Oral Sciences PhD Program Enrollment, Graduates, and Placement: 1994 to 2016.

For decades, dental schools in the United States have endured a significant faculty shortage. Studies have determined that the top 2 sources of dental faculty are advanced education programs and private practice. Those who have completed both DDS and PhD training are considered prime candidates for dental faculty positions. However, there is no national database to track those trainees and no evidence to indicate that they entered academia upon graduation. The objective of this study was to assess outcomes of dental school-affiliated oral sciences PhD program enrollment, graduates, and placement between 1994 and 2016. Using the American Dental Association annual survey of advanced dental education programs not accredited by the Commission on Dental Accreditation and data obtained from 22 oral sciences PhD programs, we assessed student demographics, enrollment, graduation, and placement. Based on the data provided by program directors, the average new enrollment was 33, and graduation was 26 per year. A total of 605 graduated; 39 did not complete; and 168 were still in training. Among those 605 graduates, 211 were faculty in U.S. academic institutions, and 77 were faculty in foreign institutions. Given that vacant budgeted full-time faculty positions averaged 257 per year during this period, graduates from those oral sciences PhD programs who entered academia in the United States would have filled 9 (3.6%) vacant faculty positions per year. Therefore, PhD programs have consistently generated only a small pipeline of dental school faculty. Better mentoring to retain talent in academia is necessary. Stronger support and creative funding plans are essential to sustain the PhD program. Furthermore, the oral sciences PhD program database should be established and maintained by dental professional organizations to allow assessments of training models, trends of enrollment, graduation, and placement outcomes.

NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

New paradigms on the transport functions of maturation-stage ameloblasts.

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.

Chymotrypsin C (caldecrin) is associated with enamel development.

Two main proteases cleave enamel extracellular matrix proteins during amelogenesis. Matrix metalloprotease-20 (Mmp20) is the predominant enzyme expressed during the secretory stage, while kallikrein-related peptidase-4 (Klk4) is predominantly expressed during maturation. Mutations to both Mmp20 and Klk4 result in abnormal enamel phenotypes. During a recent whole-genome microarray analysis of rat incisor enamel organ cells derived from the secretory and maturation stages of amelogenesis, the serine protease chymotrypsin C (caldecrin, Ctrc) was identified as significantly up-regulated (> 11-fold) during enamel maturation. Prior reports indicate that Ctrc expression is pancreas-specific, albeit low levels were also noted in brain. We here report on the expression of Ctrc in the enamel organ. Quantitative PCR (qPCR) and Western blot analysis were used to confirm the expression of Ctrc in the developing enamel organ. The expression profile of Ctrc is similar to that of Klk4, increasing markedly during the maturation stage relative to the secretory stage, although levels of Ctrc mRNA are lower than for Klk4. The discovery of a new serine protease possibly involved in enamel development has important implications for our understanding of the factors that regulate enamel biomineralization.

Enamel matrix proteins; old molecules for new applications.

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.

Role of NBCe1 and AE2 in secretory ameloblasts.

The H(+)/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

Cellular uptake of amelogenin, and its localization to CD63, and Lamp1-positive vesicles.

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

Controlled failure mechanisms toughen the dentino-enamel junction zone.

STATEMENT OF PROBLEM: The dentino-enamel junction (DEJ) durably unites dissimilar hard brittle enamel and tough flexible dentin. In contrast to artificial bonds between restorations and dentin, the DEJ rarely fails except when it is affected by inherited disorders. Knowledge of DEJ toughening mechanisms is important in understanding inherited disorders, in biomimetic engineering of junctions between artificial restorations and teeth, and in tissue-engineering a DEJ. PURPOSE: The purpose of this study was to identify specific DEJ-zone failure mechanisms and to survey the fracture toughness of the human DEJ zone. MATERIAL AND METHODS: Fracture toughness indentations were made at 3 sites across the DEJ zone of 10 human incisor teeth. Failure modes identified using optical microscopy and fracture toughness (MPa.m(1/2)) were calculated following Vickers microindentation. Site mean values were then calculated and compared using 1-way analysis of variance (alpha=.05). RESULTS: The DEJ did not undergo catastrophic interfacial delamination; instead, damage was distributed over a broad zone. The primary damage mode involved cracking and damage dispersion in the specialized first-formed enamel close to the DEJ. Multiple, somewhat convoluted and sometimes branching, cracks spread and diffused damage over a wide area of adjacent enamel rather than producing catastrophic interfacial failure. Other secondary mechanisms included short microcracks in the DEJ adjacent dentin with possible cracked bridging, as well as plastic deformation of the DEJ without delamination. A DEJ-zone fracture toughness of approximately 0.8 to 0.9 MPa.m(1/2) was calculated. CONCLUSION: DEJ-zone damage occurred primarily within the adjacent layer of specialized first-formed enamel, and the optical DEJ interface resisted delamination.

Tooth developmental biology: disruptions to enamel-matrix assembly and its impact on biomineralization.

Dental enamel is a composite bioceramic material that is the hardest tissue in the vertebrate body, containing long, thin crystallites of substituted hydroxyapatite (HAP). Over a lifetime of an organism, enamel functions under repeated and immense loads, generally without catastrophic failure. Enamel is a product of ectoderm-derived cells called ameloblasts. Recent investigations on the formation of enamel using cell and molecular approaches are now being coupled to biomechanical investigations at the nanoscale and mesoscale levels. For amelogenin, the principal structural protein for forming enamel, we have identified two domains that are required for its proper self-assembly into supramolecular structures referred to as nanospheres. Nanospheres are believed to control HAP crystal habit. Other structural proteins of the enamel matrix include ameloblastin and enamelin, but little is known about their biological importance. Transgenic animals have been prepared to investigate the effect of overexpression of wild-type or mutated enamel proteins on the developing enamel matrix. Amelogenin transgenes were engineered to contain deletions to either of the two self-assembly domains and these alterations produced significant defects in the enamel. Additional transgenic animal lines have been prepared and studied and each gives additional insights into the mechanisms for enamel biofabrication. This study summarizes the observed enamel phenotypes of recently derived transgenic animals. These data are being used to help define the role of each of the enamel structural proteins in enamel and study how each of these proteins impact on enamel biomineralization.

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11).

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.

Ultrastructure of forming enamel in mouse bearing a transgene that disrupts the amelogenin self-assembly domains.

The mouse X-chromosomal amelogenin gene promoter was used to drive the expression of mutated amelogenin proteins in vivo. Two different transgenic mouse lines based on deletions to either the amino-terminal (A-domain deletions) or to the carboxyl-region (B-domain deletions) were bred. In the molars of newborn A-domain deleted transgenic mice the formation of the initial layer of aprismatic enamel was delayed. There were severe structural alterations in the enamel of incisors of newborn mice bearing the A-domain deletion which were not apparent in animals bearing the B-domain deletion. In the A-domain-deleted animals, stippled material accumulated throughout the entire thickness of the forming enamel apparently causing a disruption of the normal rod-to-inter-rod relationship. This stippled material was likened to and interpreted as being groupings of amelogenin nanospheres. In the B-domain-deleted animals the stippled material was detected only in minute defects of the forming enamel. These data suggest significant differences in nanosphere assembly properties for animals bearing either the A-domain or the B-domain-deleted transgene. The present in vivo experimental approach suggests that at early stages of enamel formation, the A-domain plays a greater role than does the B-domain in amelogenin self-assembly, and consequently in enamel architecture and structure.

Regulated gene expression dictates enamel structure and tooth function.

Enamel is a complex bioceramic tissue. In its final form, enamel is a reflection of the unique molecular and cellular activities occurring during organogenesis. From the ectodermal origins of ameloblasts, their gene activity and protein expression profiles exist for the sole purpose of producing a mineralized shell, almost entirely devoid of protein, deposited over the 'bone-like' dentine. The interface between enamel and dentine is referred to as the dentine enamel junction and it is also unique in its biology. This review article is narrow in its scope. We restrict our review to selected advances in our understanding of the genetic, molecular and structural aspects of enamel biology. We present a model of enamel formation that relates gene expression to the assembly of an extracellular protein matrix that in turn controls the structural hierarchy and mechanical aspects of enamel and the tooth organ.

Biological organization of hydroxyapatite crystallites into a fibrous continuum toughens and controls anisotropy in human enamel.

Enamel forms the outer surface of teeth, which are of complex shape and are loaded in a multitude of ways during function. Enamel has previously been assumed to be formed from discrete rods and to be markedly aniostropic, but marked anisotropy might be expected to lead to frequent fracture. Since frequent fracture is not observed, we measured enamel organization using histology, imaging, and fracture mechanics modalities, and compared enamel with crystalline hydroxyapatite (Hap), its major component. Enamel was approximately three times tougher than geologic Hap, demonstrating the critical importance of biological manufacturing. Only modest levels of enamel anisotropy were discerned; rather, our measurements suggest that enamel is a composite ceramic with the crystallites oriented in a complex three-dimensional continuum. Geologic apatite crystals are much harder than enamel, suggesting that inclusion of biological contaminants, such as protein, influences the properties of enamel. Based on our findings, we propose a new structural model.

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy.

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG epitope ("A"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.

A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts.

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.

Enamel biomineralization defects result from alterations to amelogenin self-assembly.

Enamel formation is a powerful model for the study of biomineralization. A key feature common to all biomineralizing systems is their dependency upon the biosynthesis of an extracellular organic matrix that is competent to direct the formation of the subsequent mineral phase. The major organic component of forming mouse enamel is the 180-amino-acid amelogenin protein (M180), whose ability to undergo self-assembly is believed to contribute to biomineralization of vertebrate enamel. Two recently defined domains (A and B) within amelogenin appear essential for this self-assembly. The significance of these two domains has been demonstrated previously by the yeast two-hybrid system, atomic force microscopy, and dynamic light scattering. Transgenic animals were used to test the hypothesis that the self-assembly domains identified with in vitro model systems also operate in vivo. Transgenic animals bearing either a domain-A-deleted or domain-B-deleted amelogenin transgene expressed the altered amelogenin exclusively in ameloblasts. This altered amelogenin participates in the formation an organic enamel extracellular matrix and, in turn, this matrix is defective in its ability to direct enamel mineralization. At the nanoscale level, the forming matrix adjacent to the secretory face of the ameloblast shows alteration in the size of the amelogenin nanospheres for either transgenic animal line. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization due to perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly in forming a competent enamel organic matrix and that alterations to the matrix are reflected as defects in the structural organization of enamel.

Protein-to-protein interactions: criteria defining the assembly of the enamel organic matrix.

Enamel crystallites form in a protein matrix located proximal to the ameloblast cell layer. This unique organic extracellular matrix is constructed from structural protein components biosynthesized and secreted by ameloblasts. To date, three distinct classes of enamel matrix proteins have been cloned. These are the amelogenins, tuftelin, and ameloblastin, with recent data implicating ameloblastin gene expression during cementogenesis. The organic enamel extracellular matrix undergoes assembly to provide a three-dimensional array of protein domains that carry out the physiologic function of guiding enamel hydroxyapatite crystallite formation. Using the yeast two-hybrid system, we have surveyed these three known enamel gene products for their ability to direct self-assembly. We measured the capacity of the enamel gene products to direct protein-to-protein interactions, a characteristic of enamel proteins predicated to be required for self-assembly. We provide additional evidence for the self-assembly nature of amelogenin and tuftelin. Ameloblastin self-assembly could not be demonstrated, nor were protein-to-protein interactions observed between ameloblastin and either amelogenin or tuftelin. Within the limits of the yeast two-hybrid assay, these findings constrain the emerging model of enamel matrix assembly by helping to define the limits of enamel matrix protein-protein interactions that are believed to guide enamel mineral crystallite formation.

Fluoride use in periodontal therapy: a review of the literature.

Although the relationship between fluoride and dental caries has been widely studied and definitively determined, the relationship between fluoride and periodontal health and disease is not clear. Conflicting studies in the literature either suggest routine fluoride usage as an adjunct to conventional periodontal therapy or argue against topical fluoride use on periodontally involved teeth. This review summarizes the literature that addresses the utility of fluoride in patients with periodontal disease and aims to advance a rational criterion for the use of fluorides in the management of periodontal diseases.

Identification of tuftelin- and amelogenin-interacting proteins using the yeast two-hybrid system.

Biomineralization of enamel is a complex process that involves the eventual replacement of an extracellular protein matrix by hydroxyapatite crystallites. To date four different enamel matrix proteins have been identified; the amelogenins, tuftelin, enamelin and ameloblastin. Assembly of the enamel extracellular matrix from these component proteins is believed to be critical in producing a matrix competent to undergo mineral replacement. Enamel formation is a complex process and additional proteins are likely to have a role in the assembly of the extracellular matrix. In order to identify additional proteins involved in the assembly process, the yeast two-hybrid system developed by Fields and Song (1989) has been implemented. This system allows for the identification of unknown proteins that interact with proteins of interest. Typically a known protein is used as "bait" to screen a cDNA expression library of interest. In our studies, tuftelin or amelogenin have been used to screen a mouse tooth library produced from one day old pups. A library screening of six million clones with amelogenin as bait resulted in eleven positive clones all of which show high homology to the human leukocyte antigen-B (HLA-B) associated transcript (BAT) family of genes. A library screening of one million clones using tuftelin as the bait identified twenty-one tuftelin-interacting proteins. Ten of these proteins are either keratin K5 or keratin K6, four are constitutively expressed and the remaining seven are novel. Further characterization of the proteins shown to interact with amelogenin or tuftelin may shed additional light on this complex process of enamel matrix assembly.

Transgene animal model for protein expression and accumulation into forming enamel.

Understanding the cellular and molecular events that regulate the formation of enamel is a major driving force in efforts to characterize critical events during amelogenesis. It is anticipated that through such an understanding, improvements in prevention, diagnosis and treatment-intervention into heritable and acquired diseases of enamel could be achieved. While knowledge of the precise role of an enamel-specific protein in directing the formation of inorganic crystallites remains refractory, progress has been made with other aspects of amelogenesis that can be brought to bear on the subject. One such area of progress has been with the identification of an ameloblast-lineage specific amelogenin gene promoter. This promoter can be used to direct the expression of enamel-specific proteins, as well as the expression of proteins foreign to amelogenesis, into the enamel extracellular matrix where their effect on biomineralization can be ascertained in a prospective manner. The resulting enamel from such animals can be examined by morphologic and biochemical modalities in order to identify the effect of the transgene protein on enamel crystallite formation and subsequent biomineralization. This manuscript outlines such a strategy with the potential for enhancing our understanding of amelogenesis.