Putting science to work for women's health.

The Office of Research on Women's Health (ORWH)'s whole health paradigm expands the scope of women's health research, incorporating a life-course perspective that recognizes the profound influences of sex and gender on health. From childhood through adulthood, external and societal factors along with internal factors and biology shape women's health and influence access to quality healthcare. This comprehensive approach integrates data-driven sex- and gender-aware strategies to prevent, diagnose, and treat disease, focusing on the unique needs of women. Acknowledging the historical lack of timely research and data on women's health, an initiative led by First Lady Dr. Jill Biden and the White House Gender Policy Council, ushers in a new era of women's health research that offers unprecedented opportunities to enhance the health of women through biomedical and behavioral research. The initiative fosters interdisciplinary collaboration, supporting research on autoimmune diseases, menopause, oral health, and chronic pain conditions. ORWH serves as the focal point for National Institutes of Health (NIH) women's health research. With a commitment to advancing holistic outcomes, ORWH engages in partnerships, outreach, and educational initiatives to disseminate critical research findings and support women's health researchers. Here we describe the convergence of this initiative with the National Institute of Dental and Craniofacial Research's work to advance the understanding of sex as a biological variable for conditions such as Sjogren's disease and temporomandibular disorder. This transformative approach to women's health research propels the United States toward innovative solutions, ensuring that science works for the health and well-being of every woman.

Tendon-associated gene expression precedes osteogenesis in mid-palatal suture establishment.

Orthodontic maxillary expansion relies on intrinsic mid-palatal suture mechanobiology to induce guided osteogenesis, yet establishment of the mid-palatal suture within the continuous secondary palate and causes of maxillary insufficiency remain poorly understood. In contrast, advances in cranial suture research hold promise to improve surgical repair of prematurely fused cranial sutures in craniosynostosis to potentially restore the obliterated signaling environment and ensure continual success of the intervention. We hypothesized that mid-palatal suture establishment is governed by shared principles with calvarial sutures and involves functional linkage between expanding primary ossification centres with the midline mesenchyme. We characterized establishment of the mid-palatal suture from late embryonic to early postnatal timepoints. Suture establishment was visualized using histological techniques and multimodal transcriptomics. We identified that mid-palatal suture formation depends on a spatiotemporally controlled signalling milieu in which tendon-associated genes play a significant role. We mapped relationships between extracellular matrix-encoding gene expression, tenocyte markers, and novel suture patency candidate genes. We identified similar expression patterns in FaceBase-deposited scRNA-seq datasets from cranial sutures. These findings demonstrate shared biological principles for suture establishment, providing further avenues for future development and understanding of maxillofacial interventions.

Spatial Multiomics Reveal the Role of Wnt Modulator, Dkk2, in Palatogenesis.

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which constitutes the most common among the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study utilized the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis.

The terminal differentiation of osteoblasts and subsequent formation of bone marks an important phase in palate development that leads to the separation of the oral and nasal cavities. While the morphogenetic events preceding palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms driving the formation of this bony union of the fusing palate. Through bulk, single-nucleus, and spatially resolved RNA-sequencing analyses of the developing secondary palate, we identify a shift in transcriptional programming between embryonic days 14.5 and 15.5 pinpointing the onset of osteogenesis. We define spatially restricted expression patterns of key osteogenic marker genes that are differentially expressed between these developmental timepoints. Finally, we identify genes in the palate highly expressed by palate nasal epithelial cells, also enriched within palatal osteogenic mesenchymal cells. This investigation provides a relevant framework to advance palate-specific diagnostic and therapeutic biomarker discovery.

Integrated spatiotemporal transcriptomic resolution of embryonic palate osteogenesis.

The differentiation of osteoblasts and the subsequent formation of bone marks an important terminal phase in palate formation that leads to the separation of the oral and nasal cavities. While the developmental events that precede palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms that lead to the bony union of fusing palatal shelves. Herein, the timeline of osteogenic transcriptional programming is unveiled in the embryonic palate by way of integrated bulk, single-cell, and spatially resolved RNA-seq analyses. We define spatially restricted expression patterns of key marker genes, both regulatory and structural, that are differentially expressed during palatal fusion, including the identification of several novel genes ( Deup1, Dynlrb2, Lrrc23 ) spatially restricted in expression to the palate, providing a relevant framework for future studies that identify new candidate genes for cleft palate anomalies in humans as well as the timing of mammalian embryonic palatal osteogenesis.

Profiles of Wnt pathway gene expression during tooth morphogenesis.

Mouse and human genetic studies indicate key roles of the Wnt10a ligand in odontogenesis. Previous studies have identified effectors and regulators of the Wnt signaling pathway actively expressed during key stages of tooth morphogenesis. However, limitations in multiplexing and spatial resolution hindered a more comprehensive analysis of these signaling molecules. Here, profiling of transcriptomes using fluorescent multiplex in situ hybridization and single-cell RNA-sequencing (scRNA-seq) provide robust insight into the synchronized expression patterns of Wnt10a, Dkk1, and Sost simultaneously during tooth development. First, we identified Wnt10a transcripts restricted to the epithelium at the stage of tooth bud morphogenesis, contrasting that of Sost and Dkk1 localization to the dental mesenchyme. By embryonic day 15.5 (E15.5), a marked shift of Wnt10a expression from dental epithelium to mesenchyme was noted, while Sost and Dkk1 expression remained enriched in the mesenchyme. By postnatal day 0 (P0), co-localization patterns of Wnt10a, Dkk1, and Sost were observed in both terminally differentiating and secreting odontoblasts of molars and incisors. Interestingly, Wnt10a exhibited robust expression in fully differentiated ameloblasts at the developing cusp tip of both molars and incisors, an observation not previously noted in prior studies. At P7 and 14, after the mineralization of dentin and enamel, Wnt10a expression was limited to odontoblasts. Meanwhile, Wnt modulators showed reduced or absent signals in molars. In contrast, strong signals persisted in ameloblasts (for Wnt10a) and odontoblasts (for Wnt10a, Sost, and Dkk1) towards the proximal end of incisors, near the cervical loop. Our scRNA-seq analysis used CellChat to further contextualize Wnt pathway-mediated communication between cells by examining ligand-receptor interactions among different clusters. The co-localization pattern of Wnt10a, Dkk1, and Sost in both terminally differentiating and secreting odontoblasts of molars and incisors potentially signifies the crucial ligand-modulator interaction along the gradient of cytodifferentiation starting from each cusp tip towards the apical region. These data provide cell type-specific insight into the role of Wnt ligands and mediators during epithelial-mesenchymal interactions in odontogenesis.

Harnessing Bilayer Biomaterial Delivery of FTY720 as an Immunotherapy to Accelerate Oral Wound Healing.

Orofacial clefts are the most common craniofacial congenital anomaly. Following cleft palate repair, up to 60% of surgeries have wound healing complications leading to oronasal fistula (ONF), a persistent connection between the roof of the mouth and the nasal cavity. The current gold standard methods for ONF repair use human allograft tissues; however, these procedures have risks of graft infection and/or rejection, requiring surgical revisions. Immunoregenerative therapies present a novel alternative approach to harness the body's immune response and enhance the wound healing environment. We utilized a repurposed FDA-approved immunomodulatory drug, FTY720, to reduce the egress of lymphocytes and induce immune cell fate switching toward pro-regenerative phenotypes. Here, we engineered a bilayer biomaterial system using Tegaderm™, a liquid-impermeable wound dressing, to secure and control the delivery of FTY720- nanofiber scaffolds (FTY720-NF). We optimized release kinetics of the bilayer FTY720-NF to sustain drug release for up to 7d with safe, efficacious transdermal absorption and tissue biodistribution. Through comprehensive immunophenotyping, our results illustrate a pseudotime pro-regenerative state transition in recruited hybrid immune cells to the wound site. Additional histological assessments established a significant difference in full thickness ONF closure in mice on Day 7 following treatment with bilayer FTY720-NF, compared to controls. These findings demonstrate the utility of immunomodulatory strategies for oral wound healing, better positing the field to develop more efficacious treatment options for pediatric patients. One Sentence Summary: Local delivery of bilayer FTY720-nanofiber scaffolds in an ONF mouse model promotes complete wound closure through modulation of pro-regenerative immune and stromal cells.

Innovative Molecular and Cellular Therapeutics in Cleft Palate Tissue Engineering.

Clefts of the lip and/or palate are the most prevalent orofacial birth defects occurring in about 1:700 live human births worldwide. Early postnatal surgical interventions are extensive and staged to bring about optimal growth and fusion of palatal shelves. Severe cleft defects pose a challenge to correct with surgery alone, resulting in complications and sequelae requiring life-long, multidisciplinary care. Advances made in materials science innovation, including scaffold-based delivery systems for precision tissue engineering, now offer new avenues for stimulating bone formation at the site of surgical correction for palatal clefts. In this study, we review the present scientific literature on key developmental events that can go awry in palate development and the common surgical practices and challenges faced in correcting cleft defects. How key osteoinductive pathways implicated in palatogenesis inform the design and optimization of constructs for cleft palate correction is discussed within the context of translation to humans. Finally, we highlight new osteogenic agents and innovative delivery systems with the potential to be adopted in engineering-based therapeutic approaches for the correction of palatal defects. Impact statement Tissue-engineered scaffolds supplemented with osteogenic growth factors have attractive, largely unexplored possibilities to modulate molecular signaling networks relevant to driving palatogenesis in the context of congenital anomalies (e.g., cleft palate). Constructs that address this need may obviate current use of autologous bone grafts, thereby avoiding donor-site morbidity and other regenerative challenges in patients afflicted with palatal clefts. Combinations of biomaterials and drug delivery of diverse regenerative cues and biologics are currently transforming strategies exploited by engineers, scientists, and clinicians for palatal cleft repair.

Pax9's Interaction With the Ectodysplasin Signaling Pathway During the Patterning of Dentition.

In these studies, we explored for the first time the molecular relationship between the paired-domain-containing transcription factor, Pax9, and the ectodysplasin (Eda) signaling pathway during mouse incisor formation. Mice that were deficient in both Pax9 and Eda were generated, and the status of dentition analyzed in all progeny using gross evaluation and histomorphometric means. When compared to wildtype controls, Pax9+/-Eda-/- mice lack mandibular incisors. Interestingly, Fgf and Shh signaling are down-regulated while Bmp4 and Lef1 appear unaffected. These findings suggest that Pax9-dependent signaling involves the Eda pathway and that this genetic relationship is important for mandibular incisor development. Studies of records of humans affected by mutations in PAX9 lead to the congenital absence of posterior dentition but interestingly involve agenesis of mandibular central incisors. The latter phenotype is exhibited by individuals with EDA or EDAR mutations. Thus, it is likely that PAX9, in addition to playing a role in the formation of more complex dentition, is also involved with EDA signaling in the initiation of odontogenesis within the incisal domain.

An Msx2-Sp6-Follistatin Pathway Operates During Late Stages of Tooth Development to Control Amelogenesis.

BACKGROUND: Ameloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both Msx2 and Sp6 transcription factors play important role. Msx2 and Sp6 mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis. METHODS: Two different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of Msx2 or Sp6 or both. Msx2 mutant mouse embryos or pups were used for in vivo studies. In situ hybridization, semi-quantitative and quantitative real time PCR were employed to study gene expression pattern. MatInspector was used to identify several potential putative Msx2 binding sites upstream of the murine Sp6 promoter region. Chromatin Immunoprecipitation (chIP) was used to confirm the binding of Msx2 to Sp6 promoter at the putative sites. RESULTS: Using the above methods we identified that (i) Msx2 and Sp6 exhibit overlapping expression in secretory ameloblasts, (ii) Sp6 expression is reduced in the Msx2 mouse mutant secretoty ameloblasts, and (iii) that Msx2, like Sp6 inhibits follistatin expression. Specifically, our loss-of function studies by silencing Msx2 and/or Sp6 in mouse dental epithelial (LS8) cells showed significant downregulation of Sp6 but upregulation of Fst expression. Transient transfection of Msx2 overexpression plasmid, up-regulated Sp6 and downregulated Fst expression. Additionally, using MatInspector, we identified several potential putative Msx2 binding sites, 3.5 kb upstream of the murine Sp6 promoter region. By chIP, we confirmed the binding of Msx2 to Sp6 promoter at these sites, thus suggesting that Sp6 is a direct target of Msx2. CONCLUSION: Collectively, these results show that Sp6 and Msx2 work in a concerted manner to form part of a network of transcription factors that operate during later stages of tooth development controlling ameloblast life cycle and amelogenesis.

Modeling Hypoxia Induced Factors to Treat Pulpal Inflammation and Drive Regeneration.

INTRODUCTION: The ability to resolve pulpal inflammation to achieve predictable regeneration of the dentin-pulp complex has remained elusive and presents a challenge for clinicians and researchers. Although the dentin-pulp complex can react naturally to injury by forming a bridge of reparative dentin that protects the pulp from further damage, this process is significantly impaired if inflammation persists. Because the secretion of inflammatory cytokines by injured pulpal cells causes significant pain and discomfort to patients, it is critical to resolve pulpal inflammation in a timely manner so as to create a microenvironment conducive for pulpal healing and reparative dentin formation. The emergent field of regenerative endodontics has encouraged the development and application of biologically driven therapies that take advantage of the intrinsic healing capacities of host cells within dental pulp and the periapical complex. METHODS: These studies were designed to test the hypothesis that exposure to hypoxic conditions can modulate the production of inflammatory cytokines/factors by mesenchymal cells in vitro. A multi-domain peptide hydrogel system that is highly conducive for the growth and differentiation of tooth-derived stem cells was used for these studies. Stem cells from human exfoliated deciduous teeth (SHEDs) were first cultured within 3-dimensional hydrogel constructs and then challenged with hypoxic stresses via addition of H2O2. RESULTS: MDP constructs were successfully generated, challenged with H2O2, decellularized and lyophilized, forming a potential biomaterial containing hypoxia induced repair molecules. The ability of cell-derived factors to convert the phenotype of lipopolysaccharide-primed macrophages from a proinflammatory to a pro-resolving state was examined in the presence of the lyophilized SHED cell constructs. CONCLUSIONS: Our data suggest that hypoxia induced SHED cell products can be captured within the hydrogel system and may be useful in the resolution of pulpal inflammation to create a favorable microenvironment for regeneration of the dentin-pulp complex.