NIDCR - National Institute of Dental and Craniofacial Research Grants

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary/Abstract The proposed R13 grant seeks support for a conference entitled Harnessing Knowledge from the Social and Behavioral Sciences to Develop Sociobehavioral Interventions to Reduce Children’s Oral Health Inequities to be held at the University of Washington School of Dentistry in Seattle, WA in Spring 2026. The objective of the proposed 3-day conference is to gather a community of scholars, connecting social and behavioral scientists with applied dental researchers. We will convene 24 researchers (1 applied dental researcher paired with either a social or behavioral scientist from outside of dentistry). Each pair will engage in preparatory work on their topic before the conference, with the goals of (a) presenting a critical paper on the current state of science on their topic and proposed immediate next steps relevant to sociobehavioral interventions needed to move the field forward and (b) integrating relevant findings from the conference into a final paper. The papers presented during the conference will be part of a special issue on sociobehavioral interventions, co-edited by the MPIs (Dr. Donald Chi and Dr. Cameron Randall) and published in the International Journal of Paediatric Dentistry. Using a model that drives interdisciplinary collaborations at the Center for Advanced Study in the Behavioral Sciences (CASBS) at Stanford University, the R13 grant will bring together social and behavioral scientists and applied researchers in dentistry to advance promising interventions that address the key social and behavioral causes of children’s oral health inequities, reduce unnecessary pain and suffering, and improve the oral health and quality of life of socioeconomically vulnerable children. Children’s oral health inequities will be used as a case study to generate a more broadly generalizable model on how to systematically harness knowledge from the social and behavioral sciences to address health inequities through sociobehavioral interventions.

NIDCR - National Institute of Dental and Craniofacial Research

SUMMARY Temporomandibular joint (TMJ) osteoarthritis (OA) is characterized by degeneration of the condylar cartilage. No effective therapies exist for restoring the damaged cartilage to accommodate pediatric patients' craniomaxillofacial growth. Regenerative therapies are needed to repair TMJ cartilage. Microfracture is a technique where perforations are created through the subchondral bone to allow for bone marrow mesenchymal stem cell (MSC) infiltration to create fibrocartilaginous repair tissue. However, MSCs are difficult to localize to the damaged area without a scaffold, and their phenotype is prone to calcification. Matrix-autologous chondrocyte implantation (MACI) is the most effective knee repair approach, where autologous knee chondrocytes are seeded on a collagen scaffold and then implanted in the defect. However, MACI is costly, requires two invasive procedures, and may not be applicable to the narrow TMJ for chondrocyte extraction. Additionally, the collagenous scaffolds that are used for knee repairs do not recapitulate the native TMJ extracellular matrix environment. The objectives of this F32 training grant are to develop regenerative approaches to heal damaged TMJ cartilage for pediatric patients and train me in these approaches to complement my tissue engineering background. Using fibro-elastic cartilage of porcine meniscus, we will use our Meniscal Decellularized (MEND) scaffold to help localize and inform progenitor cells, such as ear cartilage progenitor cells (eCPCs) or MSCs. We hypothesize that regenerative therapy adapting microfracture and MACI/matrix-induced chondrogenesis can be used to repair the damaged TMJ condylar cartilage. We will first compare the in vitro chondrogenic potential and phenotypic stability of eCPCs and MSCs in MEND. Outcomes will include biochemical assays, mechanical testing, immunohistochemistry, histology, and gene expression. Then, to assess in vivo phenotypic stability of the constructs, we will subcutaneously implant cell-seeded MEND in immunocompromised mice and analyze outcomes via biochemical assays, immunohistochemistry, histology, and gene expression. Next, we will compare the repair of adapted microfracture (simulated by an MSC injection) and MACI (simulated by empty or eCPC MEND) for porcine and human TMJ condylar cartilage regeneration in vivo using the semi-orthotopic mouse model and analyze outcomes via biochemical assays, immunohistochemistry, histology, and gene expression. These studies will uncover whether microfracture regenerates TMJ condylar cartilage and if a scaffold, potentially cellular, is needed to improve condylar regeneration. Results will inform development of regenerative therapies for degenerated TMJ condylar cartilage and produce preliminary data for a K99 application to support my training as an independent researcher in the TMJ oral and craniofacial research field.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY The lower jaw evolved as a structure composed of many bones the largest of which, the dentary, persists as a single lower jawbone in modern mammals, and is referred to as the mandible in humans. Mandibular disorders, often resulting in small jaws, are among the most common human birth defects. These disorders can dramatically affect quality of life, and are often associated, or compound problems with airway obstruction, speech, and feeding. During embryogenesis, development of the mandible is preceded by and associated with a tubular cartilage rod called Meckel's cartilage (MC) and anomalies of MC have been associated with mandibular disorders. Development of MC in modern mammals is complex: the anterior part contributes to the formation of the mandibular symphysis, its posterior part forms cartilages that mineralize endochondrally to form two middle ear bones, and the posterior half of the middle region forms ligaments. Less is known about the anterior half of the middle region of MC, which is transient, present during a small window of embryonic development before it disappears. Established assessments describe MC as a template for the formation of the mandible, but evidence of this is lacking and little is known of the relationship between the midportion of MC and mandibular mineralization, size, and shape or the processes of MC growth in length, perichondrial ossification, and disappearance of MC. We present data demonstrating that MC does not serve as a template in the way cartilaginous models function in endochondral ossification and hypothesize a new role for the mid portion of MC in determining mandibular length, mineralization of the perichondrium, mineralization of the mandible, and its disappearance. Because our findings challenge the traditional role of MC, we have designed this project to validate the developmental events that take place as the midportion of MC disappears and the mandible forms through a detailed analysis of four processes: 1) initiation and growth in length of MC; 2) mineralization of MC perichondrium; 3) mineralization of the mandible; and 4) disappearance of MC. Our approach is based on knowledge we have gained through preliminary investigations of these processes occurring at different times along the length of MC, and spanning the buccal to lingual aspects of the interior of MC. We couple 3D imaging with tissue and cellular analyses of embryonic mutant Sox9flox/flox;Col2a1-CreERT, and Sox9flox/flox mice to precisely define the changing cellular dynamics of the lower jaw in developmental time and anatomical space. These data are used in turn to inform our RNA-seq analyses of the developing MC and mandible directed at recovering the underlying transcriptome by differential gene expression, pathway, and network analyses. We plan cell lineage tracing experiments to determine the fate of cells from the intermediate region of MC, those that initiate MC perichondrial mineralization, and mandible mineralization. Our integrative approach is designed to bring new understanding to lower jaw development and open novel research areas to advance strategies for bone repair, regeneration, and prevention in mandibular disease.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary: The dental pulp is the vital microenvironment in the tooth, harboring blood vessels and nerves, not to mention odontoblasts that interface with the dentinal tubules. Trauma or bacterial infection may inflame the dental pulp, creating extreme pain. Extirpating the inflamed pulp (and potentially replacing it with inert materials) ameliorates the pain, but the procedure leaves a devitalized tooth. An alternative is possible in juvenile patients, called over-instrumentation (OI). During OI, the pulpal chamber is exposed to the peripheral circulation post-pulpectomy. As long as the apical papilla is intact, some tissue regeneration takes place in the pulpal canal subsequently — although the disorganized tissue does not mimic native soft tissue. In adults in particular, OI results in non-functional pulpal ossification. Another concern in endodontic procedures is occurrence/recurrence of colonization by oral bacteria. Such infections may prolong and exacerbate pulpal inflammation. A material- based formulation is proposed that can (a) promote vascularized soft-tissue regeneration in the pulp, while (b) resisting bacterial infection. Our strategy rests on self-assembling peptide hydrogels — a class of supramolecular materials that can be injected in vivo while keeping their gel-like properties. The materials consist of canonical amino acids and are biocompatible. Such materials need to provide both mechanical support and biological cues for tissue ingrowth. Somewhat counter-intuitively, a self-assembling peptide hydrogel, without added growth factors or exogenous cells, demonstrated formation of vascularized soft-tissue in a canine pulpectomy model in 28 days. In a separate study, a different cationic amphiphilic hydrogel belonging to the same platform, showed efficacy in inhibiting bacterial growth via membrane permeabilization. In this proposal, a combinatorial treatment modality will be tested for its effectiveness in achieving the dual goals described above. A mechanistic puzzle that these projects would help solve is the lineage/source of infiltrating cells and evolution of the cellular milieu in the pulpal canal after pulpectomy and implantation of soft biomimetic hydrogels. Characterization of the long- term maturation of the vascularized soft tissue promoted by such hydrogels is another target. The multi- disciplinary project proposed in this Bioengineering Research Grant application would bring together a chemist and bioengineer (PI V.A.K., an early-stage investigator), a specialist in oral bacterial colonies (co-I C.C.), and an endodontist (co-I E.S.), to solve an enduring challenge: regenerating biomimetic vascularized soft tissue post- pulpectomy. In vitro mechanistic analyses, in vivo characterization of infiltrating cells, and histologic/radiographic identification of long-term evolution of the pulpal soft tissue and the pulp-dentin complex would build on published studies and extensive preliminary data. Even if the proposed experiments are only partially successful, we would learn about tissue-material interaction in the context of dental pulp. Success of the aims would produce compelling data for a cell-free, growth-factor-free, off-the-shelf material formulation ideal for application in endodontic settings and improve clinical outcomes in millions of patients needing pulpectomy.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary Schneckenbecken dysplasia (SBD) is a rare congenital disease characterized by severe skeletal dysplasia, caused by mutations in the nucleotide-sugar transporter gene, SLC35D1. SLC35D1 resides in the endoplasmic reticulum and facilitates transport of UDP-sugars for post-translational modification. UDP-sugars transported through SLC35D1 are crucial for biosynthesis of chondroitin sulfate, a proteoglycan in the extracellular matrix. In Slc35d1 knockout mice, chondrogenesis is disrupted and chondroitin sulfate chain length is decreased in chondrogenic regions. However, mechanistic insights into how changes in chondroitin sulfate chain length impair chondrogenesis are lacking for models of SBD and little is known about the developmental etiology. To address these knowledge gaps, we propose to use Xenopus tropicalis to generate CRISPR models of patient-specific variants of SBD to understand the underlying cause of aberrant chondrogenesis, with a specific focus on the neural-crest derived craniofacial cartilage. In the proposed aims, we will 1) uncover when neural crest (progenitors of facial skeleton) development is disrupted and assess how patient-specific SBD variants affect facial phenotypes and 2) determine how loss of Slc35d1 changes extracellular matrix composition and affects mechanical and biochemical inductive ques during chondrogenesis. This work will provide critical mechanistic insights into the role of the ECM in craniofacial development and establish tractable disease models for SBD which can be used for the development of therapeutic approaches for SBD.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary Craniofacial differences are among the most common congenital anomalies, occurring with a frequency of 1 in 700 live births. The biomedical burden for treating these conditions is over 700 million dollars a year in the US alone. Thus, assembling a group of discovery-based research scientists, geneticists, clinicians, and surgeons all focused on understanding the genetic, molecular, and cellular mechanisms essential for understanding craniofacial development and disease is of great value to public health. To address this need we have formulated a scientific program that will focus on integrating our knowledge of genetics, model systems, and biological mechanisms, with the end goal of improving craniofacial health. A keynote session will explore rare disease genetics from both a human geneticist and research perspective. Challenges and solutions related to linking gene discovery to functional genomics will be identified, with a focus on advancing the craniofacial field into the era of stem cell and organoid medicine. Molecular and biochemical mechanisms that contribute to patterning the building blocks of the face will be discussed, alongside how these mechanisms go awry under pathological conditions. Cross-disciplinary collaboration will be promoted to ensure translationally relevant data is shared, facilitating the realization of the bench-to-bedside vision from diagnosis to therapeutics. Open discussion within the community will aim to establish a consistent set of protocols for data comparison and reproducibility across laboratories, enhancing the efficiency and impact of craniofacial research. Beyond sharing cutting-edge, unpublished research, the meeting will provide opportunities for scientists at all career levels to network and form collaborations with colleagues from various disciplines to advance craniofacial research.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY (FROM ORIGINAL APPLICATION) Human papillomavirus (HPV)-driven oropharyngeal squamous cell cancer (OPSCC) has been rising in incidence in the US and worldwide over the last four decades, particularly among young men. People with human immunodeficiency virus (HIV) have an increased HPV-OPSCC risk as compared to HIV-negative individuals. However, we have a very poor understanding of the mechanisms of persistent oral HPV infections and their synergy with HIV co-infection, as well as the role of HIV in HPV-OPSCC induction and pathogenesis. The inability to histologically characterize the early stages of HPV-induced OP tumorigenesis continues to hinder efforts to diagnose OP precancerous lesions and assess cancer risk. Currently, there is no animal model for HPV-induced oral infections and malignancies in the context of HIV-induced immunosuppression. In previous collaborative efforts, the Ozbun and Traina-Dorge labs have successfully modeled cervical HPV infections and neoplasia in female rhesus macaques (RMs) using Macca mulatta papillomavirus type 1 (MmPV1), a close relative of HPV16, which causes 90-97% of HPV-OPSCC. In this R21 proposal, we will build on our prior work and collective expertise with MmPV1, simian immunodeficiency virus (SIV), and non-human primate immunology to establish a Rhesus macaque model of persistent oral HPV and HIV co-infection with which to study oral MmPV1 infections, persistence, precancer, and OPSCC induction. This model is innovative and advantageous because both MmPV1 and SIV recapitulate their respective infections and pathogenesis in RMs like their human counterparts: MmPV1 is carcinogenic in its natural RM host, and these animals develop immunosuppression following SIV infection. As HPV-OPSCCs are more common in males, we will expose immunosuppressed SIV-infected male RMs to high-titer infectious MmPV1 virions in the tongue base, the palatine tonsil, and the soft palate. In AIM 1, we will determine the oral infectivity and persistence of MmPV1 virions in SIV-infected and immunosuppressed RMs. At timed intervals and in post-mortem tissues, we will assess MmPV1 viral loads and DNA persistence at the sites of infection, and we will screen for localized viral shedding, preneoplastic oropharyngeal MmPV1 lesions, and disease progression. We will also measure circulating tumor MmPV1 DNA over time as a potential biomarker of the onset of preneoplasia and/or OPSCC. In AIM 2, we will evaluate SIV- and MmPV1-induced changes in RM biospecimens. At timed intervals, we will quantify plasma and saliva cytokine/chemokine levels by Mesoscale immunoassays and assess HPV-disease-associated markers in cytobrush exfoliated OP cells and endpoint post-mortem tissues by immunohistochemistry. These assays will identify biological pathways altered by both viruses and determine potential disease correlates. Establishing a non-human primate model for OP PV infections and precancers will facilitate expanded follow-up studies aimed at revealing mechanisms of MmPV1 infection, persistence, and progression from precancer to malignancy in the context of SIV-induced immunosuppression; it will also promote efforts to diagnose and treat OP precancers.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary The oral mucosa, and specifically, the periodontal sulcus, is considered a defense barrier between specialized immune cells and polymicrobial communities. In healthy condition, balanced interactions among the epithelium, microbiota, and immune cells are maintained within the depth of the periodontal sulcus. However, persistent inflammation within the sulcus disrupts the equilibrium, facilitating the growth of pathogenic bacteria and leading to periodontal diseases (gingivitis, periodontitis). At present, the ways in which polymicrobial community influence host physiology and how the innate immune system balance host-pathogen interactions in healthy and disease state are not yet well understood. Moreover, disease trajectory studied by using clinical, animal, or in- vitro models are limited. Specifically, research strategies have failed to mimic key elements of the gingiva, such as anatomical complexity (i.e, sulcus depth at different stages of the disease), polymicrobial native conditions (oxygen and pH levels), and immune components. Thus, there is a compelling need to improve current culture technologies to provide a more sustained in vivo-like environments to investigate host-pathogen interactions in acute and chronic conditions. Therefore, I am proposing a sustained in vitro gingival tissue model, resembling the gingival sulcus anatomy, capable of recreating different periodontal states (healthy, gingivitis, periodontitis), physical properties (i.e., oxygen gradient), and metabolic conditions. Acute and chronic states will be studied with the addition of primary human neutrophils to investigate early dysbiosis clinical fingerprints and monitoring the cytokine profiles in comparison to gingival exudates from patients.

NIDCR - National Institute of Dental and Craniofacial Research

Abstract Misshapen teeth are highly common in humans. In most instances, they are due to genetic mutations in genes controlling the morphogenesis of teeth. This is the case for the Runx2 gene, which once mutated leads to cleidocranial dysplasia (CCD) an autosomal dominant genetic syndrome presenting with peg-like teeth in humans. While many factors have been identified in the process of tooth morphogenesis, currently, little is known about how cell signaling participates in establishing organ shape during odontogenesis. Our previous work has identified the signaling molecule retinoic acid (RA) to be one of the main actors of tooth induction in fish and recent data suggest that RA also plays a role during tooth morphogenesis. Fish and zebrafish, in particular, are good models to study genetics, cell and molecular biology, and organogenesis of the tooth in vertebrates. The objective of the proposed studies is to understand the roles played by retinoic acid during tooth morphogenesis in different fish species, to develop a fish model of CCD, to clarify the role played by Runx2 during tooth morphogenesis, and to understand its link with RA signaling. Finally, this project proposes to identify novel genes implicated in tooth morphogenesis and to study their function by gene knock-outs in zebrafish. By exposing fish embryos and larvae to exogenous RA and RA inhibitor during tooth morphogenesis we will be able to understand the mechanism of action of RA signaling during tooth morphogenesis in fish. Our preliminary data identified that the levels of RA in different cells of the tooth germ are controlled by the timing and level of expression of the RA degrading enzyme cyp26b1 in a subset of cells of the developing tooth germ. Modifying the onset of cyp26b1 expression in the tooth germ will, therefore, change the level of RA available in the tooth germ ultimately modifying the shape of the tooth. We will study the cis-regulatory changes responsible for evolutionary changes in the timing of cyp26b1 expression during tooth morphogenesis between two closely related fish species, the zebrafish and the mountain minnow, that bear dramatically different shape of teeth in adults and during embryonic development. We have in hands the zebrafish runx2b (the zebrafish ortholog of the human Runx2 gene that is expressed in the tooth germ) loss-of-function mutant. This mutant will be used to perform a phenotypic analysis of tooth morphogenesis and to understand the relationship between RA signaling and Runx2 expression during tooth morphogenesis. To identify novels genes playing a role during tooth morphogenesis, we will select by cell sorting, tooth germ cells exposed to exogenous RA signaling and compare their transcriptome to control developing tooth cells at the same developmental stage. The resulting genes differentially expressed will be subjected to phenotypic analysis by gene knock-out using the CRISPR/cas9 technology. This work will reveal more about how RA and genes it regulates, including Runx2, controls tooth morphogenesis in development, diseases (cleidocranial dysplasia) and evolution.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary/Abstract Psychological stress is an established contributor to bone and tooth loss and impaired bone growth. In the US, more than 50 million people currently experience bone loss while a similar number is afflicted by anxiety (40 million) and/or depressive disorders (16 million). Bone and tooth loss resulting from psychological stress has been observed in all age populations. A murine model of stress, repeated social defeat (RSD), recapitulates key physiological, immunological, and behavioral alterations in humans exposed to psychosocial stress such as bullying and loss of social status. RSD activates the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system to create a state in which primed, pro-inflammatory monocytes traffic from the bone marrow to the brain to generate neuroinflammation and anxiety-like behavior. In addition, RSD rapidly induces bone loss through increased activity of osteoclasts, and bone growth plate reduction. However, the precise mechanisms by which RSD influences bone have not been identified. Therefore, the overall goal of this project is to exploit a rodent model of psychological stress to better explore the relationships between immunological processes, mental and bone health. This will be accomplished in three Specific Aims. Aim 1 will examine the kinetics of bone loss and growth plate reduction in adolescent male and female mice following a period of RSD. Aim 2 will investigate a central role for osteoclast activation and chemokine (CXCL12) signaling in RSD-induced monocyte mobilization. Aim 3 will investigate mechanisms of RSD-induced growth plate reduction. Outcomes of this project will help achieve the long-term goal of developing more specific interventions to treat psychological stress-related disorders of skeletal physiology.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT: The National Dental Practice-Based Research Network (Network) is a consortium of more than 8,000 dentists, hygienists, and other professionals committed to advancing dental practice. The NDPBRN embeds research in dental practices to conduct impactful research in oral and oral- systemic health. Practice-based research is invaluable for identifying trends in incidence and prevalence of oral (and other) health conditions, understanding current practitioner attitudes and practices, and studying the effectiveness of dental care delivery strategies in real-world settings. The CHR Data Coordinating Center serves as the Network Coordinating Center (NCC). During Cycle 3, the NCC seeks to optimize the productivity and impact of Network research and to impart and sustain rigor and high-quality data management practices. The NCC has created a “Year 8” work plan for the No Cost Extension (NCE) period to complete work associated with the Aims of the NCC’s parent award. Funds requested for this supplement will fill the NCC funding gap resulting from expanded resource requirements for study support and system development and from extended study start-up, recruitment and monitoring timelines. Year 8 NCC workplan activities are focused on maintaining HUB functionality and access; continuing provider enrollment and engagement; completing data analysis and dissemination; preparation of public use datasets and study datasets for study PIs; and updating and archiving important study documentation. The requested funds will serve to extend the timeframe and the capacity of NCC staff such that we will be able to sustain these network activities and resources for the full 12 months of the NCE period. The NCC has a 25-year history of successfully leading and participating in numerous collaborative multi- center studies and infrastructure development projects funded by NIH (NHLBI, NIDA, NIDCR), the Agency for Healthcare Research and Quality (AHRQ), the Health Resources and Services Administration (HRSA), the Office of the National Coordinator for Health Information Technology (ONC), the Patient-Centered Research Outcomes Institute (PCORI) and industry. The NCC has managed data collection and other study- related activities for 17 NDPBRN studies since 2019.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY / ABSTRACT FaceBase is an NIH-supported data resource that curates and distributes craniofacial and dental research data to the international scientific community, including controlled-access human-subject datasets governed under the NIH Genomic Data Sharing policy. Recent NIH policy (NOT-OD-24-157 and the accompanying Security Best Practices for Controlled-Access Data Repositories) requires all NIH-supported controlled-access data repositories to attest to compliance with NIST Special Publication 800-53 Revision 5 at the Moderate baseline, with attestation deadlines beginning in 2025 and continuing through 2026. In parallel, NIH expects integration with its Researcher Auth Service (RAS) to provide federated, GA4GH-compliant identity and authorization for researchers accessing controlled data across NIH-supported repositories. Neither requirement existed when the parent FaceBase IV award was reviewed and funded, and neither is achievable within the parent budget. This administrative supplement supports the focused, time-bounded program of work required to bring FaceBase into compliance with these new requirements and to preserve uninterrupted access to its controlled-access datasets for the craniofacial research community. The work is performed by USC Information Sciences Institute research software engineers in partnership with an external contractor specializing in NIST SP 800-53 compliance for federally-funded research data systems. The contractor facilitates production of compliance artifacts — the System Security Plan, the Plan of Action and Milestones, and supporting documentation — while USC ISI engineers perform the technical hardening of the FaceBase platform and the RAS integration engineering. The supplement period of performance is seven months, running from June 1, 2026 through December 31, 2026. During Months 1 to 3 (June through August 2026) the team will define the FaceBase authorization boundary, perform a gap analysis against the 800-53 Moderate baseline, implement the highest-priority technical and operational controls, and produce drafts of the System Security Plan and Plan of Action and Milestones. During Months 4 to 7 (September through December 2026) the team will finalize the System Security Plan and Plan of Action and Milestones, deliver the attestation submission package to NIH by the September 2026 deadline, advance the RAS technical integration on the NIH Center for Information Technology onboarding timeline, begin remediation of items captured in the Plan of Action and Milestones, and stand up the continuous monitoring activities the System Security Plan commits to. Because the underlying Deriva data management platform supports multiple NIH-funded data resources, the documentation patterns, software components, and lessons learned developed under this supplement will be released to benefit other NIH controlled-access repositories facing the same compliance obligation.

NIDCR - National Institute of Dental and Craniofacial Research

Abstract Periodontal regeneration and bone repair involve classical elements of wound healing and have been perennially met with challenges of inconsistency and predictability. Among the gaps in knowledge regarding periodontal regeneration is how macrophage polarization and function influence periodontal regeneration and bone repair. We and others have defined a central role for the macrophage in the successful regeneration of bone and the periodontium. However, there remains an undefined role for bone morphogenetic protein 2 (BMP2) and nuclear BMP2 – an under characterized BMP2 isoform – in macrophage immunomodulation that supports successful regeneration. We have observed extensive nuclear localization of BMP2 in macrophages cultured on nanoscale topographies and noted that nuclear BMP2-positive macrophages have an M2-like (regenerative) phenotype. In addition, nBMP2 knockout leads to a decrease in STAT6 expression in macrophages and an increase in ligature-induced periodontal bone loss. Combined with Alphafold 3 predictive results and our initial immunoprecipitation data, we will investigate the overarching hypothesis that macrophage nBMP2 and BMP2 expression positively influences macrophage M2 polarization and tissue regeneration via modulation of STAT6 activity. The specific aims are: AIM 1: to characterize the regulation of macrophage nBMP2/BMP2 expression. AIM 2: to functionally dissect nBMP2-regulated STAT6 signaling pathways in macrophages. AIM 3: to demonstrate the role of macrophage nBMP2/BMP2 on tissue repair and regeneration. Using cell culture and knockout mouse models of macrophage nBMP2 and BMP2 expression, and defined models of periodontal regeneration and bone repair, the present study will define the mechanism by which nuclear BMP2 controls macrophage polarization and how macrophage nBMP2/BMP2 expression contributes to bone and periodontal regeneration. The proposed mechanistic studies will be critical for understanding how macrophages contribute to immunomodulation in tissue regeneration, particularly in periodontal regeneration and bone repair. Furthermore, new insights into the physiologic regulation of macrophage function and polarization can extend beyond periodontal regeneration to other tissues, as well as their role in cancer as tissue-associated macrophages.

NIDCR - National Institute of Dental and Craniofacial Research

Head and neck cancer (HNC), including cancers of the oral cavity and oropharynx, is the sixth most common cancer and the ninth leading cause of cancer-related deaths globally. Despite advances in PET imaging, a critical tool in staging and treatment planning, the detection of cervical lymph node metastases remains challenging due to the limited resolution of conventional whole-body PET scanners. This limitation results in high false-negative rates, particularly for small (<7 mm) metastases in clinically node-negative patients. Due to the high prevalence (20-30%) of occult lymph node metastases, many HNC patients undergo elective neck dissections to remove potentially affected nodes, despite the associated risks and morbidity. Addressing this unmet need, the Head PETcoil is a portable, high-resolution, time-of-flight (TOF) PET system designed for brain, head, and neck imaging. Compared to conventional PET systems, the Head PETcoil offers nearly six times finer spatial resolution and three times higher photon sensitivity, improving the detection of tumors and small lymph node metastasis. To realize this improved resolution in patient studies, it is critical to correct for image blurring due to head motion, which can cause displacements that range from 2 to 5 mm. This Phase I project aims to develop and validate real-time motion correction software for the Head PETcoil. Aim 1 is to develop ultra-fast data processing and image reconstruction software capable of generating short timeframe (1-5 seconds) PET images in real time during data acquisition. Aim 2 is to create real-time motion estimation and correction software to calculate motion from short timeframe images and produce motion-corrected images within 5 minutes after scan completion. Aim 3 will verify the accuracy of the motion correction method using phantoms with simulated head motion. The goal is to ensure motion-corrected images meet performance metrics (e.g., spatial resolution, contrast-to-noise ratio) comparable to static images. By the end of Phase I, a robust software package capable of generating motion-corrected images for the portable, high-resolution TOF PET system within 5 minutes post- scan will be delivered. Phase II will refine the real-time motion correction software in imaging HNC patients and compare the diagnostic accuracy of the Head PETcoil to commercial whole-body PET systems. The Head PETcoil has the potential to transform HNC imaging by improving early-stage disease detection, enhancing treatment planning, and expanding access to advanced TOF PET imaging.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary/Abstract Disturbances in the molecular pathways that control lip and palate development during embryogenesis can lead to oral clefts, but knowledge of the regulatory interactions involved in modulating the expression of genes in these pathways remains limited. Because enhancers are critical to the spatiotemporal regulation of gene expression during embryogenesis, those that are active in craniofacial tissues during lip and palate development are likely to control the expression of genes relevant to oral clefts. The overall goal of the proposed research is to identify interactions between enhancers, transcription factors that bind the enhancers, and target genes of the enhancers in lip and palate development and oral clefts. Aim 1 is to identify the subset of enhancers that drive gene expression overall in human embryonic palatal mesenchyme cells. The transcription factors with binding site motifs that are enriched in the enhancers will be identified, and binding of the transcription factors within the enhancers in human embryonic palatal mesenchyme cells will be confirmed using assays of chromatin immunoprecipitation followed by sequencing. Aim 2 is to identify enhancers that regulate transcription specifically within human embryonic palatal mesenchyme cells, because such enhancers are more likely to be involved in craniofacial-specific processes during development. Enhancers that can distinguish between human embryonic palatal mesenchyme cells and other types of tissues and cells will be considered palate-specific enhancers. The palate specificity of a subset of the enhancers will be confirmed using in vitro assays.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT Enzymes are the primary functional molecules in cells, providing enormous rate enhancements, specificity and regulation to the diverse chemical reactions that are necessary for life. Enzymes, like all biological macromolecules, are the products of evolution: all enzymes have evolved to operate within the complex environment of the organism/cell in specific environmental niches(s). Thus, an understanding of enzyme function and evolution is fundamental to biology. Enzymes also have tremendous potential in medicine (e.g., as targets for anti-cancer, antimicrobial and antiviral drugs and as therapeutics for metabolic disorders) and in industry (e.g. to make important commodity chemicals and as catalysts for bioremediation). Our central premise is that a quantitative, mechanistic understanding of enzyme function and its relationship to organism fitness is critically needed to precisely manipulate enzymes and to deeply understand biology. To generate this level of understanding, we need: (1) a quantitative, chemical, and physical knowledge of enzyme function, and (2) mechanistic data describing how and when these physical principles contribute to enzyme function within the complex environments where enzymes operate. An enhanced understanding of the relationships between protein sequence, protein function and cellular/organismal fitness will have profound impacts across biology and medicine, from improving our ability to predict how mutations will influence the virulence and drug susceptibility of human pathogens, to enhancing precision medicine by accurately predicting the consequences of allelic variants, to enabling the design of next-generation protein and cellular therapeutics. Achieving this understanding requires new tools and a new conceptual paradigm. Enzymes are highly interconnected, their functions are multifaceted, and their cellular environments are complex. Traditional biochemistry is enormously powerful, allowing for the intensive study of a few individual enzymes in vitro (10s) and providing detailed knowledge of their chemical mechanisms. But identifying the many residues that matter for enzyme function requires investigation of residues beyond the active site at a scale far beyond that of traditional biochemistry. Furthermore, this biochemical information then needs to be translated to organism fitness in vivo in a quantitative manner. Here we will overcome these challenges. We will first use evolutionary sequence information to direct enzyme variant design towards functionally important areas of sequence space. We will adapt high-throughput microfluidic technologies to quantitively measure the biochemical properties (e.g., kcat, Km, Ki, and ∆GFold) of this library of 104 enzyme variants in vitro (Aim 1). Then we will determine how each of these variants affects organismal fitness in vivo using pooled competition and barcode sequencing assays (Aim 2). Finally, we will use this sequence-function-fitness map to test long-standing models in biochemistry and evolution and reveal the biochemical determinants of fitness important for industry and medicine (Aim 3). Such a comprehensive and quantitative mapping of biochemical function to fitness has never been achieved.

NIDCR - National Institute of Dental and Craniofacial Research

Abstract Keratinocyte Differentiation Factor 1 (KDF1) is an ectodermal dysplasia (ED) disease gene with a wide range in phenotype severity. Individuals with putatively damaging heterozygous variants in KDF1 present with ED and severe tooth agenesis, or milder non-syndromic tooth agenesis. Identifying KDF1 genotype-phenotype correlations that will predict the severity of oral phenotypes will result in earlier intervention with dental implants and improved prognosis for affected families. Our work has broad future applications, as five other ED disease genes display the same range in severity of syndromic and non-syndromic tooth agenesis (NSTA) phenotypes, yet the mechanism driving this variable expression remains unclear. Investigating the mechanism underlying the range in phenotype expression in KDF1-disease biology will have a profound impact on diagnostic variant interpretation in the clinic and individualized treatment plans for individuals with pathogenic variants in KDF1. Preliminary analyses suggest that individuals with pathogenic missense variants within the central domain (CD) of the KDF1 protein develop ED and severe tooth agenesis, while patients with missense variants outside the CD develop the attenuated NSTA phenotype with milder tooth agenesis. The CD is critical for KDF1 to bind and deubiquitinate IKKα, resulting in IKKα stabilization and downstream transcriptional regulation of keratinocyte and tooth germ development. Understanding the functional consequences of KDF1 variation is critical to inform disease management and potential therapeutic development. My central hypothesis is that the position of variants within KDF1 mediates the phenotype of ED or NSTA through impacts on IKKα stability. To address this hypothesis, I will first determine if severe tooth agenesis and the presence of ED phenotypes are dependent upon variant position within KDF1 by statistically interrogating well-characterized cohorts of severe and more tolerated KDF1 variation. I will then quantify the effects of KDF1 variants associated with syndromic tooth agenesis on keratinocyte differentiation and IKKα stability and abundance by creating and characterizing CRISPR/Cas9-edited cell lines and performing co-immunoprecipitation assays and western blot analysis. The findings of these studies will elucidate the mechanism driving severe KDF1-disease, advance our understanding of ED pathogenesis, and inform predictive disease management practices. The research proposed in this application aims to foster my development in becoming a successful independent researcher and clinical molecular geneticist. My research environment within the Posey laboratory, the Texas Medical Center, and the Baylor College of Medicine GREGoR Consortium research center offers an exceptional foundation for achieving these aims.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Recurrent caries is the leading cause of dental restoration failure, costing the U.S. over $5 billion annually. Enhancing the seal of dental materials bonded to hard tissues is critical for preventing recurrent caries, disease progression, and tooth loss. Eliminating bacteria at the restoration margins can prevent the biochemical degradation of these materials to deter seal damage and thus extending the clinical lifespan of restorations. We have developed a novel antibacterial composites and adhesives with piezoelectric properties. Piezoelectric biomaterials have proven antimicrobial effects. This innovative approach significantly improves upon current technologies by using a single filler that provides long-lasting antibacterial effects without antibiotic resistance issues. Sound or acoustic waves (such as the human voice) can stimulate piezo- adhesives to produce the necessary electrical charges to elicit antimicrobial effects that prevent bond degradation and extend the durability of dental restorations. In this study, we propose to investigate how the properties of the human voice (such as pitch, intensity in dB, duration, angulation) influence charge production and antimicrobial effects in restorations (aim 1). Additionally, this project will assess the degradation of the tooth-restoration interface under representative oral conditions and with piezo-adhesives stimulated by the human voice and mastication forces. This research will provide crucial insights into how smart adhesives can improve the durability of dental composite restorations.

NIDCR - National Institute of Dental and Craniofacial Research

LrgA/B (membrane proteins found in a wide variety of bacteria) has been recently identified as a pyruvate importer in Bacillus subtilis, Staphylococcus aureus, and Streptococcus mutans. An increased appreciation for extracellular pyruvate’s non-traditional and diverse roles in metabolism and stress resistance has emerged in recent years. Although our published and preliminary data demonstrate key roles for LrgAB and/or extracellular pyruvate in modulating S. mutans physiology, oxidative stress resistance, and virulence, the precise mechanism(s) by which extracellular pyruvate exerts these functions in S. mutans remain unknown. Amongst oral streptococci, lrgAB and its upstream two-component regulator lytST, are notably absent from the genomes of non-cariogenic streptococcal species, and likewise extracellular pyruvate supplementation does not confer a strong stationary phase growth advantage in non-cariogenic oral streptococci. Collectively, these observations support the hypothesis that extracellular pyruvate serves as a metabolic signal that enhances stress resistance and metabolic flexibility of S. mutans, contributing to survival in fluctuating host environments. This would provide a distinct competitive advantage to S. mutans in dental plaque biofilm and other pyruvate-replete environments such as the bloodstream. This hypothesis will be addressed by two specific aims. In Aim 1, the metabolic fate of extracellular pyruvate in S. mutans planktonic cultures will be tracked and identified by 13C-labeled pyruvate feeding experiments and NMR spectroscopy. Time-resolved metabolomic and transcriptomic analyses will also be employed to characterize pyruvate-, LytST-, and LrgAB-driven gene expression and metabolic shifts during growth-phase transitions. In Aim 2, fluorescence-based reporters and confocal microscopy will be used to examine the influence of lrgAB and extracellular pyruvate supplementation on S. mutans niche competition and behavior within dual-species biofilms grown in human saliva. Pyruvate supplementation, LrgAB, and LytST effects on S. mutans oxidative stress resistance and survival in human whole blood, plasma and serum will also be assessed. Understanding the interactions between extracellular pyruvate, LytST, and LrgAB could provide new insights into the pathogenesis of S. mutans, its persistence in the oral cavity and bloodstream, and its interactions with other microbial species. This could lead to new targeted therapeutic strategies to control S. mutans infections, both within the oral cavity and in systemic conditions such as infective endocarditis.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT Periodontal diseases are among the most prevalent on earth. Cementum is a root-covering mineralized periodontal tissue, which is critical for tooth attachment. Disorders that disrupt cementum formation or cause its destruction can result in periodontal ligament (PDL) detachment, periodontal dysfunction, and tooth loss. However, gaps in knowledge about factors that regulate cementum during development have, to date, limited therapeutic advances for cementum repair and regeneration. Inorganic pyrophosphate (PPi) is a physiological regulator of mineralization that works to prevent hydroxyapatite (HA) mineral growth. Modulation of PPi levels is a powerful approach to regulate cementogenesis. Through a series of experiments on human disorders, genetically engineered mouse models, and in vitro cell work, we established that PPi is perhaps the most important molecular regulator of cementum formation. Local levels of PPi are controlled by a few key proteins. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) increases PPi levels, limiting mineralization. ENPP1 loss-of-function in development reduces PPi levels and dramatically increases cementum growth. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes and decreases PPi levels, promoting mineralization. Loss-of-function mutations in ALPL, which encodes TNAP, cause hypophosphatasia (HPP). HPP is a mineralization disorder that contributes to substantial dental mineralization defects, including loss of fully rooted deciduous and/or permanent teeth is pathognomic due to cementum defects. Limitations in the current HPP enzyme replacement therapy have prompted research into additional treatment strategies. Pharmacological targeting of ENPP1 function represents a promising alternative approach to reduce PPi in HPP and promote cementum growth, periodontal attachment, and tooth retention. Pilot studies using dietary administration of an ENPP1 inhibitor showed dramatic improvements in multiple skeletal defects in a mouse model of HPP. However, this therapeutic approach represents a much bigger opportunity where insights gained from studying a rare disease can more broadly impact oral health in the general population. The strategy to improve cementum in HPP can also be applied to periodontal disease, where cementum-PDL-alveolar bone structures must be regenerated to restore periodontal function. Our central hypothesis is that ENPP1 inhibition will improve cementum repair and periodontal function in models of HPP and periodontal disease. We will test this hypothesis by two aims: (1) Test pharmacological ENPP1 inhibition to ameliorate periodontal defects in a mouse model of HPP; (2) Define ability of pharmacological ENPP1 inhibition to re-establish cementum in a mouse model of periodontal regeneration. Expected outcomes of these proof-of-principle experiments include data to support future translational work employing ENPP1 inhibition to promote cementum repair, potentially scaling up to larger animal models and different models of periodontal disease.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Salivary gland (SG) regeneration involves a coordinated interplay of self-renewal, cell fate determination, and differentiation programs in stem/progenitor epithelial cells. Identifying the transcriptional and signaling networks that govern these complex processes is crucial for understanding the underlying molecular mechanisms driving cellular regeneration. By leveraging extensive genomic and epigenomic datasets generated by our group and others, we have identified master transcription factors (TFs) that are enriched and highly expressed in the mouse SG. Based on our preliminary results from a well-defined ductal ligation model of SG regeneration we hypothesize that Six1 functions as a key transcriptional regulator of stem/progenitor cell activity during gland regeneration. We further hypothesize that Six1 directs distinct gene expression programs necessary for the differentiation of specialized SG cell populations and in shaping the dynamic chromatin landscape crucial for cell fate decisions in the pivotal regeneration stages. Thus, we posit that examination of the Six1-driven gene and signaling regulatory networks, particularly in the context of epigenomics, and regeneration after injury, is a key step towards advancing our understanding of SG biology. To achieve this, we propose to use a Six1 conditional knockout (cKO) mouse model and a systems biology approach to pursue two major areas of interest. First, we will use Six1cKO animals to assess the relative contribution of Six1+ve cells during SG regeneration. Furthermore, we will probe mechanistically how Six1 shapes the myriad cellular identities and fate trajectories within the 3-D ecosystem of the SG by performing single-cell (sc) RNA-sequencing (Aim1). Secondly, we will examine the cell-type specific molecular mechanism by which Six1 acts as a pioneer factor to modulate the epigenome and gene expression programs during specific stages of regeneration following ductal ligation in the SG utilizing scATAC-seq and scCUT&Tag with histone markers for active regulatory elements (Aim 2). Collectively, our proposed studies will not only reveal the role of Six1 in governing regeneration programs to maintain and restore the distinct cellular subtypes the SG, but also enrich existing molecular paradigms of cell- specific transcriptional regulatory networks and signaling pathways. Long term, such knowledge will impact therapeutic interventions in human patients who suffer from SG dysfunction.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT Periodontitis, caries, and halitosis are highly prevalent, have a large financial burden, and negatively impact quality of life. These conditions are each driven by the community of microbes in the mouth, especially oral pathogens. In contrast, during oral health, the oral microbiota is dominated by commensal, non-pathogenic species that are thought to modulate health, including through their interactions with pathogens. Select oral microbes are well characterized in the lab, but the behavior of these organisms in the human oral cavity is not well described. Over the last decade, researchers have used RNA sequencing directly from the human oral cavity (metatranscriptomics) to define the overall bacterial behavior in subgingival plaque, in supragingival plaque, and on the tongue. These studies were instrumental in uncovering broad changes in bacterial gene expression between health and diseased states. However, there remains a lack of understanding of the behavior of individual taxa in the human oral cavity. We recently started to address this gap in knowledge by characterizing the gene expression of the pathogen Porphyromonas gingivalis during periodontitis in 93 human metatranscriptomes. This proposal expands on our approach by leveraging 697 publicly-available human oral metatranscriptomes to identify the gene expression of key oral pathogens, pathobionts, and commensals across oral sites and disease states. This study will be supported by pangenomic approaches to capture the gene expression of diverse genotypes and an in silico validation approach to ensure high specificity and sensitivity. Our findings will uncover the biology of each microbe during oral health and disease, including virulence factor expression, metabolic processes, and other host-microbe and microbe-microbe interactions. Also, we will examine the variation in behavior for each taxon of interest across hosts, oral sites, and disease states. Finally, these analyses will help researchers identify key genes to study based on the expression levels in the human oral cavity. To this end, we will share our data using interactive interfaces in the Human Oral Microbiome Database, an expertly-curated and highly-used resource for oral microbiologists. In sum, this project will be instrumental in describing the gene expression of oral microbes in the human oral cavity, uncovering new biology of these important bacteria, and empowering researchers to study genes relevant to human health and disease.

NIDCR - National Institute of Dental and Craniofacial Research

ABSTRACT Despite aggressive treatments, the five-year overall survival rate for advanced head and neck squamous cell carcinoma (HNSCC) remains below 50%. The global burden of HNSCC is influenced by tobacco-derived carcinogens and excessive alcohol consumption, with oropharyngeal tumors increasingly linked to oncogenic human papillomavirus (HPV) infections. Current treatments for locoregionally advanced HNSCC include surgery followed by adjuvant external beam radiation therapy (XRT) or definitive concurrent chemoradiation, but radiation resistance is common, leading to poor outcomes. Novel strategies are needed for better detection, treatment, and monitoring of treatment responses. Our research is based on the discovery of radiation-inducible antigens that are upregulated on cancer cell surfaces post-XRT. We identified Tax-interacting protein 1 (TIP1) as a tumor- associated antigen upregulated following XRT. Anti-TIP1 antibodies undergo endocytosis, delivering payloads specifically to tumor cells and show XRT-induced tumor-specific delivery in vivo. We aim to leverage XRT- induced TIP1 expression to guide theranostic (therapy+diagnostic) radioimmunoconjugates for detection, treatment, and monitoring response to treatment. Radiopharmaceuticals are emerging as effective treatments for various cancers. In our recent publication, we developed human anti-TIP1 antibodies (L111) and labeled them with [89Zr]Zr, demonstrating specific cancer detection by PET imaging in preclinical cancer models. We propose radiolabeling L111 antibodies with the β-emitter [177Lu]Lu and evaluating their therapeutic potential for HNSCC. We hypothesize that Immuno-PET using [89Zr]Zr-L111 will monitor HNSCC response to treatment and that [177Lu]Lu-L111 will treat residual tumors unresponsive to XRT. Our objectives include assessing [89Zr]Zr- L111 for non-invasive PET imaging of HNSCC tumors after XRT and [177Lu]Lu-L111 therapy and determining if this strategy applies to both HPV+ and HPV- HNSCC subtypes. Aim 1 will evaluate the efficacy of TIP1-targeted radioimmunoconjugates in vitro using HPV+ and HPV- HNSCC cell lines, exploring XRT-driven TIP1 upregulation, enhanced binding, internalization, and cell killing efficacy of [177Lu]Lu-L111. We will also optimize radiation doses and schedules. Aim 2 will evaluate these conjugates in vivo using syngeneic orthotopic murine models treated with XRT, studying biodistribution, PET imaging, dosimetry, maximum tolerated dose, and therapeutic efficacy. Our study aims to revolutionize HNSCC management by improving detection, treatment, and monitoring, potentially leading to tailored therapies based on HPV status and enhancing survival rates through personalized interventions, marking a significant advancement in clinical practice.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT The calvaria bones of the skull begin to form bone marrow (BM) after birth, and this BM continues to expand with age in both mice and humans. Recent studies show that calvaria BM is not static but dynamically remodels in responses to local and systemic conditions, including pregnancy and disorders of skeletal, hematological, and neurological origin. BM niche formation is thought to be regulated by the intercommunication of skeletal cells (SKCs), vascular endothelial cells (ECs), and hematopoietic cells (HCs), but crosstalk mechanisms between them are complex and largely unknown. BM residing SKCs express platelet-derived growth factor (PDGF) receptors, PDGFR and PDGFRβ. The PDGFRs regulate the proliferation, migration, and differentiation of various mesenchyme-derived cell types in organ development, homeostasis, and tissue repair. Increased receptor signaling has been implicated in diseases such as fibrosis, cardiovascular disease, and cancer. Therefore, PDGF receptor activation and its downstream signaling have been proposed as potential therapeutic targets. Recent work in mice suggests a novel role for platelet-derived growth factor receptor beta (PDGFR) signaling in calvaria BM formation. Mice with a gain-of-function (GOF) mutation in PDGFR exhibit dramatically increased calvaria bone and BM size by 2-3 weeks of age, compared to wild type mice that do not generate significant calvaria BM until full adulthood. This precocious BM growth is accompanied by an expanded vascular network. Because PDGFR is expressed in SKCs but not in ECs or HCs, these results point to SKCs as the cellular origin of signals that increase calvaria vasculature and BM. The central hypothesis is that skeletal PDGFR promotes BM formation through an angiogenesis-directed mechanism. This project will address the central hypothesis with the following aims: 1) Characterize how PDGFR GOF causes dramatic expansion of calvaria vasculature and BM in juvenile mice, and 2) Determine the extent to which angiogenesis mediates BM formation in wild type and PDGFR mutants. In Aim 1, a skeletal-specific Cre recombinase mouse line will be used to conditionally induce a GOF Pdgfrb knock-in allele. The developmental processes of BM niche formation in wild type and PDGFRβ mutants will be defined using multiphoton light sheet imaging at 3 dimensions and transcriptomics. In Aim 2, experiments will focus on evaluating the contribution of ECs to BM formation with pharmacological angiogenic inhibition. The importance of skeletal and vascular coordination is evident from a close relationship between skeletal disorders, vascular malformation, and hematopoietic defects. The results of this project will generate a new mouse model to investigate calvaria bone/BM regeneration in future R01s and provide novel insights into the cell-cell interaction mechanisms that rebuild calvaria bone and BM.