In the span of just a few decades, the advent of recombinant DNA technology as well as other tools of biotechnology have revolutionized the health sciences-dentistry, medicine, nursing and pharmacy. The original federal investment in basic science research, primarily through the creation of the National Institutes of Health and several other federal agencies, has delivered truly incredible dividends. Currently, this nation has 1,200 biotechnology companies which contribute over $11 billion a year to the nation's economy. These sales are expected to reach approximately $50 billion in the next decade. In addition to the creation of nearly 100,000 highly skilled jobs and contribution to the nation's economy, biotechnology provides the promise and some uncertainty to vastly improve the quality of life throughout the world. Yet do we have a strategy which encompasses federal, state and private sector policies and an infrastructure sufficient to sustain a substantial basic science investment to fuel the engine of technology and a process that enhances the quality of life for all Americans?

Biotechnology↗

PubMed1996

Co-culture of contiguous developmental fields in a serumless, chemically-defined medium: an in vitro model permissive for coordinate development of the mouse ear.

Pattern formation is intrinsically hierarchical, increasing in complexity from the first early embryonic inductive tissue interactions to the eventual integration of multiple organ systems. Viewed as a problem in pattern formation, the vertebrate ear is an exceedingly complex organ system in which normal morphogenesis requires multiple inductive interactions between a variety of adjacent tissues. In order to model the process of higher level pattern formation, we have developed a method for organ culture of the embryonic murine ear. E10.5 mouse embryos (38 to 42 somite pairs) were microdissected into explants that consist of the first and second branchial arches, the otocyst, and the adjacent neural tube. The growth of these explants in a serumless, chemically-defined medium was compared to medium supplemented with 10% fetal calf serum. After 6 days in culture using serumless medium, we observed that this environment was permissive for the formation of pinnae, rudimentary semicircular canals and cochlear ducts, chondrogenesis of the otic capsule and elongation of the endolymphatic ducts. Posterior elements of Meckel's and Reichert's cartilages were identified as ossicular anlagen. All of these structures maintained appropriate anatomic interrelationships during in vitro development. Furthermore, no significant differences were observed in explants grown in serum-supplemented medium. We conclude that during ear development several histogenetic and morphogenetic processes, including aspects of higher level pattern formation, are mediated primarily by paracrine and/or autocrine factors. The development of an organ culture model using serumless medium should facilitate the discovery of intrinsic factors which regulate the coordinate development of inner, middle and external ear structures.

Animals↗

PubMed1995

Recombinant DNA technology and oral medicine.

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Animals↗

PubMed1995

Initial enamel crystals are not spatially associated with mineralized dentine.

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adjacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.

Animals↗

PubMed1995

A review of the aggregation properties of a recombinant amelogenin.

The present paper reviews some recent data on the aggregation properties of a recombinant amelogenin using dynamic light scattering, transmission electron microscopy, atomic force microscopy, and size exclusion chromatography. It was found that the recombinant amelogenin M179 molecules in solution form spherical monodisperse aggregates (15-20 nm) which are predominantly stabilized by intermolecular hydrophobic interactions while their surfaces are charged, carrying the hydrophilic carboxy-terminal sequence. We concluded that the spherical aggregates represent the "stippled materials" secreted by the ameloblasts at the mineralization front. We further speculate that the self-assembly process in the formation of amelogenin aggregates may play a primary role in the structural organization of mineralizing enamel.

Agglutination↗

PubMed1995

Molecular biology experimental strategies for craniofacial-oral-dental dysmorphology.

This paper is a minireview of molecular biology experimental strategies for problems within craniofacial-oral-dental biology. Many of these strategies have already made remarkable contributions towards understanding the complex developmental processes associated with craniofacial biology. For example, the utilization of these strategies has resulted in the successful mapping of approximately 70 genes related to craniofacial anomalies (e.g., Pax, retinoic acid receptors, cadhedrins, aggrecan, cell adhesion molecules, substrate adhesion molecules, etc.), 30 genes related to dental tissue disorders (e.g., BMPs, bone morphogenetic proteins; dentin phosphoproteins, dentin sialoglycoproteins, enamelins, amelogenins), 20 genes related to facial clefting defects (e.g., Hox genes, transforming growth factor alpha), and 3 genes related to craniosynostosis (e.g., Msx-2). This minireview highlights selected examples of scientific progress derived from the following experimental strategies: (i) molecular approaches to the organization of the mouse and human chromosomes, with the mapping of specific gene sequences linked to human diseases (e.g., amelogenesis imperfecta, Boston type craniosynostosis, Rieger's syndrome, Treacher Collins syndrome); (ii) reverse genetic approaches for studies of gene function; (iii) homologous recombinations and the advances from "knock-out" transgenic mouse models for human craniofacial-oral-dental diseases; (iv) mutational analyses of congenital craniofacial-oral-dental dysmorphogenesis; (v) structural biology studies using computer-assisted molecular modeling for protein-protein, protein-nucleic acid and protein-inorganic interactions; (vi) computer modeling of genetic paradigms; and (vii) a cluster of newer methodologies including computer-assisted morphometry, new microinjection techniques, new cell membrane and intracellular dyes, and a number of new RNA and DNA viral constructs for the delivery of genes to enhance the resolution of cell fate maps, cell lineage studies and gene therapy approaches to human diseases.

Animals↗

PubMed1995

Characterization of protein kinases involved in dentinogenesis.

Protein phosphorylation and dephosphorylation control many different cell functions as well as responses to internal and external signals. It has also been shown that highly phosphorylated acidic proteins have an important role in matrix mediated biomineralization, perhaps functioning as nucleators for crystal formation. Dentine phosphoprotein (DPP) is one of such proteins which is exclusively synthesized by the odontoblast cells and therefore a likely candidate to play a significant role in normal and abnormal dentine biomineralization. These studies are directed at characterizing the protein kinases involved in dentinogenesis and in particular the enzyme(s) responsible for DPP phosphorylation. In this report we present data which indicate that there are several different types of kinases in the odontoblast-enriched dental papilla mesenchyme (DPM), some of which can phosphorylate DPP, such as casein kinase I and II. However, a different DPP-kinase activity was identified. This enzyme(s) appears to be different from other reported kinases, and it is the only kinase that can phosphorylate both phosphorylated DPP and enzymatically dephosphorylated DPP.

Acid Phosphatase↗

PubMed1995

Antisense oligonucleotides: an experimental strategy to advance a causal analysis of development.

A variety of modified and unmodified oligonucleotides (ODNs) have been examined as antisense inhibitors of gene expression. Of particular interest has been the application of antisense inhibitory experimental strategies to advance a suggested causal relationship between signal transduction and inductive epithelial-mesenchymal interactions during mandibular morphogenesis, early tooth development, tooth enamel formation, lung branching morphogenesis, kidney, muscle and heart development. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and a number of transforming growth factor beta (TGF-beta) isotype mediated signal transductions have been demonstrated to regulate inductive processes associated with significant processes in development including mouse molar tooth morphogenesis. Antisense strategies have also been useful in studies designed to associate a specific morphogen signal with homeobox (HOX) gene regulation in several embryonal carcinoma cell lines. The application and results from a number of antisense inhibitory strategies serve to support the utility of this experimental paradigm for future investigations of tooth development. This review discusses the experimental strategy, a number of technical issues and the rationale for future investigations of tooth development.

Animals↗

PubMed1995

The tooth as a model in organogenesis. An interview with Professor Harold C. Slavkin. Interview by Juan Arechaga.

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Career Choice↗

PubMed1995

Control of ameloblast differentiation.

This review highlights a number of advances towards understanding the sequential developmental cascade of events beginning in the oral ectodermally-derived odontogenic placode and culminating in the formation of the mineralized enamel extracellular matrix. Recent discoveries of growth factors, growth factor receptors and transcription factors associated with instructive epithelial-mesenchymal interactions and subsequent controls for ameloblast cell differentiation are reviewed. The relationship between ameloblast cytology, terminal differentiation and biochemical phenotype are discussed. The tissue-specific gene products characteristic of the ameloblast phenotype as well as their possible functions in formation of the enamel matrix are analyzed as well as the role of maturation-stage ameloblast cells in controlling enamel biomineralization. Finally, pathological conditions in which alterations in the ameloblast or specific gene products result in an abnormal enamel phenotype are reviewed. Clearly, the scientific progress achieved in the last few years concerning the molecular determinants involved in tooth development has been remarkable. However, there remains considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. Anticipated progress continues to require increased international cooperation and collaborations as well as increased utilization of structural biology investigations of enamel extracellular matrix proteins.

Ameloblasts↗

PubMed1995

The role of platelet-derived growth factor in the development of mouse molars.

Platelet-derived growth factor (PDGF) is a potent mitogen that functions in cytodifferentiation and wound healing. PDGF receptor-alpha (PDGFR-alpha) is required for the normal development of the dental ectomesenchyme (Stephenson et al., Proc. Natl. Acad. Sci. USA 88: 6-10, 1991). To investigate the regulatory potential of PDGF on tooth development, the expression of PDGF-AA was determined by reverse transcription-polymerase chain reaction (RT-PCR), the PDGF ligands and receptors were localized by immunohistochemistry. The growth-promoting effects of exogenous PDGF-AA on molar explants were determined by assaying for tritiated thymidine incorporation, the presence of type I collagen and amelogenin messenger RNA transcripts, and total DNA, RNA and protein accumulation. Based upon the temporal and spatial localization, PDGFs and their receptors are present in the enamel epithelia and pulpal mesenchyme of developing mouse molars. Exogenous PDGF-AA administration increased the total protein accumulation of mouse molar explants but produces no discernible effect on amelogenin and type I collagen expression.

Animals↗

PubMed1994

Translocation of enamel proteins from inner enamel epithelia to odontoblasts during mouse tooth development.

The developmental problem of how dental epithelia and/or dental papilla ectomesenchyme induce and/or up- or down-regulate tooth formation are as yet unresolved issues. We have designed studies to map the synthesis and fate pathways of secreted amelogenin proteins from Kallenbach differentiation zones II-IV during in vivo and in vitro mouse mandibular first molar tooth development (M1). Tooth organs from cap, bell, and crown stages were processed for reverse transcriptase/polymerase chain reaction (RT-PCR) and high resolution Protein A immunocytochemistry using anti-amelogenin and anti-peptide antibodies. Cap stage M1 were cultured for periods ranging from 10-21 days in vitro using either serum-less, or 15% fetal calf sera-supplemented, chemically-defined medium. Amelogenin transcripts are expressed in the mouse embryonic molar from E15 through early postnatal development. Amelogenin antigens were first detected in Kallenbach's differentiation zone II. Amelogenin proteins secreted from preameloblasts were identified along cell processes and cell surfaces of odontoblasts adjacent to forming mantle dentine extracellular matrix (ECM) prior to biomineralization. Amelogenin proteins were restricted to forming endocytotic vesicles, clathrin-coated vesicles, and lysosomes within odontoblasts. At later stages (e.g. 2 days postnatal development), enamel proteins were not identified in odontoblasts or predentine matrix following mineralization. Comparable observations for stages of development were noted for in vitro cultured tooth explants. Preameloblasts synthesize and secrete amelogenin proteins which bind to odontoblast cell surfaces possibly through the process of receptor-mediated endocytosis. We conclude that amelogenin proteins secreted from preameloblasts, prior to the initiation of biomineralization, were translocated to odontoblasts to serve as yet unknown biological functions.

Amelogenin↗

PubMed1994

Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering.

Recombinant murine amelogenins M179 and M166 were expressed in Escherichia coli and purified. The aggregation properties of these amelogenins have been investigated in aqueous solutions as well as acetonitrile-containing solutions using dynamic light scattering. Dynamic light scattering provides direct measurement of the translational diffusion coefficient and hydrodynamic radius, and of an estimate of the molecular weight. Polydispersity and statistical parameters of how to interpret the analysis are also provided. Amelogenin aggregation was examined in solutions of a range of pH, ionic strengths, and protein concentrations. It was shown that at pH 7.8-8 and ionic strength of 0.02-0.05M the M179 molecules form monodispersed aggregates with hydrodynamic radii ranging from 15 to 19 nm. Analysis of hydrodynamic radii and size distribution of M179 aggregates in acetonitrile-containing solvents compared to that in aqueous solutions indicated a primary role for hydrophobic interactions in the association process of amelogenin molecules to form aggregates. Comparison between the aggregates formed by M179 and M166, which lacks the hydrophilic carboxy-terminal 13 residue sequence of M179, suggested that the self-assembly of amelogenin molecules to form stable and monodisperse aggregates requires the presence of the hydrophilic carboxy-terminal sequence of M179.

Amelogenin↗

PubMed1994

Specific transforming growth factor-beta subtypes regulate embryonic mouse Meckel's cartilage and tooth development.

Members of the transforming growth factor-beta (TGF-beta) superfamily have emerged as critical regulators for cell growth and differentiation. Whereas the different TGF-beta subtypes are equipotent in the majority of biological assays using cell lines cultured in vitro, there are indications that in more complex systems involving epithelial-mesenchymal interactions, the TGF-beta subtypes differ in their biological activities. To test the hypothesis that TGF-beta subtypes specifically regulate either Meckel's cartilage or tooth morphogenesis, we designed experiments to compare loss of function effects of TGF-beta 1, TGF-beta 2, and TGF-beta 3 subtypes using a serumless, chemically defined medium to culture embryonic mouse E10 (42-44 somite pairs) mandibular explants. The major effect of loss of function resulting from abrogation of TGF-beta 1 using antisense treatment resulted in a 20% increase (P < 0.05) in chondrocyte number, a decrease in extracellular matrix, and dysmorphology of the rostral region of Meckel's cartilage. Exogenous TGF-beta 1 provided indistinguishable recovery to the normal phenotype. TGF-beta 2 antisense treatment produced a threefold enlargement (P < 0.05) of tooth organs and advanced their development to the cap stage. TGF-beta 2 provided recovery to the normal phenotype (e.g., reduced tooth size and development to the bud stage), whereas TGF-beta 1 or TGF-beta 3 polypeptides had no effect. TGF-beta 3 antisense treatment resulted in a reduction of approximately 15% in the length of Meckel's cartilage. We interpret these results to suggest that TGF-beta 1 functions to regulate the number of chondrogenic cells, the amount of extracellular matrix, and the rate of developmental assembly of the rostral to posterior segments in forming Meckel's cartilage. TGF-beta 2 appears to regulate tooth size and stage of development without affecting cartilage. TGF-beta 3 appears to regulate Meckel's cartilage size without altering tooth size or shape. The results are discussed in terms of the regulatory functions of the TGF-beta subtypes during embryonic craniofacial morphogenesis.

Animals↗