for recent DDS graduates that leads to a PhD in Biomedical Sciences and MS degrees in either Clinical Research, Health Informatics or Public Health. Non-degree fellowship training for PhD graduates is also offered; Track V: Short-term research training for dental faculty. Trainees can select projects from three research focus areas (i) Biomimetics: Development, Genetics, and Bioengineering (ii) Molecular Pathology: Immunology, Infectious Diseases, and Cancer (iii) Patient-oriented Research and Health Informatics. A highly innovative and integrative core and discipline-specific curriculum with journal clubs, seminars, and research symposia are proposed to facilitate an exchange of information among students in various disciplines and to allow each trainee to acquire a broad mix of critical thinking skills. Students and faculty at the University of Mississippi Dental School will have access to training opportunities provided by UT-TORCH through a formal partnership. The two institutions will also collaborate on the recruitment of minority applicants into UT-TORCH. Activities with T32 trainees at the two other Texas Dental Schools (UTHSC-San Antonio and Baylor College of Dentistry-Dallas) are planned. Support is requested for a total of 23 highly qualified trainees (8 full-time positions) and two DDS/PhD candidates for Year 1 of the award. UT-TORCH will be administered by a cohesive and experienced group of faculty: The program director, Dr. Rena D'Souza and co-director, Dr. George Stancel will work closely with the Steering Team composed of the five track coordinators. An internal advisory panel of faculty with extensive experience in the proposed tracks of training as well as an External Review Board will provide oversight for UT-TORCH. Graduate trainees of UT-TORCH will be able to (a) Interpret new scientific information from an insightful perspective and teach with scholarly credibility (b) Provide knowledgeable dialog with student and faculty clinicians (c) Lead cutting edge craniofacial, dental, and oral biology research in sustained NIH and other extramurally-supported research programs (d) Publish reports in peer-reviewed journals, and (e) Pass muster with UT-Houston promotion and tenure committees. Strong institutional support exists for this new Dental Branch endeavor that is designed to train dental academicians for craniofacial and oral biology research-oriented careers.

[unreadable] DESCRIPTION (provided by applicant): In this competing renewal, we propose to continue our research on the role of Runx2 in tooth development. Data from our work in the previous award period indicated key roles for Runx2 in directing the fate of Dental epithelium during morphogenesis and in controlling the onset of odontoblast differentiation. Our studies point to the critical need to learn how Runx2 activities are precisely regulated during tooth morphogenesis and cell differentiation and whether its role in these processes is modulated through interactions with other molecules. The nuclear protein Twist-1 is of particular interest as a regulatory protein partner for Runx2. Our rationale for studying if Runx2, a cell differentiation factor, interacts with Twist-1, a cell survival factor, is derived from studies in our and other laboratories that suggest that these interactions between Runx2 and Twist-1 occur at the protein level. Our experiments will directly test the hypothesis that Runx2's key functions in odontoblast differentiation are regulated by Twist-1 at the level of protein- protein interactions that are functionally antagonistic in nature. The selective and transient blocking of Runx2 function by Twist-1 provides a means to restrain odontoblast differentiation until morphogenesis is complete. We further propose that interactions between Runx2 and Twist-1 are not mutually antagonistic as Twist-1 can mediate cell proliferation during morphogenesis via FGF-mediated epithelial mesenchymal signaling. Hence, the presence of supernumerary teeth in human CCD and accessory buds in Runx2(-/-) mice likely reflect increased activity of Twist-1 rather than a direct effect of decreased levels of Runx2. Aim 1 will determine if the patterns of Runx2 and Twist-1 (mRNA and protein) expression are compatible with their proposed partnership during tooth development and will correlate these patterns with the expression of molecular markers of tooth morphogenesis and odontoblast differentiation. Aim 2 will assess with mouse genetic loss-of-function and gain-of-function approaches whether alterations in Twist-1 expression affects tooth morphogenesis and odontoblast differentiation. Aim 3 will study the molecular basis of Runx2 - Twist-1 protein interactions in Dental mesenchyme and the functional consequences of this interaction on Runx2 functions in odontoblast differentiation, and Aim 4 will test whether the bHLH domain of Twist-1 can mediate tooth morphogenesis via FGF-signaling that is independent of its interactions with Runx2. These studies will increase our understanding of how Runx2 achieves its selective functions in tooth development through its partnership with Twist-1. Importantly, they will explain how supernumerary teeth form and if odontoblast differentiation is determined by the release of an inhibition. Such data will also provide a framework for understanding the pathogenesis of Cleidocranial Dysplasia and Saethre-Chotzen Syndrome, 2 human genetic disorders that threaten dentition. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a new NRSA Institutional Research Training Grant (T32) application for Baylor's Scientific Training Program for Dental Academic Research Scholars (B-STARS). The application is in direct response to the revised training initiatives of the NIDCR and requests: (i) Continued support for a highly successful short-term program for the training of dental research scholars. The program has been intensified to increase the commitment of trainees toward academic research careers by early identification and continued engagement in research throughout dental school;(ii) New funding for an integrated dual-degree DDS/PhD program. This program was initiated through institutional funds and a successful series of NIDCR Individual Predoctoral Dentist Scientist Fellowships (F30 awards);and (iii) Support for a well-crafted series of short- and long-term postdoctoral training experiences for faculty and fellows. These programs will provide the skills needed to address new opportunities in craniofacial research. B-STARS draw from the expanding scientific, intellectual and physical resources at Baylor College of Dentistry (BCD). A full spectrum of research training opportunities is enhanced by new institutional initiatives and NIDCR R24/U24 awards for the enhancement of research infrastructure at BCD. Strategic alliances with UT Southwestern Medical Center (UTSW), Texas A&M's Institute of Biosciences and Technology (IBT), Rice University, and the hiring of new BCD researchers have led to the selection of a team of 52 outstanding faculty mentors. This group offers interdisciplinary training in the following focus research areas: Genes and Development;Matrix Biology and Tissue Engineering;Neurosciences and Molecular Pathology;and Clinical Research. BCD's participation in an NIH Roadmap K12 and CTSA awards to UTSW offers multidisciplinary training to B-STARS Clinical Research Scholars. A highly innovative and integrative core and discipline-specific curriculum with journal clubs, seminars and research symposia will facilitate an exchange of information among students and faculty in various disciplines and allow each trainee to acquire extensive critical thinking skills. Graduate trainees of B-STARS will be able to (a) interpret new scientific information from an insightful perspective and teach with scholarly credibility;(b) engage in knowledgeable dialog with student and faculty clinicians;(c) lead cutting-edge craniofacial, dental and oral biology research in NIH and other extramural research programs;(d) publish reports in peer reviewed journals;and (e) sustain successful careers in academic dentistry. B-STARS benefits from unequivocal institutional support and dynamic leadership. A program evaluation plan will critically assess the success of the training program on the development of future dental academicians. The leadership team of B-STARS is committed to work with the NIDCR to improved performance and outcomes that will develop the best practices for the training, education and career development of dentist-scientists.

DESCRIPTION (provided by applicant): This proposed project, entitled "Baylor's Program in Bioengineering Sciences and Translational Research (B-BEST)", will provide support for the hiring and development of two new faculty with expertise in Bioengineering Sciences and Translational Research. These first-time faculty will add expertise in the area of Tissue Regeneration needed to bridge the gap between basic and clinical research. Their skills will complement and add to existing strengths of BCD's Department of Biomedical Sciences (BMS), whose faculty engage in clinically-relevant, translational research that is interdisciplinary in nature. The P30-supported junior faculty will develop their own studies in tissue regeneration by increasing their knowledge of the underlying biological mechanisms leading to the normal and abnormal development/structure/function of craniofacial tissues. By drawing from the strong infrastructure for mentorship within BMS, the P-30-supported faculty will be cross-appointed in either the Depts. of Periodontics or Oral and Maxillofacial Surgery, and the Department of Bioengineering/Biomedical Engineering Graduate Program at UT-Arlington to foster multi-disciplinary bioengineering applications in the area of mineralized and soft tissue regeneration. The network of established basic, translational and clinical researchers in BMS and BCD will assist P-30 faculty in driving new technologies for biomimetic and/or nanostructured scaffolds, use of stem cells, and the delivery of bioactive factors/drugs. Such approaches will provide the framework for future independent research project grants that will further the mission of BMS/BCD. The long-term aims of this project are the following: 1) Strengthen research capacity through recruitment and retention of additional outstanding faculty, improved research resources, and expanded research collaborations at BCD;2) Provide additional focus, directions, and successful outcomes to the research programs in BMS/BCD in areas spanning the basic, translational, and clinical research spectrum.;3) Increase opportunities to collaborate with existing biomedical engineering research groups at UTA, SMU, and UTSW in the area of dental and craniofacial health;and 4) Provide training opportunities for predoctoral dental trainees interested in bioengineering and translational research. PUBLIC HEALTH RELEVANCE: This research grant will provide funding for the hiring of two first-time faculty members with expertise in tissue regeneration in the Dept. of Biomedical Sciences at Baylor College of Dentistry. These faculty will work with the present faculty who perform basic and translational research on craniofacial tissues. Advances in tissue regeneration research will bring about treatments for various kinds of craniofacial maladies that afflict many in the population.

DESCRIPTION (provided by applicant): The development of dentition involves a complex series of epithelial-mesenchymal signaling interactions. It is not surprising that such a process is prone to disturbances which then manifest as congenital tooth agenesis in up to 10% of the population and impose significant functional, emotional and financial burdens on patients. Mutations in the paired domain transcription factor, PAX9, contribute to human tooth agenesis. Genetic and molecular studies in mice also indicate a key role for Pax9 in tooth development. Similar to other Pax family members that act in a highly tissue-specific manner, Pax9 is likely to mediate its tooth-specific functions through its interactions with other proteins. Multiple studies point to an important partnership between Pax9 and the Msx1 homeprotein in regulating gene expression in dental mesenchyme. Our long-term goal is to understand how transcription factors like Pax9 mediate key signaling actions in tooth development and how aberrations in Pax9 functions lead to tooth agenesis. The objective of this proposal, which is the next step to accomplish this goal, is to study how Pax9 achieves its selective functions in dental mesenchyme. Based on our preliminary data, we hypothesize that Pax9 maintains the inductive potential of dental mesenchyme through its transactivation functions and protein interactions within a positive feedback loop involving Msx1, Bmp4, and other partner genes. We will achieve our goals by testing the central hypothesis in three specific aims. Aim 1 will define the molecular basis for the relationship between Pax9, Msx1 and Bmp4. Studies in Aim2 will assess if other candidate genes that are coordinately expressed with Pax9 are involved in the Pax9- Msx1 signaling pathway with Bmp4. Aim3 will use a human genetics approach to identify additional genes that are responsible for human tooth agenesis and may partner with Pax9 during tooth development. The proposed work is innovative as it capitalizes on a new means to uncover the molecular functions of Pax9 by use of biochemical and human genetics approaches uniquely available in our laboratory. The work will positively impact the field of tooth development by deepening our understanding of the network of interactions that coordinate signaling. Such knowledge may lead to innovative treatments for patients with tooth agenesis including the possibility of bioengineering new teeth. PUBLIC HEALTH RELEVANCE: Congenitally missing teeth are a consequence of gene mutations which interrupt the process of normal tooth development. Only a few of these genes have been identified. We propose to discover additional genes and show how several of these genes interact in normal tooth development and what disturbs their interaction in patients with missing teeth.

DESCRIPTION (provided by applicant): This proposal develops a nanostructured mimic of extracellular matrix prepared from a self-assembling peptide we call Multidomain Peptides or MDPs. The MDPs self-assemble into nanofibers that can be triggered to form a hydrogel. Because the peptides are easy to prepare and have a well defined design criteria many variations on the MDP architecture can be prepared which allow us to tailor 1) the conditions under which the fibers self-assemble (including conditions compatible with cell culture and in vivo applications), 2) the mechanical properties critical for handling and injectability, 3) the presentation of chemical information for cells, and 4) controlled biodegradation. This exceptional combination of properties will be developed here to create biomimetic scaffolds for the entrapment and delivery of cells, proteins and small molecule drugs. Our work will culminate with an in vivo application which uses this nanostructured hydrogel for dental regeneration. It is expected that the nanofibers will prove to be suitable as an injectable, localized, simultaneous delivery method for cells, proteins and small molecule drugs which will actively assist in directing cellular activity. Finally, after the MDP matrix has played its role it will degrade leaving behind only regenerated tissue. Such a matrix will play a critical role in future tissue engineering strategies (including, but not limited to, dental regeneration) which require a smart scaffolding material to organize the constituent cells and drugs until the body's own regenerative ability can take over. Our proposal is organized into four aims. Aim 1 will determine the design, flexibility and methods used to prepare MDP nanofibers. Aim 2 will optimize MDP nanofibers for three dimensional cell entrapment, cell delivery and cell mediated biodegradation. Aim 3 will develop a series of nanofibrous gels which will deliver growth factors and other small molecules localized in time and space. Aim 4 will test the above developed nanofibrous hydrogels ability to promote dental regeneration in vivo. This highly translational research will apply novel tissue engineering and nanotechnology concepts to the design of multidomain peptide hydrogels intended as an all-purpose scaffold for the regeneration tissue (tested here on the regeneration of the dentin-pulp complex). By combining expertise in chemistry, materials sciences, nanotechnology, cell biology and clinical dentistry we will generate data that will provide the framework for further studies testing these hydrogels in human clinical trials. PUBLIC HEALTH RELEVANCE: One of the most exciting areas of medical research is tissue engineering which promises to supplement our own regenerative ability to allow us to replace or re-grow damaged or diseased tissues and organs. Researchers use appropriate cell lines, growth factors and drugs which are organized by a synthetic matrix. In this proposal we describe an interdisciplinary approach to the design and optimization of a nanostructured matrix made from synthetic proteins. This matrix will entrap cells, proteins and drugs and allow them to be delivered together by syringe to the site necessary.

? DESCRIPTION (provided by applicant): Tooth agenesis, the congenital absence of one or more permanent teeth, is the most common inherited disorder in humans, affecting up to 10% of the population, even when third molars are excluded. The condition occurs as an isolated anomaly or as part of dozens of syndromes and imposes significant functional, emotional and financial burdens on patients and their families. Since restoring esthetics and function for patients with tooth agenesis is complex and expensive, new therapies are warranted. Thus a central challenge relates to the critical need to translate the fruits of several decades of basic and clinical research on tooth development and tooth agenesis into tangible therapies that can benefit these patients. The long-range goals of this research are to develop replacement therapies that are safe and effective in correcting non-syndromic tooth agenesis in humans. We propose translational approaches that will test the hypotheses that the timely administration of select recombinant proteins or small molecule activators/inhibitors of major developmental signaling pathways will be able to replace missing or mutated gene products needed for tooth development. Furthermore, replacement proteins do not have to be identical with the tooth agenesis-causing protein as downstream targets/effectors of the mutated gene can also achieve the desired effect, either singly or in combination with other downstream effectors. Our approaches are inspired by the recent success in using recombinant Ectodysplasin A (rEDA) protein to treat X-linked hypohidrotic ectodermal dyslpasia (ED) in mice and dogs, results that led to clinical trials that are now assessing the safety and efficacy of rEDA therapy in humans with ED. Three aims are proposed as follows: (1) To explore the expanded use of EDA therapeutics for non-syndromic tooth agenesis. (2) To develop and evaluate novel Wnt therapeutics for non-syndromic tooth agenesis. (3) To identify new tooth agenesis genes through the use of human genetics approaches. The project addresses an important problem of high clinical relevance for which there are no cures. Data from these translational studies will provide the proof of principles needed to test whether such therapies can be used to restore tooth development in humans affected by non-syndromic tooth agenesis. In the broadest sense, this research will impact concepts and technologies that drive the field of therapies for other single gene disorders affecting the craniofacial complex and other systems.

PROJECT SUMMARY The development of dentition is a highly complex process that involves a series of reciprocal epithelial- mesenchyme interactions that are regulated by five conserved signaling pathways, namely Bmp, Fgf, Wnt, Eda and Shh. That such a precise process is often perturbed is not surprising. Indeed, tooth agenesis is one of the most commonly inherited human disorders that affects up to 10% of the population and imposes significant burdens on patients and their families. Mutations in PAX9, a paired-domain transcription factor that is specifically expressed in dental mesenchyme, cause human tooth agenesis. The deletion of Pax9 in mice leads to tooth arrest at the bud stage, thus underscoring its key inductive role within dental mesenchyme. Better understanding the molecular actions of Pax9 in dental mesenchyme during the induction phase of tooth morphogenesis offers hope for the development of tangible therapies that can benefit patients with tooth agenesis. Our microarray analyses of Pax9-/- tooth organs show that Wnt signaling genes are most markedly altered along with the Bmps, Fgfs, Shh and Eda-related genes. The results of our human genetic analyses and data from other groups confirm that mutations in WNT10A are responsible for the majority of cases of human tooth agenesis. Significantly, our preliminary experiments suggest that Wnt agonists, when administered to pregnant Pax9+/- mothers, are able to rescue the mutant phenotype of cleft palate and tooth arrest. Despite these advances there is little understood about the precise molecular relationship of Pax9 with the Wnt signaling pathway in dental mesenchyme and how such basic science knowledge can be translated into new advances for the treatment of human tooth agenesis. Taken together, our data provide the framework for studies that will systematically test the hypothesis that Pax9 is a key modulator of signaling events in dental mesenchyme during early tooth morphogenesis through its regulation of genes in the Wnt pathway. The restoration of Wnt signaling in Pax9 and Wnt10a mutant dental mesenchyme is hence likely to normalize tooth morphogenesis. Aim 1 studies will use multipronged approaches to provide new data on the molecular relationship of Pax9 with genes that regulate Wnt signaling activities in dental mesenchyme during early morphogenesis since this relationship is not as well studied as that with the Bmp and Fgf pathways. Aim 2 will test how human tooth agenesis-causing mutations in Pax9 and Wnt10A affect the functional relationship of these genes to result in an arrest in tooth development. Aim 3 will confirm the upstream relationship of Pax9 by assessing whether novel Wnt-based therapeutics when administered in-vivo, can correct the Pax9-/- tooth agenesis phenotype through a restoration of Wnt function. Data from these basic science and translational studies will advance our understanding about the signaling molecules in dental mesenchyme and will provide the framework for developing and testing non-invasive therapies to restore tooth development in humans affected by non-syndromic tooth agenesis, an important problem of high clinical relevance and for which there are no cures.

PROJECT SUMMARY The development of dentition is a highly complex process that involves a series of reciprocal epithelial- mesenchyme interactions that are regulated by five conserved signaling pathways, namely Bmp, Fgf, Wnt, Eda and Shh. That such a precise process is often perturbed is not surprising. Indeed, tooth agenesis is one of the most commonly inherited human disorders that affects up to 10% of the population and imposes significant burdens on patients and their families. Mutations in PAX9, a paired-domain transcription factor that is specifically expressed in dental mesenchyme, cause human tooth agenesis. The deletion of Pax9 in mice leads to tooth arrest at the bud stage, thus underscoring its key inductive role within dental mesenchyme. Better understanding the molecular actions of Pax9 in dental mesenchyme during the induction phase of tooth morphogenesis offers hope for the development of tangible therapies that can benefit patients with tooth agenesis. Our microarray analyses of Pax9-/- tooth organs show that Wnt signaling genes are most markedly altered along with the Bmps, Fgfs, Shh and Eda-related genes. The results of our human genetic analyses and data from other groups confirm that mutations in WNT10A are responsible for the majority of cases of human tooth agenesis. Significantly, our preliminary experiments suggest that Wnt agonists, when administered to pregnant Pax9+/- mothers, are able to rescue the mutant phenotype of cleft palate and tooth arrest. Despite these advances there is little understood about the precise molecular relationship of Pax9 with the Wnt signaling pathway in dental mesenchyme and how such basic science knowledge can be translated into new advances for the treatment of human tooth agenesis. Taken together, our data provide the framework for studies that will systematically test the hypothesis that Pax9 is a key modulator of signaling events in dental mesenchyme during early tooth morphogenesis through its regulation of genes in the Wnt pathway. The restoration of Wnt signaling in Pax9 and Wnt10a mutant dental mesenchyme is hence likely to normalize tooth morphogenesis. Aim 1 studies will use multipronged approaches to provide new data on the molecular relationship of Pax9 with genes that regulate Wnt signaling activities in dental mesenchyme during early morphogenesis since this relationship is not as well studied as that with the Bmp and Fgf pathways. Aim 2 will test how human tooth agenesis-causing mutations in Pax9 and Wnt10A affect the functional relationship of these genes to result in an arrest in tooth development. Aim 3 will confirm the upstream relationship of Pax9 by assessing whether novel Wnt-based therapeutics when administered in-vivo, can correct the Pax9-/- tooth agenesis phenotype through a restoration of Wnt function. Data from these basic science and translational studies will advance our understanding about the signaling molecules in dental mesenchyme and will provide the framework for developing and testing non-invasive therapies to restore tooth development in humans affected by non-syndromic tooth agenesis, an important problem of high clinical relevance and for which there are no cures.