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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.

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.

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).

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.

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.

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.

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.

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.

Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH2- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH2 termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp181 (corresponding to Asp197 of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH2 terminus of Asp197 of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp197 is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp197 with Ala197 by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH2 terminus of Asp197 is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH2-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser74 in rat DMP1 (Ser89 in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser89, we substituted Ser89 by Gly89. Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser89 in mouse DMP1.

Supernumerary teeth are defined as those that are present in excess of the normal complement of human dentition and represent a unique developmental anomaly of patterning and morphogenesis. Despite the wealth of information generated from studies on normal tooth development, the genetic etiology and molecular mechanisms that lead to congenital deviations in tooth number are poorly understood. For developmental biologists, the phenomenon of supernumerary teeth raises interesting questions about the development and fate of the dental lamina. For cell and molecular biologists, the anomaly of supernumerary teeth inspires several questions about the actions and interactions of transcription factors and growth factors that coordinate morphogenesis, cell survival and programmed cell death. For human geneticists, the condition as it presents itself in either syndromic or non-syndromic forms offers an opportunity to discover mutations in known or novel genes. For clinicians faced with treating the dental complications that arise from the presence of supernumerary teeth, knowledge about the basic mechanisms involved is essential. The purpose of this manuscript is to review current knowledge about how supernumerary teeth form, the molecular insights gained through studies on mice that are deficient in certain tooth signaling molecules and the questions that require further research in the field.

Primary cells in culture have a limited capacity to divide and soon reach a non-proliferative state. This cellular senescence limits the investigation of cells derived from human pulp concerning cellular pathways, gene regulation, mechanisms of dentin formation, or responses to material exposure. To overcome this problem, primary human pulp-derived cells were established and transfected with a plasmid containing coding sequences of Simian Virus 40 (SV40) large T-antigen. This resulted in the establishment of several cell clones showing an extension of life span. Expression of T-antigen transcripts and protein was verified by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Primary human pulp cells were cultured until senescence (i.e. up to passage 7) and transfected cells could be cultured to passage 18 after transfection, when a cellular crisis with massive cell death occurred. One clone escaped from crisis and has been maintained in culture for 55 wk. Experiments were performed to characterize transfected cells in comparison to primary cells. Cell morphology and proliferation were analyzed, and expression of cell-specific gene transcripts and proteins (including collagen types I and III, alkaline phosphatase, bone sialoprotein, osteocalcin, and dentin sialophosphoprotein and dentin matrix protein I) was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining many of the phenotypic characteristics observed in primary cells.

Pax9 belongs to the Pax family of transcriptional regulators that are defined by a highly conserved DNA-binding region, the paired domain. Drosophila, mouse and human genetics have shown that Pax proteins play multiple roles in tissue patterning and organogenesis by mediating their functions in a highly tissue-specific manner. Members of the Pax family, Pax9 and Pax1, act synergistically during vertebral formation. However, only Pax9 is essential for tooth formation. Furthermore, mutations of PAX9 are associated with human tooth agenesis. The highly tooth-specific molecular functions of Pax9 suggest that its activity is tightly regulated. Most likely, this occurs through interactions with other protein factors. Among the regulatory molecules that are expressed in dental mesenchyme, the Msx1 homeoprotein is of particular interest. The closely overlapping expression patterns of Pax9 and Msx1 are consistent with a role in epithelial-mesenchymal interactions. To demonstrate that Pax9 interacts with Msx1 physiologically in vivo and in vitro, we performed co-immunoprecipitation and GST interaction assays. Our results indicate that there is a physical association between the two proteins. Our biochemical data, coupled with human genetic studies and expression analysis in a mouse model, indicate a functional relationship between Pax9 and Msx1 during tooth development.

The need for women in academia to unite was seen in 1881, when Professors Marion Talbot and Ellen Richards founded the American Association of University Women. During this “Progressive Era”, women suffragists fought for and gained the right to vote, first in New Zealand (1893) and later in the United States (1920). As this first wave of feminism grew, it was highlighted by French philosopher Simone de Beauvoir in her book Second Sex, on gender inequality in a patriarchic society (Beauvoir, 1949). Betty Friedan’s The Feminine Mystique sparked the second wave of feminism, presenting the needs of women trapped in a life that they did not necessarily choose (Friedan, 1963). These movements culminated with The Equal Pay Act (1963) and The Equal Rights Amendment (1972), denouncing pay and sex discrimination for women, currently ratified in 35 U.S. states (113th Congress Joint Resolution 10, 2013). These advances led to the Women in Science and Technology Equal Opportunity Act, ensuring that “men and women have equal opportunity in education, training, and employment in scientific and technical fields” (Carter, 1980) and resulting in increased numbers of female investigators funded by the National Science Foundation (24%) and the National Institutes of Health (27%) (NSF, 2012; NIH, 2012). While these social and civil achievements improved the status of women in academia, there are several career challenges remaining related to an increasingly complex work environment. Although the Council of Graduate Education report shows that women are earning more doctoral degrees (52.4%) than men (Allum et al., 2012), women are still held back in achieving successful careers in academics (Shen, 2013). The “Beyond Bias and Barriers” report (National Research Council, 2007) clearly demonstrated that women are less likely to achieve tenure in academic institutions and are often expected to meet higher standards than their male counterparts. Generally, tenure policies lack flexibility for women during their junior faculty/childbearing years (Marchant et al., 2007). Departmental climates have also been shown to influence higher attrition rates among women since they tend to be less satisfied with their jobs and thus are more likely to quit (Nesbitt et al., 2003). Consequently, the conflicts between personal and professional life develop and strongly affect women in terms of faculty promotion and retention, with 40% of women more likely to exit the tenure track for adjunct positions (National Research Council, 2007). When examining global gender issues, the World Economic Forum (Hausmann et al., 2008) measured the gender gap according to 4 distinct criteria: economic participation and opportunity, educational attainment, political empowerment, and health and survival. The Nordic countries are on the top of the list, confirming their consistent gender equality priorities, with the United States ranked 27th, showing modest improvements in wage inequality and political empowerment (Hausmann et al., 2008). These results correlate with the European Union data on gender and research funding (European Commission, 2009), where the Nordic countries are considered proactive, with policies that secure women’s participation in research by encouraging grant applications from women, increasing numbers of women on review boards, and promoting funding transparency. Unlike the United States, the European Commission has been mandating 40% women participation on advisory boards for research funding programs (Vernos, 2013), striving to eliminate bias and enhance the funding success of women academicians in all 27 countries. Despite the Equal Pay Act, salary inequality between men and women has been recognized across all fields and academic appointment levels (West and Curtis, 2006), with women medical scientists earning 80% of male median wages (Goldberg Dey and Hill, 2007). This pay gap, established as early as one year post-bachelor’s degree, widens by 10 years post-degree (69%) and it is consistent for the level of education: Master’s degree (76%), professional degree (72%), and doctoral degree (80%) (Goldberg Dey and Hill, 2007). Among dental faculty, men are consistently compensated at a higher level than women (ADEA, 2011, 2013) (Table). In an examination of the mean salaries for women vs. men in senior dental school positions, where men hold positions at a 3:1 ratio over women, the differentials currently range from $2,806 to $93,443 (Table). The comparative Table shows that the salary gap between women and men has not only not improved in the past two years but has also widened (ADEA, 2011, 2013). Table. Guaranteed Annual Salary of Full-time Faculty by Primary Appointment and Gender in 2008-2009 and 2010-2011 Since social models have changed and continue to evolve, it is hoped that women will not have to face dilemmas in balancing careers with child-rearing (Ledford et al., 2013). “As more women lean in their careers, more men lean in their families” (Sandberg, 2013). To increase women’s participation in academic leadership, paid maternity/paternity leave and a delayed ‘tenure clock’, as well as consideration and selection of women as finalists for leadership positions, the presence of women on research committees and scientific review and editorial boards, should not be just mandates but an institutional strategic priority. Clearly, issues facing women in academia are multiple and complex, influenced by many personal/perceptual as well as social or state/federal barriers to equality. Recent evidence showed that several dental schools did not offer paid maternity or paternity leave (3.1%), an automatically delayed ‘tenure clock’ with childbirth or adoption (13.3%), dual career appointments (21.9%), and mandatory representation on search committees (15.6%) (Dannels et al., 2009). Analysis of these data stresses the need for critical institutional policies that will promote the career advancement of women in dental academics. Today, there are numerous leadership programs preparing women academicians for better navigation of the labyrinth of academic life and to meet their fullest potential, such as the American Dental Education Association Leadership Institute and Executive Leadership in Academic Medicine (ELAM). Both programs have been successfully promoting the career advancement of women, with currently 15.3% of U.S. dental school deans being women, of whom 60% were ELAM graduates (ADA, 2013). Moreover, over the past decades, the number of women dental students has increased from 23.80% in the early 1980s to 46.6% in 2010 (Gonzalez et al., 2011), resulting in increased enrollment of women in advanced education programs (40.2%) (ADA, 2012) and increased participation in dental academia (ADEA, 2013). Further, Drs. Martha Somerman and Isabel Garcia currently serve as the Director and Deputy Director, respectively, of the National Institute of Dental and Craniofacial Research (NIDCR), demonstrating the presence of women in dental research and leadership at the national level. In 2011, the International Association for Dental Research (IADR) established the “Women in Science” Network with a mission to focus on women’s issues related to career development and health research. The network’s goals are to examine dental academia policies and benefits related to key issues, such as recruitment and retention, promotion and tenure, salary, and childcare, as well as mentorship of junior women faculty and graduate trainees. The Women in Science Network is supportive of our academic institutions developing, encouraging, empowering, and equally promoting faculty regardless of their gender. The network, currently with 181 active and student members, has an active presence in every American Association for Dental Research (AADR) and International Association for Dental Research (IADR) meeting, organizing symposia and workshops with a focus on increasing women’s participation in dental academia, holding business and brainstorming meetings, as well as mentoring graduate students and junior faculty. Dr. Marie Nylen provided a legacy for change through her leadership as the first female president of both the AADR (1979-1980) and the IADR (1981-1982). Since the mid-1990s, the role of women in leadership positions in IADR/AADR has expanded, with 7 out of 15 female IADR presidents (46.7%) and 6 out of 16 female AADR Presidents (37.5%) – excellent statistics for progressive organizations that continue to foster the development of women scientists, clinicians, and educators for future leadership roles.

Pax9 is a paired domain-containing transcription factor that plays an essential role in the patterning of murine dentition. In humans, mutations in PAX9 are associated with unique phenotypes of familial tooth agenesis that mainly involve posterior teeth. Among these, a frameshift mutation (219InsG) within the paired domain of PAX9 produces a protein product associated with a severe form of molar agenesis in a single family. The objectives of this study were to gain new insights into the molecular pathogenesis of the 219InsG mutation and its role in tooth agenesis. Here we describe functional defects in DNA binding and transactivation of mutant 219InsGPax9. Although wild type Pax9 binds to the high affinity paired domain recognition sequences, e5 and CD19-2(A-ins), the 219InsGPax9 mutant protein was unable to bind to these cognate DNA-binding sites. In co-transfection assays, wild type Pax9 activated reporter gene transcription although the mutant was transcriptionally inactive. Immunolocalization data show that Pax9 and 219InsGPax9 proteins are synthesized in mammalian cells but that the nuclear localization of the mutant Pax9 protein is altered. Furthermore, transactivation by the full-length Pax9 protein from paired domain binding sites was not impaired by the 219InsGPax9 mutant. The latter did not alter the DNA binding activities of wild type Pax9 in gel mobility shift assays. The combined defects in DNA binding activities and transactivation function of mutant 219InsGPAX9 likely alter the selective activation and/or repression of PAX9 effector genes during odontogenesis. This loss-of-function of PAX9 most likely results in its haploinsufficiency during the patterning of dentition and the subsequent loss of posterior teeth.

Oligodontia, the congenital absence of six or more permanent teeth, is a common developmental anomaly of human dentition whose genetic basis is poorly understood. We recently reported a nonsyndromic form of oligodontia involving mostly all permanent molars in a large kindred, caused by a frameshift mutation in exon 2 of the human PAX9 gene [3]. To better understand the genotype/phenotype correlation in non-syndromic familial oligodontia, we identified additional families with a similar pattern of molar oligodontia. We hypothesize that molar oligodontia is due to allelic heterogeneity involving different mutations in PAX9. To test this hypothesis, we performed pedigree analysis followed by mutational analysis. Pedigree analysis revealed that the oligodontia trait is inherited in an autosomal dominant fashion. Mutational analysis of PAX9 thus far excludes the presence of the previously identified frameshift mutation.

Cell-based treatments are being developed as a novel approach for the treatment of many diseases in an effort to repair injured tissues and regenerate lost tissues. Interest in the potential use of multipotent progenitor or stem cells has grown significantly in recent years, specifically the use of mesenchymal stem cells (MSCs), for tissue engineering in combination with extracellular matrix-based scaffolds. An area that warrants further attention is the local or systemic host responses toward the implanted cell-biomaterial constructs. Such immunological responses could play a major role in determining the clinical efficacy of the therapeutic device or biomaterials used. MSCs, due to their unique immunomodulatory properties, hold great promise in tissue engineering as they not only directly participate in tissue repair and regeneration but also modulate the host foreign body response toward the engineered constructs. The purpose of this review was to summarize the current state of knowledge and applications of MSC-biomaterial constructs as a potential immunoregulatory tool in tissue engineering. Better understanding of the interactions between biomaterials and cells could translate to the development of clinically relevant and novel cell-based therapeutics for tissue reconstruction and regenerative medicine.

Here we report three new nanofibrous, self-assembling multidomain peptide (MDP) sequences and examine the effect of sequence on the morphology and expansion of encapsulated Stem cells from Human Exfoliated Deciduous teeth (SHED). We modified our previously reported set of serine-based MDPs, changing the serine residues in the amphiphilic region to threonine. The three new threonine-based sequences self-assemble into antiparallel β-sheet nanofibers, confirmed by CD and IR. AFM and negative-stained TEM show that the nanofibers formed by the new sequences are more curved than their serine-containing predecessors. Despite this change in nanofiber morphology, SEM illustrates that all three new sequences still form porous hydrogels. K(TL)2SLRG(TL)3KGRGDS, with a designed cleavage site, is able to be degraded by Matrix Metalloprotease 2. We then examine SHED cell response to these new sequences as well as their serine-based predecessors. We observe faster cell attachment and spreading in hydrogels formed by K2(SL)6K2GRGDS and K(SL)3RG(SL)3KGRGDS. By day 3, the SHEDs in all of the serine-based sequences exhibit a fibroblast-like morphology. Additionally, the SHED cells expand more rapidly in the serine-based gels while the cell number remains relatively constant in the threonine-based peptides. In hydrogels formed by K2(TL)6K2GRGDS and K(TL)2SLRG(TL)3KGRGDS, this low expansion rate is accompanied by changes in morphology where SHEDs exhibit a stellate morphology after 3 days in culture; however, by day 7 they appear more fibroblast-shaped. Throughout the duration of the experiment, the SHED cells encapsulated in the K2(TL)6K2 hydrogels remain rounded. These results suggest that the basic MDP structure easily accommodates modifications in sequence and, for SHED cells, the threonine-containing gels require the integrin-binding RGDS sequence for cell attachment to occur, while the serine-based gels are less selective and support an increase in cell number, regardless of the presence or absence of RGDS.

An epidemiologic study from the year 2008 found a highly significant increase of congenital tooth agenesis in women with ovarian cancer suggesting that a common genetic etiology may predispose women to both conditions. The finding was reminiscent of a previously described family harboring an AXIN2 mutation which could be shown to segregate with both the tooth agenesis and the predisposition to colon cancer transmitted in this family. Since tooth agenesis as a marker for susceptibility to ovarian cancer would be of great relevance to both oncologists and women with inborn missing teeth, the relationship between the two disorders requires a thorough assessment. We examined DNA samples from the ovarian cancer patients who participated in the original study, to look for a possible genetic connection between their ovarian malignancies and tooth agenesis. MSX1, PAX9, AXIN2, EDA, WNT10A, BARX and BRCA1 genes were selected for sequence analysis as they may cause tooth agenesis, are expressed in the female reproductive system, and/or are involved in tumorigenesis in general or specifically in the ovary. Our study revealed evidence that one half of the dually affected patients had an independent causation of the two conditions, thus reducing the previously estimated ovarian cancer risk for women with congenital tooth agenesis quite significantly.

A frameshift mutation recently identified within the paired domain of the transcription factor, PAX9, has been linked to a unique form of oligodontia in a single, multigenerational family (Stockton et al., 2000). We now describe the phenotypic and segregation analyses of this remarkable kindred, the initial approach taken to identify a candidate gene involved in this form of oligodontia, and the power of this single-family pedigree to generate significant linkage in a genome search. Of the 43 family members enrolled in this study, 21 individuals were affected with several congenitally missing permanent teeth. The pattern of inheritance of the oligodontia trait suggested the involvement of a single gene bearing a dominant mutation. To various degrees, affected members lacked permanent first, second, and third molars in all four quadrants. Several individuals with missing molars also lacked second premolars-most commonly, maxillary second premolars and mandibular central incisors. To the best of our knowledge, this pattern of non-syndromic, familial tooth agenesis has not been previously described in the literature. Since a missense mutation in the homeobox gene, MSX1, was previously linked to tooth agenesis in a single family lacking second premolars and third molars, we performed a mutational analysis of MSX1 by PCR. The absence of a mutation in exons 1 and 2 of MSX1 suggested that allelic mutations in the coding region of MSX1 are not associated with this phenotypically distinct form of oligodontia. Computer simulation of linkage analysis further proved that this pedigree alone was sufficient to generate a significant result for a total genome scan.

The majority of tooth agenesis cases are mild (hypodontia) and typically not associated with the gene mutations linked to oligodontia. From this, we hypothesise that most cases of tooth agenesis fit a polygenic mode of inheritance, where several genes with small effects cause a variety of varying phenotypes. In this study, we looked at 18 not typically studied genes in this condition, to ascertain their contribution to hypodontia. Our study subjects consisted of 167 patients with hypodontia and their parents from two cohorts (one from Brazil and one from Turkey). An additional 465 DNA samples (93 cases with hypodontia and 372 controls without family history for tooth agenesis or oral clefts) from Brazil were also available for this study. Ninety-three single nucleotide polymorphisms that maximally represent the linkage disequilibrium structure of the genes for the 18 genes were selected and genotyped using Taqman chemistry. Chi square was used to test if genotype distributions were in Hardy–Weinberg equilibrium, and 24 markers that were in Hardy–Weinberg equilibrium and had allele frequencies higher than 5 % in a panel of 50 CEPH samples were further tested. Association between hypodontia and genetic variants was tested with the transmission disequilibrium test within the programme Family-Based Association Test (FBAT) and by using Chi square and Fisher’s exact tests. Alpha at a level of 0.05 was used to report results. Results suggest possible associations between several genes and hypodontia in the three populations. In the Turkish cohort (n = 51 parent–affected child trios) the most significant results were as follows: FGF3 rs1893047, p = 0.08; GLI3 rs929387, p = 0.03; GLI3 haplotype rs929387-rs846266, p = 0.002; and PAX9 rs2073242, p = 0.03. In the Brazilian cohort (n = 116 parent–affected child trios), the results were as follows: DLX1 rs788173, p = 0.07; FGF3 rs12574452, p = 0.03; GLI2 rs1992901, p = 0.03; and PITX2 rs2595110, p = 0.01. The second Brazilian cohort also suggested that FGF3 (rs12574452, p = 0.01) is associated with hypodontia and added EDAR (rs17269487, p = 0.04), LHX6 (rs989798, p = 0.02), and MSX1 (rs12532, p = 0.003). Our results suggest that several genes are potentially associated with hypodontia and their individual contributions may be modest. Hence, these cases may not be explained by inactivating mutations such as many oligodontia cases segregating in a Mendelian fashion but rather are influenced by one or more susceptibility alleles in multiple small effect genes.

Root canal therapy is common practice in dentistry. During this procedure, the inflamed or necrotic dental pulp is removed and replaced with a synthetic material. However, recent research provides evidence that engineering of dental pulp and dentin is possible by using biologically driven approaches. As tissue engineering strategies hold the promise to soon supersede conventional root canal treatment, there is a need for customized scaffolds for stem cell delivery or recruitment. We hypothesize that the incorporation of dental pulp-derived stem cells with bioactive factors into such a scaffold can promote cell proliferation, differentiation, and angiogenesis. In this study, we used a cell adhesive, enzyme-cleavable hydrogel made from self-assembling peptide nanofibers to encapsulate dental pulp stem cells. The growth factors (GFs) fibroblast growth factor basic, transforming growth factor β1, and vascular endothelial growth factor were incorporated into the hydrogel via heparin binding. Release profiles were established, and the influence of GFs on cell morphology and proliferation was assessed to confirm their bioactivity after binding and subsequent release. Cell morphology and spreading in three-dimensional cultures were visualized by using cell tracker and histologic stains. Subcutaneous transplantation of the hydrogel within dentin cylinders into immunocompromised mice led to the formation of a vascularized soft connective tissue similar to dental pulp. These data support the use of this novel biomaterial as a highly promising candidate for future treatment concepts in regenerative endodontics.

For tissue engineering strategies, the choice of an appropriate scaffold is the first and certainly a crucial step. A vast variety of biomaterials is available: natural or synthetic polymers, extracellular matrix, self-assembling systems, hydrogels, or bioceramics. Each material offers a unique chemistry, composition and structure, degradation profile, and possibility for modification. The role of the scaffold has changed from passive carrier toward a bioactive matrix, which can induce a desired cellular behavior. Tailor-made materials for specific applications can be created. Recent approaches to generate dental pulp rely on established materials, such as collagen, polyester, chitosan, or hydroxyapatite. Results after transplantation show soft connective tissue formation and newly generated dentin. For dentin-pulp-complex engineering, aspects including vascularization, cell-matrix interactions, growth-factor incorporation, matrix degradation, mineralization, and contamination control should be considered. Self-assembling peptide hydrogels are an example of a smart material that can be modified to create customized matrices. Rational design of the peptide sequence allows for control of material stiffness, induction of mineral nucleation, or introduction of antibacterial activity. Cellular responses can be evoked by the incorporation of cell adhesion motifs, enzyme-cleavable sites, and suitable growth factors. The combination of inductive scaffold materials with stem cells might optimize the approaches for dentin-pulp complex regeneration.

For many years the molecular mechanisms governing bone morphogenetic protein 4 (Bmp4) expression in tooth bud mesenchyme could be explained by an uncomplicated model involving the interaction of the homeobox gene Msx1 and the paired domain gene Pax9 and a limited proximal promoter segment of Bmp4. New insights have led to major revisions, but we are still far from understanding the role of Msx1 and Pax9 in the complex processes that result in the expression of Bmp4 in the mesenchymal layer of the developing tooth bud. The objective of these studies was to gain further insight into the molecular relationship between Pax9, Msx1, and Bmp4 in dental mesenchyme and explore its association with nonsyndromic tooth agenesis in humans.

Mutations in the transcription factors PAX9 and MSX1 cause selective tooth agenesis in humans. In tooth bud mesenchyme of mice, both proteins are required for the expression of Bmp4, which is the key signaling factor for progression to the next step of tooth development. We have previously shown that Pax9 can transactivate a 2.4-kb Bmp4 promoter construct, and that most tooth-agenesis-causing PAX9 mutations impair DNA binding and Bmp4 promoter activation. We also found that Msx1 by itself represses transcription from this proximal Bmp4 promoter, and that, in combination with Pax9, it acts as a potentiator of Pax9-induced Bmp4 transactivation. This synergism of Msx1 with Pax9 is significant, because it is currently the only documented mechanism for Msx1-mediated activation of Bmp4. In this study, we investigated whether the 5 known tooth-agenesis-causing MSX1 missense mutations disrupt this Pax9-potentiation effect, or if they lead to deficiencies in protein stability, protein-protein interactions, nuclear translocation, and DNA-binding. We found that none of the studied molecular mechanisms yielded a satisfactory explanation for the pathogenic effects of the Msx1 mutations, calling for an entirely different approach to the investigation of this step of odontogenesis on the molecular level.

Direct application of dentin bonding agents onto the exposed pulp has been advocated, but in vivo studies indicate a lack of reparative dentin formation. Our objective was to investigate the role of triethylene glycol dimethacrylate (TEGDMA), a commonly used compound in dentin bonding agents, as a potential inhibitor of mineralization. Human pulp cells were exposed to different concentrations of TEGDMA, and expression of the mineralization-related genes collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein was analyzed. Gene expression studies by real-time polymerase chain-reaction revealed a concentration- and time-dependent decrease of mineralization markers. A subtoxic TEGDMA concentration (0.3 mM) reduced expression levels by 5 to 20% after 4 hrs and by 50% after 12 hrs. Furthermore, alkaline phosphatase activity and calcium deposition were significantly lower in dental pulp cells treated with TEGDMA over 14 days. These findings indicate that even low TEGDMA concentrations might inhibit mineralization induced by dental pulp cells, thus impairing reparative dentin formation after pulp capping with dentin bonding agents.

Stem cells derived from the dental pulp of extracted human third molars (DPSCs) have the potential to differentiate into odontoblasts, osteoblasts, adipocytes, and neural cells when provided with the appropriate conditions. To advance the use of DPSCs for dentin regeneration, it is important to replicate the permissive signals that drive terminal events in odontoblast differentiation during tooth development. Such a strategy is likely to restore a dentin matrix that more resembles the tubular nature of primary dentin. Due to the limitations of culture conditions, the use of ex vivo gene therapy to drive the terminal differentiation of mineralizing cells holds considerable promise. In these studies, we asked whether the forced expression of TWIST1 in DPSCs could alter the potential of these cells to differentiate into odontoblast-like cells. Since the partnership between Runx2 and Twist1 proteins is known to control the onset of osteoblast terminal differentiation, we hypothesized that these genes act to control lineage determination of DPSCs. For the first time, our results showed that Twist1 overexpression in DPSCs enhanced the expression of DSPP, a gene that marks odontoblast terminal differentiation. Furthermore, co-transfection assays showed that Twist1 stimulates Dspp promoter activity by antagonizing Runx2 function in 293FT cells. Analysis of our in vitro data, taken together, suggests that lineage specification of DPSCs can be modulated through ex vivo gene modifications.

Multidomain peptides are a class of amphiphilic self-assembling peptides with a modular ABA block motif in which the amphiphilic B block drives self-assembly while the flanking A blocks, which are electrostatically charged, control the conditions under which assembly takes place. Previously we have shown that careful selection of the amino acids in the A and B blocks allow one to control the self-assembled fiber length and viscoelastic properties of formed hydrogels. Here we demonstrate how the modular nature of this peptide assembler can be designed for biological applications. With control over fiber length and diameter, gelation conditions, and viscoelastic properties, we can develop suitable materials for biological applications. Going beyond a simple carrier for cell delivery, a biofunctional scaffold will interact with the cells it carries, promoting advantageous cell-matrix interactions. We demonstrate the design of a multidomain peptide into a bioactive variant by incorporation of a matrix metalloprotease 2 (MMP-2) specific cleavage site and cell adhesion motif. Gel formation and rheological properties were assessed and compared to related peptide hydrogels. Proteolytic degradation by collagenase IV was observed in a gel weight loss study and confirmed by specific MMP-2 degradation monitored by mass spectrometry and cryo-transmission electron microscopy (cryo-TEM). Combination of this cleavage site with the cell adhesion motif RGD resulted in increased cell viability and cell spreading and encouraged cell migration into the hydrogel matrix. Collectively the structural, mechanical, and bioactive properties of this multidomain peptide hydrogel make it suitable as an injectable material for a variety of tissue engineering applications.

Mutations of the Ectodysplasin-A (EDA) gene are generally associated with the syndrome hypohidrotic ectodermal dysplasia (MIM 305100), but they can also manifest as selective, non-syndromic tooth agenesis (MIM300606). We have performed an in vitro functional analysis of six selective tooth agenesis-causing EDA mutations (one novel and five known) that are located in the C-terminal tumor necrosis factor homology domain of the protein. Our study reveals that expression, receptor binding or signaling capability of the mutant EDA1 proteins is only impaired in contrast to syndrome-causing mutations, which we have previously shown to abolish EDA1 expression, receptor binding or signaling. Our results support a model in which the development of the human dentition, especially of anterior teeth, requires the highest level of EDA-receptor signaling, whereas other ectodermal appendages, including posterior teeth, have less stringent requirements and form normally in response to EDA mutations with reduced activity.

Genetics gains more and more importance in all areas of health care including craniofacial surgery and dentistry. This does not mean that every patient will benefit from genetic advances, but for many health problems, we will see progress in explaining disease pathogenesis, establishing diagnosis, guiding therapy, predicting prognosis, and achieving prevention. In this report, we briefly review the roles of the PAX9, MSX1, AXIN2, and EDA genes in the causation of congenital tooth agenesis and the promise of molecular genetic research for the improvement of patient care.

Hereditary conditions are traditionally classified based either on physical/physiological attributes or using the names of the individuals credited with identifying the condition. For the 170 plus conditions classified as ectodermal dysplasias (EDs), both of these nosological systems are used, at times interchangeably. Over the past decade our knowledge of the human genome and the molecular basis of the EDs have greatly expanded providing the impetus to consider alternative classification systems. The incorporation of the molecular basis of hereditary conditions adds important information allowing effective transfer of objective genetic information that can be lacking from traditional classification systems. Molecular information can be added to the nosological system for the EDs through a hierarchical- and domain-based approach that encompasses the condition's name, mode of inheritance, molecular pathway affected, and specific molecular change. As new molecular information becomes available it can be effectively incorporated using this classification approach. Integrating molecular information into the ED classification system, while retaining well-recognized traditional syndrome names, facilitates communication at and between different groups of people including patients, families, health care providers, and researchers.

The history and the lessons learned from hypohidrotic ectodermal dysplasia (HED) may serve as an example for the unraveling of the cause and pathogenesis of other ectodermal dysplasia syndromes by demonstrating that phenotypically identical syndromes (HED) can be caused by mutations in different genes (EDA, EDAR, EDARADD), that mutations in the same gene (EDA) can lead to different phenotypes (HED and selective tooth agenesis) and that mutations in genes further downstream in the same signaling pathway (NEMO) may modify the phenotype quite profoundly (incontinentia pigmenti (IP) and HED with immunodeficiency). But it also demonstrates that diligent phenotype characterization and classification is extremely helpful in uncovering the underlying genotype. We also present a new mutation in the EDA gene which causes selective tooth agenesis and demonstrates the phenotype variation that can be encountered in the ectodermal dysplasia syndrome (HED) with the highest prevalence worldwide.

The aim of this study was to examine the particle length, width, perimeter, and aspect ratio of calcium hydroxide powder using a flow particle image analyzer (FPIA). Five sample groups each with 10 mg of calcium hydroxide were mixed with 15 mL of alcohol and sonicated. Digital images of the particle samples were taken using the FPIA and analyzed with a one-way analysis of variance. The overall averages +/- standard deviation among the five groups for particle length (microm), width (microm), perimeter (microm), and aspect ratio were 2.255 +/- 1.994, 1.620 +/- 1.464, 6.699 +/- 5.598, and 0.737 +/- 0.149, respectively. No statistical significance was observed among the groups for all parameters. When the total of 46,818 particles from all five groups were classified into the five length categories of 0.5-microm increments, there were significant differences in width, perimeter, and aspect ratio (all p values0.0001). In conclusion, calcium hydroxide particles have a size and shape that may allow direct penetration into open dentin tubules.

Previously, non-collagenous matrix proteins, such as DMP1, were viewed with little biological interest. The last decade of research has increased our understanding of DMP1, as it is now widely recognized that this protein is expressed in non-mineralized tissues, as well as in cancerous lesions. Protein chemistry studies have shown that the full length of DMP1, as a precursor, is cleaved into two distinct forms: the C-terminal and N-terminal fragments. Functional studies have demonstrated that DMP1 is essential in the maturation of odontoblasts and osteoblasts, as well as in mineralization via local and systemic mechanisms. The identification of DMP1 mutations in humans has led to the discovery of a novel disease: autosomal-recessive hypophosphatemic rickets. Furthermore, the regulation of phosphate homeostasis by DMP1 through FGF23, a newly identified hormone that is released from bone and targeted in the kidneys, sets a new direction for research that associates biomineralization with phosphate regulation.

The development of dentition is a fascinating process that encompasses a complex series of epithelial-mesenchymal interactions involving growth factors, transcription factors, signal receptors and other soluble morphogens. It is not surprising that such a complex process is prone to disturbances and may result in tooth agenesis. Initial discoveries indicating that the homeo-domain protein MSX1 and the paired-domain transcription factor PAX9 are causative genes in tooth morphogenesis were made in mice. Both genes are co-expressed in dental mesenchyme and either one, when homozygously deleted, results in an arrest at an early developmental stage. Heterozygous Pax9 or Msx1 mice have normal teeth, however, double heterozygous Pax9/Msx1 mice show a phenotype of arrested tooth development which can be rescued by transgenic expression of Bmp4, a very influential signaling factor in many developmental processes. We have obtained mounting evidence for a partnership between PAX9 and MSX1 within the tooth-specific Bmp4 signaling pathway. In humans, unlike in mice, a heterozygous mutation in either PAX9 or MSX1 suffices to cause tooth agenesis of a predominantly molar or more premolar pattern, respectively. Our laboratory and others have identified several PAX9 and MSX1 mutations in families with non-syndromic forms of autosomal dominant posterior tooth agenesis. We have also identified families with tooth agenesis in whom PAX9 and MSX1 mutations have been excluded opening up the possibilities for the discovery of other genes that contribute to human tooth agenesis.

Pax9 and Msx1 encode transcription factors that are known to be essential for the switch in odontogenic potential from the epithelium to the mesenchyme. Multiple lines of evidence suggest that these molecules play an important role in the maintenance of mesenchymal Bmp4 expression, which ultimately drives morphogenesis of the dental organ. Here we demonstrate that Pax9 is able to directly regulate Msx1 expression and interact with Msx1 at the protein level to enhance its ability to transactivate Msx1 and Bmp4 expression during tooth development. In addition, we tested how a missense mutation (T62C) in the paired domain of PAX9 that is responsible for human tooth agenesis (1) affects its functions. Our data indicate that although the mutant Pax9 protein (L21P) can bind to the Msx1 protein, it fails to transactivate the Msx1 and Bmp4 promoter, presumably because of its inability to bind cognate paired domain recognition sequences. In addition, synergistic transcriptional activation of the Bmp4 promoter was lost with coexpression of mutant Pax9 and wild-type Msx1. This suggests that Pax9 is critical for the regulation of Bmp4 expression through its paired domain rather than Msx1. Our findings demonstrate the partnership of Pax9 and Msx1 in a signaling pathway that involves Bmp4. Furthermore, the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 at the level of transcription and through formation of a protein complex determines the fate of the transition from bud to cap stage during tooth development.