Top 10 priorities for future infertility research: an international consensus development study† ‡.

STUDY QUESTION: Can the priorities for future research in infertility be identified? SUMMARY ANSWER: The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction and ethics, access and organization of care for people with fertility problems were identified. WHAT IS KNOWN ALREADY: Many fundamental questions regarding the prevention, management and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems. STUDY DESIGN, SIZE, DURATION: Potential research questions were collated from an initial international survey, a systematic review of clinical practice guidelines and Cochrane systematic reviews. A rationalized list of confirmed research uncertainties was prioritized in an interim international survey. Prioritized research uncertainties were discussed during a consensus development meeting. Using a formal consensus development method, the modified nominal group technique, diverse stakeholders identified the top 10 research priorities for each of the categories male infertility, female and unexplained infertility, medically assisted reproduction and ethics, access and organization of care. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, people with fertility problems and others (healthcare funders, healthcare providers, healthcare regulators, research funding bodies and researchers) were brought together in an open and transparent process using formal consensus methods advocated by the James Lind Alliance. MAIN RESULTS AND THE ROLE OF CHANCE: The initial survey was completed by 388 participants from 40 countries, and 423 potential research questions were submitted. Fourteen clinical practice guidelines and 162 Cochrane systematic reviews identified a further 236 potential research questions. A rationalized list of 231 confirmed research uncertainties was entered into an interim prioritization survey completed by 317 respondents from 43 countries. The top 10 research priorities for each of the four categories male infertility, female and unexplained infertility (including age-related infertility, ovarian cysts, uterine cavity abnormalities and tubal factor infertility), medically assisted reproduction (including ovarian stimulation, IUI and IVF) and ethics, access and organization of care were identified during a consensus development meeting involving 41 participants from 11 countries. These research priorities were diverse and seek answers to questions regarding prevention, treatment and the longer-term impact of infertility. They highlight the importance of pursuing research which has often been overlooked, including addressing the emotional and psychological impact of infertility, improving access to fertility treatment, particularly in lower resource settings and securing appropriate regulation. Addressing these priorities will require diverse research methodologies, including laboratory-based science, qualitative and quantitative research and population science. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations, including the representativeness of the participant sample, methodological decisions informed by professional judgment and arbitrary consensus definitions. WIDER IMPLICATIONS OF THE FINDINGS: We anticipate that identified research priorities, developed to specifically highlight the most pressing clinical needs as perceived by healthcare professionals, people with fertility problems and others, will help research funding organizations and researchers to develop their future research agenda. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Auckland Medical Research Foundation, Catalyst Fund, Royal Society of New Zealand and Maurice and Phyllis Paykel Trust. G.D.A. reports research sponsorship from Abbott, personal fees from Abbott and LabCorp, a financial interest in Advanced Reproductive Care, committee membership of the FIGO Committee on Reproductive Medicine, International Committee for Monitoring Assisted Reproductive Technologies, International Federation of Fertility Societies and World Endometriosis Research Foundation, and research sponsorship of the International Committee for Monitoring Assisted Reproductive Technologies from Abbott and Ferring. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and editor for the Cochrane Gynaecology and Fertility Group. J.L.H.E. reports being the Editor Emeritus of Human Reproduction. A.W.H. reports research sponsorship from the Chief Scientist's Office, Ferring, Medical Research Council, National Institute for Health Research and Wellbeing of Women and consultancy fees from AbbVie, Ferring, Nordic Pharma and Roche Diagnostics. M.L.H. reports grants from Merck, grants from Myovant, grants from Bayer, outside the submitted work and ownership in Embrace Fertility, a private fertility company. N.P.J. reports research sponsorship from AbbVie and Myovant Sciences and consultancy fees from Guerbet, Myovant Sciences, Roche Diagnostics and Vifor Pharma. J.M.L.K. reports research sponsorship from Ferring and Theramex. R.S.L. reports consultancy fees from AbbVie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. B.W.M. reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. E.H.Y.N. reports research sponsorship from Merck. C.N. reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring and retains a financial interest in NexHand. J.S. reports being employed by a National Health Service fertility clinic, consultancy fees from Merck for educational events, sponsorship to attend a fertility conference from Ferring and being a clinical subeditor of Human Fertility. A.S. reports consultancy fees from Guerbet. J.W. reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. A.V. reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and the journal Reproduction. His employing institution has received payment from Human Fertilisation and Embryology Authority for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. N.L.V. reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the present work. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: N/A.

Top 10 priorities for future infertility research: an international consensus development study.

STUDY QUESTION: Can the priorities for future research in infertility be identified? SUMMARY ANSWER: The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care for people with fertility problems were identified. WHAT IS KNOWN ALREADY: Many fundamental questions regarding the prevention, management, and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems. STUDY DESIGN, SIZE, DURATION: Potential research questions were collated from an initial international survey, a systematic review of clinical practice guidelines, and Cochrane systematic reviews. A rationalized list of confirmed research uncertainties was prioritized in an interim international survey. Prioritized research uncertainties were discussed during a consensus development meeting. Using a formal consensus development method, the modified nominal group technique, diverse stakeholders identified the top 10 research priorities for each of the categories male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, people with fertility problems, and others (healthcare funders, healthcare providers, healthcare regulators, research funding bodies and researchers) were brought together in an open and transparent process using formal consensus methods advocated by the James Lind Alliance. MAIN RESULTS AND THE ROLE OF CHANCE: The initial survey was completed by 388 participants from 40 countries, and 423 potential research questions were submitted. Fourteen clinical practice guidelines and 162 Cochrane systematic reviews identified a further 236 potential research questions. A rationalized list of 231 confirmed research uncertainties were entered into an interim prioritization survey completed by 317 respondents from 43 countries. The top 10 research priorities for each of the four categories male infertility, female and unexplained infertility (including age-related infertility, ovarian cysts, uterine cavity abnormalities, and tubal factor infertility), medically assisted reproduction (including ovarian stimulation, IUI, and IVF), and ethics, access, and organization of care, were identified during a consensus development meeting involving 41 participants from 11 countries. These research priorities were diverse and seek answers to questions regarding prevention, treatment, and the longer-term impact of infertility. They highlight the importance of pursuing research which has often been overlooked, including addressing the emotional and psychological impact of infertility, improving access to fertility treatment, particularly in lower resource settings, and securing appropriate regulation. Addressing these priorities will require diverse research methodologies, including laboratory-based science, qualitative and quantitative research, and population science. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations, including the representativeness of the participant sample, methodological decisions informed by professional judgement, and arbitrary consensus definitions. WIDER IMPLICATIONS OF THE FINDINGS: We anticipate that identified research priorities, developed to specifically highlight the most pressing clinical needs as perceived by healthcare professionals, people with fertility problems, and others, will help research funding organizations and researchers to develop their future research agenda. STUDY FUNDING/ COMPETING INTEREST(S): The study was funded by the Auckland Medical Research Foundation, Catalyst Fund, Royal Society of New Zealand, and Maurice and Phyllis Paykel Trust. Geoffrey Adamson reports research sponsorship from Abbott, personal fees from Abbott and LabCorp, a financial interest in Advanced Reproductive Care, committee membership of the FIGO Committee on Reproductive Medicine, International Committee for Monitoring Assisted Reproductive Technologies, International Federation of Fertility Societies, and World Endometriosis Research Foundation, and research sponsorship of the International Committee for Monitoring Assisted Reproductive Technologies from Abbott and Ferring. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and editor for the Cochrane Gynaecology and Fertility Group. Hans Evers reports being the Editor Emeritus of Human Reproduction. Andrew Horne reports research sponsorship from the Chief Scientist's Office, Ferring, Medical Research Council, National Institute for Health Research, and Wellbeing of Women and consultancy fees from Abbvie, Ferring, Nordic Pharma, and Roche Diagnostics. M. Louise Hull reports grants from Merck, grants from Myovant, grants from Bayer, outside the submitted work and ownership in Embrace Fertility, a private fertility company. Neil Johnson reports research sponsorship from Abb-Vie and Myovant Sciences and consultancy fees from Guerbet, Myovant Sciences, Roche Diagnostics, and Vifor Pharma. José Knijnenburg reports research sponsorship from Ferring and Theramex. Richard Legro reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. Ben Mol reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. Ernest Ng reports research sponsorship from Merck. Craig Niederberger reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and retains a financial interest in NexHand. Jane Stewart reports being employed by a National Health Service fertility clinic, consultancy fees from Merck for educational events, sponsorship to attend a fertility conference from Ferring, and being a clinical subeditor of Human Fertility. Annika Strandell reports consultancy fees from Guerbet. Jack Wilkinson reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. Andy Vail reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from HFEA for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. Lan Vuong reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the present work. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: Not applicable.

Human and mouse cementum proteins immunologically related to enamel proteins.

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.

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.

Progressive accretion of amelogenin molecules during nanospheres assembly revealed by atomic force microscopy.

Amelogenin proteins, the principal components of the developing dental enamel matrix, self-assemble to form nanosphere structures that are believed to function as structural components directly involved in the matrix mediated enamel biomineralization. The self-assembly behavior of a recombinant murine amelogenin (rM179) was investigated by atomic force microscopy (AFM) for further understanding the roles of amelogenin proteins in dental enamel biomineralization. Recombinant rM179 amelogenin was dissolved in a pH 7.4 Tris-HCl buffer at concentrations ranging from 12.5 to 300 microg/ml. The solutions were adsorbed on mica, fixed with Karnovsky fixative and rinsed thoroughly with water for atomic force microscopy (AFM). At low concentrations (12.5-50 microg/ml), nanospheres with diameters varying from 7 to 53 nm were identified while at concentrations ranging between 100-300 microg/ml the size distribution was significantly narrowed to be steadily between 10 and 25 nm in diameter. These nanospheres were observed to be the basic building blocks of both engineered rM179 gels and of the developing enamel extracellular matrix. The stable 15-20-nm nanosphere structures generated in the presence of high concentrations of amelogenins were postulated to be of great importance in facilitating the highly organized ultrastructural microenvironment required for the formation of initial enamel apatite crystallites.

Immunohistochemical similarities and differences between amelogenin and tuftelin gene products during tooth development.

Amelogenins and tuftelins are highly specialized proteins secreted into the developing enamel matrix during mammalian enamel formation. Both tuftelins and amelogenins have been associated with various functions during nucleation and maturation of the developing enamel matrix. In this study we conducted experiments to investigate whether tuftelins and portions of the amelogenin molecule were deposited and processed in spatially distinguished portions of the developing enamel matrix, using antibodies specific against tuftelin or amelogenins. The amelogenin antibodies were raised against recombinant and native amelogenins and also included an antibody against a polypeptide encoded by amelogenin exon 4. To compare spatial expression patterns of enamel protein epitopes, 3-day postnatal mouse molar tooth organs were processed for paraffin histology and cut into serial sections. Adjacent sections were exposed to antibodies against either tuftelin or various amelogenin epitopes. To investigate age-related changes of enamel protein expression, amelogenin and tuftelin antibodies were applied to tooth organs of developmental stages E19 and 1, 3, 5, 7, 9 and 11 postnatal days. Tuftelin was detected within the odontoblast processes during earlier stages of development (E19 and 1 day postnatal), whereas during later stages (3-11 days) it was recognized in a portion of the enamel layer adjacent to the dentine-enamel junction. In contrast, all four antibodies against amelogenins reacted with parts of the ameloblast cytoplasm and the entire enamel layer. Using immunohistochemistry, we were not able to detect any differences in the spatial distribution of the four amelogenin epitopes investigated. The spatial differences in the distribution of amelogenin and tuftelin as observed in this study may be interpreted as an indication of functional differences between both proteins during early enamel biomineralization.

Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors.

Mineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and mid- to late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 microm to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. (J Histochem Cytochem 46:911-934, 1998)

Modulation of apatite crystal growth on Bioglass by recombinant amelogenin.

The effects of a recombinant mouse amelogenin (rM179) on the growth of apatite crystals nucleated on a bioactive glass (45S5 type Bioglass) surface were investigated with a view to gaining a better understanding of the role of amelogenin protein in tooth enamel formation and of its potential application in the design of novel enamel-like biomaterials. Bioglass discs were incubated in phosphate-buffered saline (PBS) to preform a calcium phosphate surface layer and subsequently immersed in blank, bovine serum albumin (BSA)- and rM179-containing supersaturated calcification solutions (SCS(B), SCS(BSA) and SCSrM179), respectively. Calcium phosphate layers formed on all the treated samples and were characterized to be apatite by X-ray diffraction and Fourier transmission infrared spectrophotometry. Under scanning electron microscopy, plate-shaped crystals (approximately 50 nm thick and 300-600 nm across) were observed on the samples after PBS incubation. The crystals grown from SCS(B) were of the typical plate shape except for an increased thickness, while needle-shaped crystals (200-300 nm long and 50-70 nm thick) were precipitated on the SCS(BSA)-immersed samples. Interestingly, it was found that the crystals deposited on the SCSrM179-immersed samples adopted an elongated, curved shape (approximately 500 nm long and approximately 120 nm thick). Further TEM observations showed that the crystals generated by the SCSrM179 immersion appeared to be composed of bundles of lengthwise crystals (15-20 nm thick) orientated parallel to one another, much alike the long and thin crystals observed in the very early stage of enamel formation. The significant modulation by the rM179 protein of apatite crystal growth is quite different from the overall inhibition observed by BSA and most likely is relevant to the specific function of the amelogenin matrix in controlling enamel crystal growth in vivo.

Dental enamel matrix--isolation and partial sequence of a peptide component.

Chromatography of demineralized bovine fetal enamel matrix proteins on 'Biogel P6' was shown to yield a trailing peak which contained a single electrophoretic component. This polypeptide, further purified by chromatography on 'Biogel P4', was found to be homogeneous by disc electrophoresis and gel isoelectric focussing. Amino acid analyses indicated this component (designated: "E5') to be similar to some of the phosphopeptides previously described. Cyanogen bromide cleavage of E5 yielded three principal products whose amino acid compositions and N-terminal residues were investigated. A partial sequence for component E5 was proposed and comparison with previous bovine matrix isolates was made.

Dose-dependent modulation of octacalcium phosphate crystal habit by amelogenins.

In vitro studies on interactions between amelogenins and calcium phosphate crystals are critical for elucidating biomineralization mechanisms of tooth enamel. This work was aimed at investigating the effects of native porcine amelogenins on octacalcium phosphate (OCP) crystal growth in a gelatin gel. We prepared OCP mineral discs by circulating calcium and phosphate solutions on the opposite ends of the gels loaded with 0-2% amelogenin for one week. A dose-dependent modulation of OCP crystal habit by amelogenins was observed by scanning electron microscopy. While the incorporation of 0.125, 0.25, or 0.5% amelogenins showed no significant effect on the crystal morphology, in the presence of 1 and 2% amelogenins, the crystals were remarkably longer, having an average aspect ratio 3-5 times greater than that of those formed in the control gels. Transmission electron microscopy and atomic force microscopy suggested that amelogenin assemblies selectively blocked b-axial development, resulting in the c-axial elongation of OCP crystals.

Molecular composition of the protein matrix of developing human dental enamel.

The protein matrix of fetal human dental enamel was isolated and fractionated by chromatographic and electrophoretic procedures. Nine principal protein fractions were isolated and characterized. Possible inter-relationships of these proteins and their comparison with data from other species were examined. Significant differences between human proteins and those described for cow or pig enamel were identified.

Quantitative analysis of amelogenin solubility.

Amelogenins are a group of extracellular enamel matrix proteins which are believed to be involved in the regulation of the size and habits of forming enamel crystals. The aim of this study was to compare the solubility properties of several amelogenins at various pH (from 4.0 to 9.0) at constant ionic strength (IS), and to examine the influence of buffer composition, IS, and divalent metal ions (including Ca2+, Mg2+, and Zn2+) on amelogenin solubility. The solubility of the recombinant murine amelogenin ("rM179") was minimum near its isoelectric point and increased rapidly below and above, regardless of buffer composition. A similar trend was observed for the native porcine ("25K") amelogenin. Porcine "23K" amelogenin was only sparingly soluble from pH of 4.0 to 9.0, in contrast to the analogous recombinant "rM166", which was more soluble in acidic solutions. The synthetic amelogenin polypeptide "TRAP" was extremely insoluble, while synthetic LRAP was readily soluble. Porcine "20K" amelogenin solubility increased strikingly as the solution pH was lowered from 7.0 to 6.0. Increasing IS decreased the solubility of rM179. While Zn2+ reduced rM179 solubility, Ca2+ and Mg2+ showed no significant effects. We conclude that the solubility of amelogenin was dependent on the primary structure, solution pH, and IS, and the low solubility of amelogenins under physiological conditions may result from their tendency to form quaternary (aggregate) structures in vivo.

Elongated growth of octacalcium phosphate crystals in recombinant amelogenin gels under controlled ionic flow.

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.

Cloning, DNA sequence, and alternative splicing of opossum amelogenin mRNAs.

The enamel layer that covers the surfaces of teeth is thickest and most highly mineralized in mammals. The durability of mammalian enamel may have allowed for selection against the lifelong replacement of teeth that is observed in other vertebrates. Variation in enamel structure among animals is thought to be the result of evolutionary changes in the constituents of the developing enamel matrix. In placental mammals, the principal component of this matrix is amelogenin. We have determined the complete primary structures of two opossum amelogenins through a combination of protein sequencing, cloning, and DNA sequencing. RNA messages were cloned that encode 202- and 57-residue amelogenins, which are presumed to be expressed from the same gene but differ due to alternative splicing of identical pre-mRNAs. Edman degradation of the larger amelogenin ran for 42 cycles and yielded the sequence: IPLPPHPGHPGYINFS YEVLTPLKWYQSMMRQQYPSYGYEPM. The derived 202-residue amelogenin, assuming that serine 16 is phosphorylated, has an isotope-averaged molecular mass of 23,023.75 Daltons and a pI of 6.2. This is the largest amelogenin yet characterized. The increase in length is due to the presence of a 30-residue tandem repeat of QP(I/M) in exon 6 in the same position as a similar, but shorter, repeat expressed from the bovine X-chromosome. The 57-residue amelogenin, which is known from other organisms as the leucine-rich amelogenin protein (LRAP), has an isotope-averaged molecular mass of 6764.75 Daltons and a pI of 5.5. The opossum enamel protein is highly homologous to those previously characterized in eutherians and demonstrates that amelogenins were refined structurally prior to the metatherian/eutherian divergence between 100 and 150 million years ago.

Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins.

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the catalytic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated together, and the digestion products were analyzed by SDS-PAGE and mass spectrometric analyses. We assigned amelogenin cleavage products by selecting among the possible polypeptides having a mass within 2 Daltons of the measured values. The polypeptides identified included the intact protein (amino acids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46-147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 include virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that processes enamel proteins during the secretory phase of amelogenesis.

Dental enamel matrix: sequences of two amelogenin polypeptides.

The amino acid sequences of a leucine-rich amelogenin polypeptide (LRAP) and a tyrosine-rich amelogenin polypeptide (TRAP), isolated from foetal bovine enamel matrix, were determined. Both LRAP and TRAP occurred in two forms; in each case, one of the molecular species appeared to be shortened at the COOH terminus by 2 and 4 residues, respectively. A striking finding was that LRAP and TRAP had identical sequences for the first 33 residues but were almost completely different for the remaining 12 amino acids.

Specific cleavage of a recombinant murine amelogenin at the carboxy-terminal region by a proteinase fraction isolated from developing bovine tooth enamel.

A proteinase fraction of 48-70-kDa was isolated from developing bovine tooth enamel by size exclusion and reversed-phase high-pressure liquid chromatography (HPLC) techniques. Proteolytic activity in the HPLC fraction was visualized by enzymography using gelatin as substrate. A recombinant murine amelogenin (M179) composed of 179 amino acid residues (20 kDa) was used as a substrate to examine the specificity of the enzymes in the isolated fractions. Incubation of M179 with the proteinase fraction at 37 degrees C generated a major proteolytic product eluting at about 42% acetonitrile from the reversed-phase column. This product had an amino-terminal sequence Pro-Leu-Pro-Pro-His-Pro- in conformity with that of the M179 parent protein. These data indicated that the product resulted from the cleavage of the M179 recombinant protein in the carboxy-terminal region. Mass spectroscopic analysis of the product isolated by reversed-phase HPLC gave a molecular mass of 18.89 kDa. Given an intact amino-terminal sequence, this mass figure suggests that this product terminates at Pro168 of the M179 residue sequence. The presence of EDTA in proteolysis experiments when M179 was used as substrate inhibited production of the 18.89-kDa product. Antipain, aprotinin, leupeptin and 4,(amidinophenyl)methanesulphonyl fluoride, which are serine proteinase inhibitors, did not affect the proteolytic activity. In addition, replacement of Ca2+ with Zn2+, Mn2+ or Co2+ in the proteolysis buffer inhibited the enzymatic activity. It is concluded that the 'high molecular-weight' proteinase cleaving M179 at Pro168-Ala169 is a specific 'calcium-dependent metalloproteinase'.

Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth.

The primary structures, molecular genetics and biosynthesis of the amelogenin protein of the developing tooth are established, but knowledge of their subsequent post-secretory processing and its relation to enamel biomineralization is fragmentary. Preparations of tooth matrix proteins were isolated from molars (M1) of mice from birth to 15 days and analysed by SDS-PAGE and immunochemical methods. Amelogenin proteins, isolated and partially purified by HPLC, were characterized by amino acid analysis and SDS-PAGE. At birth a 26 kDa amelogenin was present that during subsequent developmental stages generated a series of 20-25 kDa amelogenins differing in apparent size by approximately 1 kDa. Amino acid analyses showed that all these amelogenins have amino-terminal TRAP sequences; analyses for both glycosylation and phosphorylation were negative. It is suggested that these post-secretory amelogenins are generated by a sequence of specific carboxy-terminal cleavages, and that the observed post-secretory processing of amelogenin is functionally linked to the structure of the enamel matrix and the control of crystallite development.

The changing face of dental education: the impact of PBL.

The past decade has seen increasing demands for reform of dental education that would produce a graduate better equipped to work in the rapidly changing world of the twenty-first century. Among the most notable curriculum changes implemented in dental schools is a move toward Problem-Based Learning (PBL). PBL, in some form, has been a feature of medical education for several decades, but has only recently been introduced into dental schools. This paper discusses the rationale for the introduction of a PBL pedagogy into dental education, the modalities of PBL being introduced, and the implications of the introduction of PBL into dental schools. Matters related to implementation, faculty development, admissions, and assessment are addressed. Observations derived from a parallel-track dental PBL curriculum at the University of Southern California (USC) are presented and discussed. This program conforms to the Barrows (1998) concept of "authentic PBL" in that the program has no scheduled lectures and maintains a PBL pedagogy for all four years of the curriculum. The USC dental students working in the PBL curriculum have attained a high level of achievement on U.S. National Dental Boards (Part I) examinations, significantly superior to their peers working in a traditional lecture-based curriculum.

Problem-based learning at the University of Southern California School of Dentistry.

Responding to the recent Institute of Medicine report on dental education, the Center for Craniofacial Molecular Biology (CCMB) of the University of Southern California School of Dentistry has developed a parallel track program in dental education leading to the D.D.S. degree. This program was proposed in May of 1995, and the first class of twelve students was admitted in September of that year. Currently two classes are enrolled and plans to admit a further twelve students (Class of 2001) are in place. The educational strategy for this program is totally problem-based. Students work in groups of six with a faculty facilitator, not necessarily a content expert. Facilitators are largely drawn from the multidisciplinary pool of research faculty at the center. All learning is mediated through biomedical and biodental problem cases. No formal lectures or classes are scheduled. The learning of clinical dental skills is promoted through focussed dental patient simulations in which students review clinical charts, radiographs, medical reports and then explore identified, hands-on learning needs using patient simulators in a clinical context. Early patient exposure is obtained through dental office visits and other special patient clinics. Initial experience with this program suggests that the problem-based learning (PBL) students learn as well (if not better) than their traditional program peers and develop excellent group and cognitive analytical skills. The absence of a pool of dentally related biomedical cases suitable for a PBL program has necessitated the use of innovative approaches to their development and presentation. It is believed that this educational approach will produce dental clinicians equipped with the self-motivated, life-long learning skills required in the ever-changing world of bio-dental sciences in the twenty-first century.