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Cleft palate is a common craniofacial disorder involving multiple genetic and environmental predisposing factors. Currently, limited insight exists into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis. This study utilized thePax9-deficient mouse genetic model of cleft palate to investigate the role ofPax9in osteogenic differentiation. Single-nucleus transcriptomics and chromatin accessibility assays validated by whole-transcriptome and single-molecule spatial transcriptomics suggest a relationship between separatePax9+and osteogenic populations. Loss ofPax9resulted in premature osteogenic differentiation and bone maturation. The spatially restricted osteogenic domains inPax9−/−mice are bounded byDkk2, which normally interfaces withPax9in the mesenchyme. Together, these results confirm a regulatory role for the Wnt pathway in patterning of palatal bone, offering novel insights into the complex nature of developmental signaling and osteodifferentiation in the palate.SUMMARY STATEMENTNovel evidence of Wnt-mediated osteogenic differentiation and patterning of palatal bone is presented in a murine cleft palate model.Dkk2is implicated as a spatial regulator of palate ossification zones, in concert withPax9.

Human Wharton's jelly stem cells (HWJSC) can be efficiently isolated from the umbilical cord, and numerous reports have demonstrated that these cells can differentiate into several cell lineages. This fact, coupled with the high proliferation potential of HWJSC, makes them a promising source of stem cells for use in tissue engineering and regenerative medicine. However, their real potentiality has not been established to date. In the present study, we carried out a systematic review to determine the multilineage differentiation potential of HWJSC. After a systematic literature search, we selected 32 publications focused on the differentiation potential of these cells. Analysis of these studies showed that HWJSC display expanded differentiation potential toward some cell types corresponding to all three embryonic cell layers (ectodermal, mesodermal, and endodermal), which is consistent with their constitutive expression of key pluripotency markers such as OCT4, SOX2, and NANOG, and the embryonic marker SSEA4. We conclude that HWJSC can be considered cells in an intermediate state between multipotentiality and pluripotentiality, since their proliferation capability is not unlimited and differentiation to all cell types has not been demonstrated thus far. These findings support the clinical use of HWJSC for the treatment of diseases affecting not only mesoderm-type tissues but also other cell lineages. Impact statement Human Wharton's jelly stem cells (HWJSC) are mesenchymal stem cells that are easy to isolate and handle, and that readily proliferate. Their wide range of differentiation capabilities supports the view that these cells can be considered pluripotent. Accordingly, HWJSC are one of the most promising cell sources for clinical applications in advanced therapies.

Orofacial clefts and their management impose a substantial burden on patients, on their families, and on the health system. Under the current standard of care, affected patients are subjected to a lifelong journey of corrective surgeries and multidisciplinary management to replace bone and soft tissues, as well as restore esthetics and physiologic functions while restoring self-esteem and psychological health. Hence, a better understanding of the dynamic interplay of molecular signaling pathways at critical phases of palate development is necessary to pioneer novel prenatal interventions. Such pathways include transforming growth factor–β ( Tgfβ), sonic hedgehog ( Shh), wingless-integrated site ( Wnt)/β-catenin, bone morphogenetic protein ( Bmp), and fibroblast growth factor ( Fgf) and its associated receptors, among others. Here, we summarize commonly used surgical methods used to correct cleft defects postnatally. We also review the advances made in prenatal diagnostics of clefts through imaging and genomics and the various in utero surgical corrections that have been attempted thus far. An overview of how key mediators of signaling that drive palatogenesis are emphasized in the context of the framework and rationale for the development and testing of therapeutics in animal model systems and in humans is provided. The pros and cons of in utero therapies that can potentially restore molecular homeostasis needed for the proper growth and fusion of palatal shelves are presented. The theme advanced throughout this review is the need to develop preclinical molecular therapies that could ultimately be translated into human trials that can correct orofacial clefts at earlier stages of development.

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.

Regeneration of the pulp-dentin complex with stem cells is a potential alternative to conventional root canal treatments. Human dental pulp stem cells (hDPSCs) have been extensively studied because of their ability to proliferate and differentiate into mineralized dental and non-dental tissues. Here we combined hDPSCs with two types of injectable poly-l-lactic acid (PLLA) microsphere with a nanofibrous or smooth surface to form bioactive injectable aggregates, and examined their ability to promote pulp regeneration in the root canal in an in vivo model. We investigated the biocompatibility, biosafety and odontogenic potential of fibrous (F-BIM) and smooth bioactive injectable microspheres (S-BIM) in vitro and in vivo. Our results demonstrated that PLLA microspheres and hDPSCs were able to form bioactive injectable aggregates that promoted dentin regeneration in both in vitro and in vivo models. Our results suggest that F-BIM and S-BIM may induce dentinogenesis upon in vivo grafting, and propose that the potential usefulness of the microsphere-hDPSC aggregates described here should be evaluated in clinical settings. Copyright © 2017 John Wiley & Sons, Ltd.

The aim of this review is to investigate the growth of diversity and inclusion in global academic dental research with a focus on gender equality. A diverse range of research methodologies were used to conduct this review, including an extensive review of the literature, engagement of key informants in dental academic leadership positions around the world, and review of current data from a variety of national and international organizations. Results provide evidence of gender inequalities that currently persist in dental academics and research. Although the gender gap among graduating dental students in North America and the two most populous countries in Europe (the United Kingdom and France) has been narrowed, women make up 30% to 40% of registered dentists in countries throughout Europe, Oceania, Asia, and Africa. In academic dentistry around the globe, greater gender inequality was found to correlate with higher ranking academic and leadership positions in the United States, United Kingdom, several countries in European Union, Japan, and Saudi Arabia. Further disparities are noted in the dental research sector, where women make up 33% of dental researchers in the European Union, 35% in North America, 55% in Brazil, and 25% in Japan. Family and societal pressures, limited access to research funding, and lack of mentoring and leadership training opportunities are reported as also contributing to gender inequalities. To continue advancing gender equality in dental academia and research, efforts should be geared toward the collection and public dissemination of data on gender-specific distributions. Such evidence-driven information will guide the selection of future strategies and best practices for promoting gender equity in the dental workforce, which provides a major pipeline of researchers and scholars for the dental profession.

Background Despite the strides made in understanding the complex network of key regulatory genes and cellular processes that drive palate morphogenesis, patients suffering from these conditions face treatment options that are limited to complex surgeries and multidisciplinary care throughout life. Hence, a better understanding of how molecular interactions drive palatal growth and fusion is critical for the development of treatment and preventive strategies for cleft palates in humans. Our previous work demonstrated that Pax9-dependent Wnt signaling is critical for the growth and fusion of palatal shelves. We showed that controlled intravenous delivery of small molecule Wnt agonists specifically blocks the action of Dkks (inhibitors of Wnt signaling) and corrects secondary palatal clefts in Pax9-/- mice. While these data underscore the importance of the functional upstream relationship of Pax9 to the Wnt pathway, not much is known about how the genetic nature of Pax9's interactions in vivo and how it modulates the actions of these downstream effectors during palate formation. Results Here, we show that the genetic reduction of Dkk1 during palatogenesis corrected secondary palatal clefts in Pax9-/- mice with restoration of Wnt signaling activities. In contrast, genetically induced overexpression of Dkk1 mice phenocopied the defects in tooth and palate development visible in Pax9-/- strains. Results of ChIP-qPCR assays showed that Pax9 can bind to regions near the transcription start sites of Dkk1 and Dkk2 as well as the intergenic region of Wnt9b and Wnt3 ligands that are downregulated in Pax9-/- palates. Conclusions Taken together, these data suggest that the molecular mechanisms underlying Pax9's role in modulating Wnt signaling activity likely involve the inhibition of Dkk expression and the control of Wnt ligands during palatogenesis.

An international advisory group met at the National Institutes of Health in Bethesda, Maryland in 2017, to discuss a new classification system for the ectodermal dysplasias that would integrate both clinical and molecular information. We propose the following, a working definition of the ectodermal dysplasias building on previous classification systems and incorporating current approaches to diagnosis: ectodermal dysplasias are genetic conditions affecting the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Genetic variations in genes known to be associated with ectodermal dysplasias that affect only one derivative of the ectoderm (attenuated phenotype) will be grouped as non-syndromic traits of the causative gene (e.g. non-syndromic hypodontia or missing teeth associated with pathogenic variants of EDA “ectodysplasin” ). Information for categorization and cataloging includes the phenotypic features, Online Mendelian Inheritance in Man number, mode of inheritance, genetic alteration, major developmental pathways involved (e.g. EDA, WNT “wingless-type”, TP63 “tumor protein p63”) or the components of complex molecular structures ( e.g. connexins, keratins, cadherins).

Hydrogels are homogenous materials that are limited in their ability to form oriented multilayered architecture in three-dimensional (3D) tissue constructs. Current techniques have led to advancements in this area. Such techniques often require extra devices and/or involve complex processes that are inaccessible to many laboratories. Here is described a one-step methodology that permits reliable alignment of cells into multiple layers using a self-assembling multidomain peptide (MDP) hydrogels. We characterized the structural features, viability, and molecular properties of dental pulp cells fabricated with MDP and demonstrated that manipulation of the layering of cells in the scaffolds was achieved by decreasing the weight by volume percentage (w/v%) of MDP contained within the scaffold. This approach allows cells to remodel their environment and enhanced various gene expression profiles, such as cell proliferation, angiogenesis, and extracellular matrix (ECM) remodeling-related genes. We further validated our approach for constructing various architectural configurations of tissues by fabricating cells into stratified multilayered and tubular structures. Our methodology provides a simple, rapid way to generate 3D tissue constructs with multilayered architectures. This method shows great potential to mimic in vivo microenvironments for cells and may be of benefit in modeling more complex tissues in the field of regenerative medicine.

Preservation of a vital dental pulp is a central goal of restorative dentistry. Currently, there is significant interest in the development of tissue engineering scaffolds that can serve as biocompatible and bioactive pulp-capping materials, driving dentin bridge formation without causing cytotoxic effects. Our earlier in vitro studies described the biocompatibility of multidomain peptide (MDP) hydrogel scaffolds with dental pulp–derived cells but were limited in their ability to model contact with intact 3-dimensional pulp tissues. Here, we utilize an established ex vivo mandible organ culture model to model these complex interactions. MDP hydrogel scaffolds were injected either at the interface of the odontoblasts and the dentin or into the pulp core of mandible slices and subsequently cultured for up to 10 d. Histology reveals minimal disruption of tissue architecture adjacent to MDP scaffolds injected into the pulp core or odontoblast space. Additionally, the odontoblast layer is structurally preserved in apposition to the MDP scaffold, despite being separated from the dentin. Alizarin red staining suggests mineralization at the periphery of MDP scaffolds injected into the odontoblast space. Immunohistochemistry reveals deposition of dentin sialophosphoprotein by odontoblasts into the adjacent MDP hydrogel, indicating continued functionality. In contrast, no mineralization or dentin sialophosphoprotein deposition is evident around MDP scaffolds injected into the pulp core. Collagen III expression is seen in apposition to gels at all experimental time points. Matrix metalloproteinase 2 expression is observed associated with centrally injected MDP scaffolds at early time points, indicating proteolytic digestion of scaffolds. Thus, MDP scaffolds delivered centrally and peripherally within whole dental pulp tissue are shown to be biocompatible, preserving local tissue architecture. Additionally, odontoblast function and pulp vitality are sustained when MDP scaffolds are intercalated between dentin and the odontoblast region, a finding that has significant implications when considering these materials as pulp-capping agents.

Delivery of small molecules and drugs to tissues is a mainstay of several tissue engineering strategies. Next generation treatments focused on localized drug delivery offer a more effective means in dealing with refractory healing when compared to systemic approaches. Here we describe a novel multidomain peptide hydrogel that capitalizes on synthetic peptide chemistry, supramolecular self-assembly and cytokine delivery to tailor biological responses. This material is biomimetic, shows shear stress recovery and offers a nanofibrous matrix that sequesters cytokines. The biphasic pattern of cytokine release results in the spatio-temporal activation of THP-1 monocytes and macrophages. Furthermore, macrophage–material interactions are promoted without generation of a proinflammatory environment. Subcutaneous implantation of injectable scaffolds showed a marked increase in macrophage infiltration and polarization dictated by cytokine loading as early as 3 days, with complete scaffold resorption by day 14. Macrophage interaction and response to the peptide composite facilitated the (i) recruitment of monocytes/macrophages, (ii) sustained residence of immune cells until degradation, and (iii) promotion of a pro-resolution M2 environment. Our results suggest the potential use of this injectable cytokine loaded hydrogel scaffold in a variety of tissue engineering applications.

To date, surgical interventions are the only means by which craniofacial anomalies can be corrected so that function, esthetics, and the sense of well-being are restored in affected individuals. Unfortunately, for patients with cleft palate—one of the most common of congenital birth defects—treatment following surgery is prolonged over a lifetime and often involves multidisciplinary regimens. Hence, there is a need to understand the molecular pathways that control palatogenesis and to translate such information for the development of noninvasive therapies that can either prevent or correct cleft palates in humans. Here, we use the well-characterized model of the Pax9−/−mouse, which displays a consistent phenotype of a secondary cleft palate, to test a novel therapeutic. Specifically, we demonstrate that the controlled intravenous delivery of a novel mouse monoclonal antibody replacement therapy, which acts as an agonist for the ectodysplasin (Eda) pathway, can resolve cleft palate defects in Pax9−/−embryos in utero. Such pharmacological interventions did not reverse the arrest in tooth, thymus, and parathyroid gland development, suggesting that the relationship of Pax9 to the Eda/Edar pathway is both unique and essential for palatogenesis. Expression analyses and unbiased gene expression profiling studies offer a molecular explanation for the resolution of palatal defects, showing that Eda and Edar-related genes are expressed in normal palatal tissues and that the Eda/Edar signaling pathway is downstream of Pax9 in palatogenesis. Taken together, our data uncover a unique relationship between Pax9 and the Eda/Edar signaling pathway that can be further exploited for the development of noninvasive, safe, and effective therapies for the treatment of cleft palate conditions and other single-gene disorders affecting the craniofacial complex.

Clefts of the palate and/or lip are among the most common human craniofacial malformations and involve multiple genetic and environmental factors. Defects can only be corrected surgically and require complex life-long treatments. Our studies utilized the well-characterized Pax9−/− mouse model with a consistent cleft palate phenotype to test small-molecule Wnt agonist therapies. We show that the absence of Pax9 alters the expression of Wnt pathway genes including Dkk1 and Dkk2, proven antagonists of Wnt signaling. The functional interactions between Pax9 and Dkk1 are shown by the genetic rescue of secondary palate clefts in Pax9−/−Dkk1f/+;Wnt1Cre embryos. The controlled intravenous delivery of small-molecule Wnt agonists (Dkk inhibitors) into pregnant Pax9+/− mice restored Wnt signaling and led to the growth and fusion of palatal shelves, as marked by an increase in cell proliferation and osteogenesis in utero, while other organ defects were not corrected. This work underscores the importance of Pax9-dependent Wnt signaling in palatogenesis and suggests that this functional upstream molecular relationship can be exploited for the development of therapies for human cleft palates that arise from single-gene disorders.

Clefts of the palate and/or lip arise in about 1/700 human live births and are caused by multiple genetic and environmental factors. Studies of mouse knockout models of cleft palate have improved our understanding of the molecular control of palatogenesis. While it is known that Pax9 regulates palatogenesis through Bmp, Fgf and Shh signaling, there is still much to learn about its precise relationship with other pathways. Here we show that alterations of Wnt expression and decreased Wnt activity inPax9-/-palatal shelves are a result of Pax9’s ability to directly bind and repress the promoters ofDkk1andDkk2,proteins that antagonize Wnt signaling. The delivery of small-molecule Dkk inhibitors (Wnt agonists) into the tail-veins of pregnantPax9+/-mice from E10.5 to E14.5 restored Wnt signaling, promoted cell proliferation, bone formation and restored the fusion of palatal shelves inPax9-/-embryos. In contrast, other organ defects inPax9mutants were not corrected. These data uncover a unique molecular relationship betweenPax9and Wnt genes in palatogenesis and offer a new approach for treating cleft palates in humans.Summary StatementThese studies demonstrate that the Pax9/Wnt genes regulate murine palatogenesis. This unique molecular relationship is proven by the correction of cleft defects inPax9-deficient mice through Wnt agonist therapies.

Mutations in the WNT10A gene were first detected in the rare syndrome odonto-onycho-dermal dysplasia (OODD, OMIM257980) but have now also been found to cause about 35–50% of selective tooth agenesis (STHAG4, OMIM150400), a common disorder that mostly affects the permanent dentition. In our random sample of tooth agenesis patients, 40% had at least one mutation in the WNT10A gene. The WNT10A Phe228Ile variant alone reached an allele frequency of 0.21 in the tooth agenesis cohort, about 10 times higher than the allele frequency reported in large SNP databases for Caucasian populations. Patients with bi-allelic WNT10A mutations have severe tooth agenesis while heterozygous individuals are either unaffected or have a mild phenotype. Mutations in the coding areas of the WNT10B gene, which is co-expressed with WNT10A during odontogenesis, and the WNT6 gene which is located at the same chromosomal locus as WNT10A in humans, do not contribute to the tooth agenesis phenotype. © 2014 Wiley Periodicals, Inc.

In dentistry, the maintenance of a vital dental pulp is of paramount importance because teeth devitalized by root canal treatment may become more brittle and prone to structural failure over time. Advanced carious lesions can irreversibly damage the dental pulp by propagating a sustained inflammatory response throughout the tissue. Although the inflammatory response initially drives tissue repair, sustained inflammation has an enormously destructive effect on the vital pulp, eventually leading to total necrosis of the tissue and necessitating its removal. The implications of tooth devitalization have driven significant interest in the development of bioactive materials that facilitate the regeneration of damaged pulp tissues by harnessing the capacity of the dental pulp for self-repair. In considering the process by which pulpitis drives tissue destruction, it is clear that an important step in supporting the regeneration of pulpal tissues is the attenuation of inflammation. Macrophages, key mediators of the immune response, may play a critical role in the resolution of pulpitis because of their ability to switch to a proresolution phenotype. This process can be driven by the resolvins, a family of molecules derived from fatty acids that show great promise as therapeutic agents. In this review, we outline the importance of preserving the capacity of the dental pulp to self-repair through the rapid attenuation of inflammation. Potential treatment modalities, such as shifting macrophages to a proresolving phenotype with resolvins are described, and a range of materials known to support the regeneration of dental pulp are presented.

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies.

Background: Understanding of the self-renewal and differentiation of dental epithelial stem cells (DESCs) is important for tooth regeneration therapies. Results: Depletion of FGF signaling suppressed self-renewal and led to differentiation of DESCs. Conclusion: FGF signaling is essential for maintenance of DESCs. Significance: The finding sheds new light on the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated. A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.

Biomimetic approaches are widely used in scaffolding designs to enhance tissue regeneration. In this study, we integrated noncollagenous proteins (NCPs) from bone extracellular matrix (ECM) with three-dimensional nanofibrous gelatin (NF-Gelatin) scaffolds to form an artificial matrix (NF-Gelatin-NCPs) mimicking both the nano-structured architecture and chemical composition of natural bone ECM. Through a chemical coupling process, the NCPs were evenly distributed over all the surfaces (inner and outer) of the NF-gelatin-NCPs. The in vitro study showed that the number of osteoblasts (MC3T3-E1) on the NF-Gelatin-NCPs was significantly higher than that on the NF-Gelatin after being cultured for 14 days. Both the alkaline phosphatase (ALP) activity and the expression of osteogenic genes (OPN, BSP, DMP1, CON, and Runx2) were significantly higher in the NF-Gelatin-NCPs than in the NF-Gelatin at 3 weeks. Von Kossa staining, backscattered scanning electron microscopy, and microcomputed tomography all revealed a higher amount of mineral deposition in the NF-Gelatin-NCPs than in the NF-Gelatin after in vitro culturing for 3 weeks. The in vivo calvarial defect study indicated that the NF-Gelatin-NCPs recruited more host cells to the defect and regenerated a higher amount of bone than the controls after implantation for 6 weeks. Immunohistochemical staining also showed high-level mineralization of the bone matrix in the NF-Gelatin-NCPs. Taken together, both the in vitro and in vivo results confirmed that the incorporation of NCPs onto the surfaces of the NF-Gelatin scaffold significantly enhanced osteogenesis and mineralization. Biomimetic engineering of the surfaces of the NF-Gelatin scaffold with NCPs, therefore, is a promising strategy to enhance bone regeneration.

The detection of gene mutations in patients with congenitally missing teeth is not very complicated; however, proving causality is often quite difficult. Here, we report the detection of a substitution mutation, A42P, within the prodomain of bone morphogenetic protein 4 (BMP4) in a small family with tooth agenesis and describe a functional alteration that may be responsible for the tooth phenotype. As BMP4 is essential for the development of teeth and also for many other organs, it would be of considerable interest to find a BMP4 mutation that is associated only with tooth agenesis. Our in vitro investigations revealed that the A42P mutation neither affected processing and secretion of BMP4 nor altered functional properties, such as the induction of alkaline phosphatase or signaling through Smad1/5/8 phosphorylation by the mature BMP4 ligand. However, immunofluorescence staining revealed that the prodomains of BMP4 which harbor the A42P substitution form fibrillar structures around transfected cells in culture and that this fibrillar network is significantly decreased when mutant prodomains are expressed. Our finding suggests that in vivo, BMP4 prodomain behavior might also be altered by the mutation and could influence storage or transport of mature BMP4 in the extracellular matrix of the developing tooth.

Implantable devices have improved the lives of many patients, but implant-associated infection remains a serious complication with significant morbidity and mortality. In recent years, the role of biofilm in the development of these infections has become increasingly recognized, and strategies to combat biofilm are beginning to proliferate in the literature. This review will explore how implant-associated infections have historically been treated, where the research currently stands, and the future trends of infection control.

This is a critical time in the history of the dental profession for it to fully embrace the responsibility to safeguard its reputation as a learned profession. In this golden era of scientific and technological advances, opportunities abound to create new diagnostics, preventions, treatments, and cures to improve oral health. Dental schools are the largest national resource entrusted with the responsibility to educate, train, and retain oral health researchers who can leverage such technologies and research opportunities that will benefit the profession at large as well as patients. This article reemphasizes the theme that research training and scholarship must be inextricably woven into the environment and culture in dental schools to ensure the future standing of the profession. An overview of the history of support provided by the National Institutes of Health and National Institute of Dental and Craniofacial Research for the training and career development of dentist-scientists is presented. In addition, new data on the outcomes of such investments are presented along with a comparison with other health professions. This overview underscores the need to expand the capacity of a well-trained cadre of oral health researchers through the reengineering of training programs. Such strategies will best prepare future graduates for team science, clinical trials, and translational research as well as other emerging opportunities. The urgent need for national organizations like the American Dental Association, American Dental Education Association, and American Association for Dental Research to create new alliances and novel initiatives to assist dental schools and universities in fulfilling their research mission is emphasized. To ignore such calls for action is to disavow a valuable legacy inherited by the dental profession. This article was written as part of the project "Advancing Dental Education in the 21st Century."

Clefts of the palate and/or lip are the most common among human craniofacial malformations and involve multiple genetic and environmental factors. Defects can only be corrected surgically and require complex life-long treatments. Our studies utilized the well-characterized Pax9–/– mouse model with a consistent cleft palate phenotype to test small-molecule Wnt agonist therapies. We first show that the absence of Pax9 alters the expression of Wnt pathway genes including Dkk1 and Dkk2, proven antagonists of Wnt signaling. The functional interactions between Pax9 and Dkk1 is shown by the genetic rescue of secondary palate clefts in Pax9–/–Dkk1f/+;Wnt1Cre embryos. The controlled intravenous delivery of small-molecule Wnt agonists (Dkk inhibitors) into pregnant Pax9+/– mice restored Wnt signaling and led to the growth and fusion of palatal shelves as marked by an increase in cell proliferation and osteogenesis in-utero while other organ defects were not corrected. This work underscores the importance of Pax9-dependent Wnt signaling in palatogenesis and suggests that such a functional upstream molecular relationship can be exploited for the development of therapies for human cleft palates that arise from single gene disorders.

The 4th Annual AADR Fall Focused Symposium (FFS), “Oral Health Disparities Research and the Future Face of America”, took place on November 3-4, 2011 in Washington, DC. The FFS strategy was developed by the AADR to help provide additional opportunities for members to engage in research discussions during the year by identifying specific research topics of interest among the 21 Scientific Groups and 4 Networks of the IADR and targeting a focused topic area for the FFS. The conference attracted an international group of approximately 120 registrants, including participants from Canada, India, Mexico, and China; 4 oral sessions and 32 poster presentations were offered.

Background and Objective Dentin sialophosphoprotein (DSPP) and its cleaved products, dentin phosphoprotein (DPP) and dentin sialoprotein (DSP), play important roles in biomineralization. Recently, we observed that DSPP is highly expressed in the alveolar bone and cementum, indicating that this molecule may play an important role in the formation and maintenance of a healthy periodontium, and its deletion may cause increased susceptibility to periodontal diseases. The objective of this investigation was to study the effects of Dspp ablation on periodontal tissues by analyzing Dspp null mice. Material and Methods Newborn to 6-mo-old Dspp null mice were examined, and the 3- and 6-mo-old Dspp null mice were characterized in detail using X-ray radiography, histology and scanning electron microscopy (backscattered as well as resin-infiltrating). Wild-type mice of the same age groups served as the normal controls. Results The Dspp null mice showed significant loss of alveolar bone and cementum, particularly in the furcation and interproximal regions of the molars. The alveolar bone appeared porous while the quantity of cementum was reduced in the apical region. The canalicular systems and osteocytes in the alveolar bone were abnormal, with reduced numbers of canaliculi and altered osteocyte morphology. The loss of alveolar bone and cementum along with the detachment of the periodontal ligaments (PDL) led to the apical migration of the epithelial attachment and formation of periodontal pockets. Conclusion Inactivation of DSPP leads to the loss of alveolar bone and cementum and increased susceptibility to bacterial infections in PDL of Dspp null mice. The fact that the loss of DSPP results in periodontal diseases indicates that this molecule plays a vital role in maintaining the health of the periodontium.

Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclearmore » DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.« less

Dentinogenesis imperfecta (DGI) is a hereditary defect of dentin, a calcified tissue that is the most abundant component of teeth. Most commonly, DGI is manifested as a part of osteogenesis imperfecta (OI) or the phenotype is restricted to dental findings only. In the latter case, DGI is caused by mutations in the DSPP gene, which codes for dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Although these two proteins together constitute the majority of noncollagenous proteins of the dentin, little is known about their transcriptional regulation. Here we demonstrate that mice overexpressing the Trps1 transcription factor (Col1a1-Trps1 mice) in dentin-producing cells, odontoblasts, present with severe defects of dentin formation that resemble DGI. Combined micro–computed tomography (µCT) and histological analyses revealed tooth fragility due to severe hypomineralization of dentin and a diminished dentin layer with irregular mineralization in Col1a1-Trps1 mice. Biochemical analyses of noncollagenous dentin matrix proteins demonstrated decreased levels of both DSP and DPP proteins in Col1a1-Trps1 mice. On the molecular level, we demonstrated that sustained high levels of Trps1 in odontoblasts lead to dramatic decrease of Dspp expression as a result of direct inhibition of the Dspp promoter by Trps1. During tooth development Trps1 is highly expressed in preodontoblasts, but in mature odontoblasts secreting matrix its expression significantly decreases, which suggests a Trps1 role in odontoblast development. In these studies we identified Trps1 as a potent inhibitor of Dspp expression and the subsequent mineralization of dentin. Thus, we provide novel insights into mechanisms of transcriptional dysregulation that leads to DGI. © 2012 American Society for Bone and Mineral Research.

Introduction Recent successes in dental pulp engineering indicate that regenerative treatment strategies in endodontics are feasible. Clinically, revascularization procedures render completion of root formation in immature teeth. The generation of a pulp-like tissue after seeding of dental pulp stem cells into dentin discs or cylinders and transplantation in vivo is possible. In this experimental setup, which mimics the situation in the root canal, the pretreatment of dentin might influence cellular behavior at the cell-dentin interface. Thus, the objective of this study was to investigate whether dentin conditioning can determine cell fate. Methods Dental pulp stem cells (DPSCs) were seeded into a growth factor–laden peptide hydrogel, transferred into dentin cylinders, and transplanted subcutaneously into immunocompromised mice. Before cell seeding, dentin cylinders were either pretreated with sodium hypochloride (NaOCl) or conditioned with EDTA. The constructs were explanted after 6 weeks and subjected to histological and immunohistochemical analysis. Results In dentin treated with NaOCl, resorption lacunae were found at the cell-dentin interface created by multinucleated cells with clastic activity. After conditioning with EDTA, DPSCs adjacent to the dentin formed an intimate association with the surface, differentiated into odontoblasts-like cells that expressed dentin sialoprotein, and extended cellular processes into the dentinal tubules. A vascularized soft connective tissue similar to dental pulp was observed inside the dentin cylinder. Conclusions Dentin conditioning considerably influences DPSC fate when seeded in close proximity to dentin. This information might be critical for optimized strategic planning for future regenerative endodontic treatment.

In the early years of the new millennium, the National Institute of Dental and Craniofacial Research of the National Institutes of Health began funding Oral Health Research Education Grants using the R25 mechanism to promote the application of basic and clinical research findings to clinical training and to encourage students to pursue careers in oral health research. This report describes the impact of an R25 grant awarded to the Texas A&M Health Science Center’s Baylor College of Dentistry (BCD) on its curriculum and faculty development efforts. At BCD, the R25 grant supports a multipronged initiative that employs clinical research as a vehicle for acquainting both students and faculty with the tools of evidence-based dentistry (EBD). New coursework and experiences in all four years of the curriculum plus a variety of faculty development offerings are being used to achieve this goal. Progress on these fronts is reflected in a nascent EBD culture characterized by increasing participation and buy-in by students and faculty. The production of a new generation of dental graduates equipped with the EBD skill set as well as a growing nucleus of faculty members who can model the importance of evidence-based practice is of paramount importance for the future of dentistry.

Dentin matrix protein 1 (DMP1), an acidic protein important to the formation of bone and dentin, primarily exists as the processed NH(2)-terminal and COOH-terminal fragments in the extracellular matrix of the two tissues. Previous in vitro studies showed that the substitution of residue Asp(213) by Ala(213) (D213A) at a cleavage site blocked the processing of mouse DMP1 in cells. In this study, we generated transgenic mice expressing mutant D213A-DMP1 (WT/D213A-Tg mice) to test the hypothesis that the proteolytic processing of DMP1 is an activation step essential to osteogenesis. By crossbreeding WT/D213A-Tg mice with Dmp1 knock-out (Dmp1-KO) mice, we obtained mice expressing D213A-DMP1 in a Dmp1-KO background; these mice will be referred to as "Dmp1-KO/D213A-Tg" mice. Biochemical, radiological, and morphological approaches were used to characterize the skeletal phenotypes of Dmp1-KO/D213A-Tg mice compared with wild-type mice, Dmp1-KO mice, and Dmp1-KO mice expressing the normal Dmp1 transgene. Protein chemistry analyses showed that DMP1 was barely cleaved in the bone of the Dmp1-KO/D213A-Tg mice, indicating that D213A substitution effectively blocked the proteolytic processing of DMP1 in vivo. While the expression of the normal Dmp1 transgene completely rescued the phenotypic skeletal changes of the Dmp1-KO mice, the expression of the mutant D213A-Dmp1 transgene failed to do so. These results indicate that the full-length form of DMP1 is an inactive precursor and its proteolytic processing is an activation step essential to the biological functions of this protein in osteogenesis.

Matrix Gla protein (MGP) is, along with osteocalcin, a skeletal member of the family of extracellular mineral-binding Gla proteins. Although the precise function of these proteins remains obscure, circumstantial evidence suggests that they play a role in endochondral ossification. As a first step toward understanding MGP function we have performed a preliminary characterization of its promoter element and studied the developmental pattern of expression of this gene. DNA transfection experiments indicate that the mouse MGP promoter functions better in cells expressing the MGP gene than in cells that do not express the gene. During mouse development, MGP gene expression is detectable as early as day 10.5 of embryonic development (E10.5), before any skeletal structures are identifiable. In situ hybridization analysis shows that MGP mRNA is initially present at the mesenchymal epithelial interphase in lung and limb buds. As development proceeds, MGP gene is predominantly expressed in cells of the chondrocytic lineage in areas that will undergo endochondral ossification as well as in areas that will remain cartilaginous, such as the trachea and bronchi. In growth plate cartilage, MGP mRNA is present in resting, proliferative, and late hypertrophic chondrocytes. Surprisingly, MGP mRNA is absent from the early hypertrophic chondrocytes and from the osteoblasts. Finally, the MGP gene is expressed at a lower level in kidney medulla and uterus smooth muscle but not in brain, spleen, or heart during development. This study demonstrates that during development MGP gene expression occurs early and is predominant at the epithelial mesenchymal interfaces, principally of lung and limb buds, and in cells of the chondrocytic lineage. This finding raises the intriguing possibility that MGP may play distinct roles during embryogenesis and in the adult organism.

To study the fate of bone cells, we used the transferase-mediated, biotin-dUTP nick end-labeling (TUNEL) assay to detect DNA fragmentation during the formation of intramembranous and endochondral bone in newly born hamsters, mice, and rats. In alveolar bone forming around the developing tooth crowns, DNA fragmentation was found in three cell types: TRAP-negative mononuclear cells at the bone surface, osteocytes, and some but not all nuclei of TRAP-positive osteoclasts. Osteoblasts did not undergo DNA fragmentation. A strong positive correlation was found between contacts of TUNEL-positive osteocytes and osteoclasts. Extracellular bone matrix also stained occasionally for the presence of DNA fragments. During endochondral bone formation, TUNEL staining was detected in late hypertrophic chondrocytes of the epiphyseal growth plate. During rapid longitudinal growth of long bones, TUNEL-positive hypertrophic chondrocytes were found coincident with or slightly after invasion of blood vessels from the diaphysis. However, during slow longitudinal growth and in secondary ossification centers, DNA fragmentation was seen in hypertrophic chondrocytes still located within their lacunae. We conclude that some of the osteocytes in deeper layers of bone die within their lacuna and disperse nuclear fragments over the extracellular matrix, that a majority of the osteocytes are phagocytosed and degraded by osteoclasts at sites of intense bone resorption, and that during endochondral ossification, substantial numbers of late hypertrophic chondrocyte cells undergo cell death.

Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the 18 mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants, the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wild-type PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.

Dental caries remains one of the most prevalent infectious diseases in the world. So far, available treatment methods rely on the replacement of decayed soft and mineralized tissue with inert biomaterials alone. As an approach to develop novel regenerative strategies and engineer dental tissues, two dental stem cell lines were combined with peptide-amphiphile (PA) hydrogel scaffolds. PAs self-assemble into three-dimensional networks of nanofibers, and living cells can be encapsulated. Cell-matrix interactions were tailored by incorporation of the cell adhesion sequence RGD and an enzyme-cleavable site. SHED (stem cells from human exfoliated deciduous teeth) and DPSC (dental pulp stem cells) were cultured in PA hydrogels for 4 weeks using different osteogenic supplements. Both cell lines proliferate and differentiate within the hydrogels. Histologic analysis shows degradation of the gels and extracellular matrix production. However, distinct differences between the two cell lines can be observed. SHED show a spindle-shaped morphology, high proliferation rates, and collagen production, resulting in soft tissue formation. In contrast, DPSC reduce proliferation, but exhibit an osteoblast-like phenotype, express osteoblast marker genes, and deposit mineral. Since the hydrogels are easy to handle and can be introduced into small defects, this novel system might be suitable for engineering both soft and mineralized matrices for dental tissue regeneration.

Axenfeld-Rieger syndrome (ARS) patients with PITX2 point mutations exhibit a wide range of clinical features including mild craniofacial dysmorphism and dental anomalies. Identifying new PITX2 targets and transcriptional mechanisms are important to understand the molecular basis of these anomalies. Chromatin immunoprecipitation assays demonstrate PITX2 binding to the FoxJ1 promoter and PITX2C transgenic mouse fibroblasts and PITX2-transfected cells have increased endogenous FoxJ1 expression. FoxJ1 is expressed at embryonic day 14.5 (E14.5) in early tooth germs, then down-regulated from E15.5-E17.5 and re-expressed in the inner enamel epithelium, oral epithelium, tongue epithelium, sub-mandibular salivary gland and hair follicles during E18.5 and neonate day 1. FoxJ1 and Pitx2 exhibit overlapping expression patterns in the dental and oral epithelium. PITX2 activates the FoxJ1 promoter and, Lef-1 and beta-catenin interact with PITX2 to synergistically regulate the FoxJ1 promoter. FoxJ1 physically interacts with the PITX2 homeodomain to synergistically regulate FoxJ1, providing a positive feedback mechanism for FoxJ1 expression. Furthermore, FoxJ1, PITX2, Lef-1 and beta-catenin act in concert to activate the FoxJ1 promoter. The PITX2 T68P ARS mutant protein physically interacts with FoxJ1; however, it cannot activate the FoxJ1 promoter. These data indicate a mechanism for the activity of the ARS mutant proteins in specific cell types and provides a basis for craniofacial/ tooth anomalies observed in these patients. These data reveal novel transcriptional mechanisms of FoxJ1 and demonstrate a new role of FoxJ1 in oro-facial morphogenesis.

The paired-domain transcription factor PAX9 plays a critical role in tooth development, as heterozygous mutations in PAX9 have been shown to be associated with human tooth agenesis. In this study, we report 2 novel missense mutations, gly6arg (G6R) and ser43lys (S43K), in the paired domain of PAX9 in Chinese patients with varying degrees of nonsyndromic tooth agenesis. Excluding third molars, the individual with the G6R mutation was missing 2 mandibular incisors and a maxillary premolar, while the phenotype of individuals with the S43K mutation consisted of peg-shaped upper lateral incisors and missing molars, premolars and canines. As these 2 mutations occur at highly conserved amino acids in the PAX gene family and between different species, we further analyzed the effects of the mutations on the function of the resulting proteins. Immunofluorescence and immunoblotting studies showed that the mutations did not alter nuclear localization in mammalian cells. Gel shift and super shift assays indicate that both mutant proteins bound DNA at a lower level than the normal protein, with G6R having a greater affinity for DNA than S43K. Likewise, the G6R protein was able to transcriptionally activate a Bmp4 promoter construct to a greater extent than S43K. Our finding that the severity of tooth agenesis in the patients was correlated to the DNA-binding capacity of the mutated PAX9 9proteins supports the hypothesis that DNA binding is responsible for the genetic defect.