The International Journal of Developmental Biology
The central problems of enamel biochemistry are the mechanisms concerned with initiation and deve... more The central problems of enamel biochemistry are the mechanisms concerned with initiation and development of the mineral crystals, together with their architectural arrangement within the tissue. These processes are mediated by the extracellular matrix as well as the composition of the mineral itself. Initial mineral deposition occurs at the dentine surface, nucleated either by dentinal components or early enamel matrix, possibly non-amelogenin molecules. The early crystals are small in size and rich in magnesium and carbonate resulting in relatively poor crystallinity. This is in spite of the fact that fluoride is high at this stage. Crystal development includes a reduction in magnesium, carbonate and fluoride as crystals increase in length following the retreating ameloblasts from the dentine. The matrix acquires increasing concentrations of amelogenin and albumin. Prismatic structure begins to develop together with some growth of crystals in width and thickness. Degradation of ame...
Enamel maturation is characterized by massive crystal growth in both width and thickness, resulti... more Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.
Objective: Molecular events defining enamel matrix removal during amelogenesis are poorly underst... more Objective: Molecular events defining enamel matrix removal during amelogenesis are poorly understood. In early reports coated vesicles were shown on the cytoplasmic surface of the apical pole of ameloblasts, suggesting that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Method: and Result: Here, using real time PCR, we show that clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most highly up-regulated of the four distinct adaptor protein complexes. Immunolocalizations confirm the presence of AP-2 and clathrin in ameloblasts, where the highest expression is noted at the apical pole. AP-2 and clathrin are also present in other enamel organ cells. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying ...
There are several mouse models for human Amelogenesis imperfecta generated by gene knockout or in... more There are several mouse models for human Amelogenesis imperfecta generated by gene knockout or induced mutation of the enamel matrix genes e.g. Masuya et al, Hum Mol Genet 14:275, 2005. However, there is little baseline data on the mouse dentition and it has largely been assumed that mouse incisor development closely follows the extremely consistent pattern seen in rats. Objectives: To correlate mineral density, microhardness, and composition with enamel architecture in the mouse incisor and to compare this to the situation in rats. Methods: Wild-type mouse lower incisors were either 1) fixed, demineralised and examined histologically, 2) embedded in methylmethacrylate, sectioned transverse to their long axis and analysed for mineral density by TMR and microhardness by microindentation, viewed by SEM and composition determined by EDS, 3) whole teeth were analysed by nanoCT. Results: Histology revealed that unlike the rat, mouse incisors retained residual matrix proteins throughout a...
Amelogenin is the principle extracellular matrix protein in developing enamel. Amelx mutations ca... more Amelogenin is the principle extracellular matrix protein in developing enamel. Amelx mutations can have severe impact on dental phenotype but the precise role of amelogenin remains unclear. Understanding the effects of specific amelogenin mutations on enamel phenotype will help clarify this role. Objectives: To identify any Amelx mutation and characterise enamel phenotype in the mouse mutant M100888. Methods: Direct sequencing of the Amelx gene was carried out. Phenotype analysis of mandibular incisors of affected and wild type (WT) animals included quantitative microradiography, immunohistology and SDS PAGE/ amelogenin western blotting of the developing enamel matrix following microdissection. Results: A point mutation in the amelogenin N-acetylglucosamine binding domain was identified (Tyr to His). Affected males had significant areas of erupted enamel missing while remaining enamel exhibited normal mineral content. Histology of the developing teeth showed disordered ameloblasts a...
Mutations in Enam result in autosomal dominant hypoplastic Amelogenesis imperfect (AI). However, ... more Mutations in Enam result in autosomal dominant hypoplastic Amelogenesis imperfect (AI). However, the resulting enamel phenotype, whether a generalised or local hypoplasia, appears to depend upon the nature of the specific mutation. There are a number of murine models for human AI and several ENU-induced mutations in Enam have been shown to result in hypoplastic AI in the mouse (Masuya et al, Hum Mol Genet 14:275, 2005). Objectives: To compare incisor enamel thickness and mineral density in mice with each of two distinct ENU-induced Enam mutations: a G to T substitution at position 6489 resulting in an amino acid change at residue 55 of enamelin protein (ENAMRgsc395) and a T to A substitution at the splicing donor site in intron 4, resulting in a frameshift giving rise to a premature stop codon (ENAMRgsc521). Methods: Lower incisors from wild-type (WT) mice and those heterozygous (+/-) and homozygous (-/-) for the mutant alleles were embedded in methylmethacrylate and sectioned trans...
Amelogenin is essential for amelogenesis and amelogenin mutations lead to X-linked amelogenesis i... more Amelogenin is essential for amelogenesis and amelogenin mutations lead to X-linked amelogenesis imperfecta (AI) in humans. Previously we showed that the Y64H amelogenin mutation causes AI by a mechanism involving increased ameloblast apoptosis when compared to wild type mice(Barron et al (2010), Hum. Mol. Genet. doi:10.1093/hmg/ddq001). Objective: To investigate the role of cellular stress in the molecular pathogenesis of AI in mice expressing the Y64H amelogenin mutation. Methods: The mandibular incisor teeth of mice expressing the Y64H amelogenin mutation were investigated by immunohistochemistry, electron microscopy and in situ hybridisation. Results: Immunohistochemistry of affected secretory ameloblasts revealed the accumulation of vesicles containing both amelogenin and ameloblastin. Vesicles were positive for the endoplasmic reticulum (ER) markers calnexin and protein disulphide isomerase but also contained the Golgi markers GM130 and COPI. Ultrastructurally, the vesicles app...
A number of murine models have appeared for human AD hypoplastic AI either by knockout or by indu... more A number of murine models have appeared for human AD hypoplastic AI either by knockout or by induced mutation in the enamelin gene. One such model involves a G to T substitution at position 6489 resulting in an amino acid change at position 55 of the translated protein (Masuya et al, Hum Mol Genet 14:275, 2005). Objectives: To correlate mineral density, microhardness, and composition with enamel architecture in mice carrying this mutation, designated ENAMRgsc395. This will then allow direct comparison between this data and that obtained for human teeth for which this is a potential model. Methods: Lower incisors from wild-type mice and those heterozygous for the mutant allele were embedded in methylmethacrylate and sectioned transverse to their long axis with a peripheral diamond disc. Sections from mature enamel were polished plano-parallel to a thickness of 100 m, etched for 15 sec with 35 % phosphoric acid and thoroughly washed. Mineral density across the full enamel thickness wa...
The 32kDa post-secretory breakdown product of enamelin (a minor component of the developing ename... more The 32kDa post-secretory breakdown product of enamelin (a minor component of the developing enamel matrix) has been predicted to have an important function in enamel biomineralisation. This view is supported by the fact that mutations in the ENAM gene are associated with mineralisation defects, resulting in amelogenesis imperfecta. Objectives: To determine the mineral nucleating potential of a recombinant 32 kDa enamelin (rEnamelin) in vitro. Methods: rEnamelin was expressed in an eukaryotic expression system and purified for use in the steady state model described by Hunter and Goldburg. rEnamelin (1 ug/mL 10 ug/mL) was added to agarose gels and incubated under steady state conditions for 7d at 37C. Agarose alone and poly-glutamate (PGA, 10 g/mL) served as negative and positive controls respectively. Mineral deposition was determined by measurement of phosphate in the gels at the end of each run. Mineral deposits were characterised using SEM. Results: rEnamelin nucleated mineral at...
Objectives: Understanding the formation of enamel is of particular interest in the study of biomi... more Objectives: Understanding the formation of enamel is of particular interest in the study of biomineralisation processes and the development of biomaterials. Amelogenin is the most abundant component of the extracellular matrix associated with enamel formation. To distinguish the role of full-length amelogenin (P173) and its processing product lacking the hydrophilic C-terminal (P148), we have studied amelogenin adsorption onto a model crystal surface. In both the outer and inner regions of developing enamel the interface between protein matrix and crystal surface is the critical juncture, therefore we have used the novel technique of neutron reflectometry (NR) to probe this interface directly. Methods: Adsorption of nascent full-length P173 amelogenin protein and its 148 residue processing product onto silica substrates was studied using NR. For each experiment, the bare substrate was first characterised, then amelogenin P173 and P148 were introduced at a concentration of 0.1 mg/ml ...
Amelogenins are self-assembling proteins comprising 90% of the developing enamel matrix. AMELX ge... more Amelogenins are self-assembling proteins comprising 90% of the developing enamel matrix. AMELX gene mutations result in X-linked amelogenesis imperfecta (AI). We recently reported an amelogenin tyrosine to histidine mutation (Y64H) in mice treated with N-ethyl-N-nitrosurea (M100888, RIKEN, Japan) and displaying an AI phenotype. Western blotting of the developing affected enamel indicated an absence of full length amelogenin from the tissue. Objectives: To express recombinant wild type (WT) and Y64H amelogenins and compare their self assembly properties and degradation by enamel proteases in vitro. Methods: Amelogenin genes were inserted into a pET 28 vector modified with the addition of a human retrovirus 3C protease site for expression in E. coli. Recombinant proteins were purified using affinity chromatography and preparative electrophoresis and characterised by SDS-PAGE, Western blotting and mass spectroscopy. Functional analysis included in vitro proteolytic degradation with ena...
Objective: Amelogenesis Imperfecta (AI) is a collective term for a heterogeneous group of enamel ... more Objective: Amelogenesis Imperfecta (AI) is a collective term for a heterogeneous group of enamel biomineralisation abnormalities. Our research group aims to identify AI-causing gene mutations that give insight into the network of genes critical to human enamel formation. This study aims to describe the genetic cause of generalised hypoplastic AI observed in three siblings from a second cousin consanguineous family. Method: Family members were assessed through clinical examination and supporting investigations. DNA and other samples were collected following informed consent and where appropriate ethics committee approval. SNP chip genotyping identified a locus consistent with inheritance identical by descent that differed from those in unaffected members. DNA from one affected individual was whole exome sequenced on an Illumina HiSeq 2500 sequencer. The sequence data was aligned to human reference genome (GRCh37) and analysed for potential relevant mutations within the homozygous int...
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research
Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early ... more Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during matur...
Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase ... more Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography-electrospray ionization-tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.
Objective: Therapeutic effects of enamel matrix derivatives have been extensively documented. But... more Objective: Therapeutic effects of enamel matrix derivatives have been extensively documented. But, the physiological expression and functions of enamel matrix peptides (EMPs) are a matter of debate. The present study explores endogenous amelogenin (AMELX) and ameloblastin (AMBN)mRNA/proteins in bones. Method: Samples. of mandibular alveolar (AB) and basal (BB) bone of growing (7 PN days) and adult (8 weeks) mice were microdissected and RNAs and proteins, extracted and analyzed. Laser-captured tissue extracts were also studied at 3 PN days. Results: RT-qPCR and Western-blotting evidenced AMELX and AMBN expression in AB and BB (about 10x less relatively to incisor enamel organ at 7PN days). These levels decreased with age and varied according to the anatomical sites (AB ≥BB). In situ studies were monitored using (1) enamel and ameloblasts as positive controls and (2) RT-qPCR/Western-blot data on their relative levels and (3) a set of negative controls (notably AMEL-KO mice –NIDCR, NIH...
ABSTRACT Enamel maturation is characterized by massive crystal growth in both width and thickness... more ABSTRACT Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.
Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomi... more Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In s...
International Journal of Paediatric Dentistry, 2006
ABSTRACT Objectives: To compare the effect of brushing versus dipping using three different conce... more ABSTRACT Objectives: To compare the effect of brushing versus dipping using three different concentrations of fluoridated toothpastes on enamel erosion in vitro using surface profilometry.Methods: In a randomised, blinded experiment six groups of seven enamel slabs each were cut and mounted into resin blocks, ground and checked for surface flatness using a scanning profilometer (Scantron Proscan 2000). Each slabs’ surface was covered with nail varnish except for a small window (1 x 2 mm). Each group was immersed under static conditions for 2 minutes, five times daily in fresh 200 ml aliquots of citric acid 0.3% (pH = 3.6). In addition, three groups were immersed in three different fluoridated toothpastes (0 ppm F, 1100 ppm F, or 1450 ppm F) twice daily morning and evening for 2 minutes each time. The other three groups were brushed using the same toothpastes twice daily for 2 minutes each time. The total cycling period lasted 16 days during which the slabs were incubated overnight and between erosive challenges in artificial saliva at 37oC. A 60-minute gap was left between daytime immersions. Before and after dipping in the erosive solutions the slabs were rinsed with de-ionised water. After the cycling period, the slabs were analysed with the scanning profilometer to measure the amount of surface loss at day 4, 8, 12, and 16.Results: Surface loss ± SD of enamel at day 16 caused by citric acid combined with dipping using non-fluoridated toothpaste was 61.19 ± 8.50 μm, 1100 ppm F was 43.44 ± 10.94 μm or 1450 ppm F was 34.98 ± 4.29 μm. Surface Loss ± SD of enamel at day 16 caused by citric acid combined with brushing using 0 ppm F, 1100 ppm F, or 1450 ppm F toothpastes was 75.62 ±10.64, 63.51 ± 5.27 and 48.94 ± 13.67 μm, respectively.Conclusion: Enamel erosion was increased significantly (CI 95%) using brushing with toothpastes compared to dipping. In addition, enamel erosion showed a dose-response to fluoridated toothpastes.Acknowledgment: This project was supported by GlaxoSmithKline.
The International Journal of Developmental Biology
The central problems of enamel biochemistry are the mechanisms concerned with initiation and deve... more The central problems of enamel biochemistry are the mechanisms concerned with initiation and development of the mineral crystals, together with their architectural arrangement within the tissue. These processes are mediated by the extracellular matrix as well as the composition of the mineral itself. Initial mineral deposition occurs at the dentine surface, nucleated either by dentinal components or early enamel matrix, possibly non-amelogenin molecules. The early crystals are small in size and rich in magnesium and carbonate resulting in relatively poor crystallinity. This is in spite of the fact that fluoride is high at this stage. Crystal development includes a reduction in magnesium, carbonate and fluoride as crystals increase in length following the retreating ameloblasts from the dentine. The matrix acquires increasing concentrations of amelogenin and albumin. Prismatic structure begins to develop together with some growth of crystals in width and thickness. Degradation of ame...
Enamel maturation is characterized by massive crystal growth in both width and thickness, resulti... more Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.
Objective: Molecular events defining enamel matrix removal during amelogenesis are poorly underst... more Objective: Molecular events defining enamel matrix removal during amelogenesis are poorly understood. In early reports coated vesicles were shown on the cytoplasmic surface of the apical pole of ameloblasts, suggesting that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Method: and Result: Here, using real time PCR, we show that clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most highly up-regulated of the four distinct adaptor protein complexes. Immunolocalizations confirm the presence of AP-2 and clathrin in ameloblasts, where the highest expression is noted at the apical pole. AP-2 and clathrin are also present in other enamel organ cells. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying ...
There are several mouse models for human Amelogenesis imperfecta generated by gene knockout or in... more There are several mouse models for human Amelogenesis imperfecta generated by gene knockout or induced mutation of the enamel matrix genes e.g. Masuya et al, Hum Mol Genet 14:275, 2005. However, there is little baseline data on the mouse dentition and it has largely been assumed that mouse incisor development closely follows the extremely consistent pattern seen in rats. Objectives: To correlate mineral density, microhardness, and composition with enamel architecture in the mouse incisor and to compare this to the situation in rats. Methods: Wild-type mouse lower incisors were either 1) fixed, demineralised and examined histologically, 2) embedded in methylmethacrylate, sectioned transverse to their long axis and analysed for mineral density by TMR and microhardness by microindentation, viewed by SEM and composition determined by EDS, 3) whole teeth were analysed by nanoCT. Results: Histology revealed that unlike the rat, mouse incisors retained residual matrix proteins throughout a...
Amelogenin is the principle extracellular matrix protein in developing enamel. Amelx mutations ca... more Amelogenin is the principle extracellular matrix protein in developing enamel. Amelx mutations can have severe impact on dental phenotype but the precise role of amelogenin remains unclear. Understanding the effects of specific amelogenin mutations on enamel phenotype will help clarify this role. Objectives: To identify any Amelx mutation and characterise enamel phenotype in the mouse mutant M100888. Methods: Direct sequencing of the Amelx gene was carried out. Phenotype analysis of mandibular incisors of affected and wild type (WT) animals included quantitative microradiography, immunohistology and SDS PAGE/ amelogenin western blotting of the developing enamel matrix following microdissection. Results: A point mutation in the amelogenin N-acetylglucosamine binding domain was identified (Tyr to His). Affected males had significant areas of erupted enamel missing while remaining enamel exhibited normal mineral content. Histology of the developing teeth showed disordered ameloblasts a...
Mutations in Enam result in autosomal dominant hypoplastic Amelogenesis imperfect (AI). However, ... more Mutations in Enam result in autosomal dominant hypoplastic Amelogenesis imperfect (AI). However, the resulting enamel phenotype, whether a generalised or local hypoplasia, appears to depend upon the nature of the specific mutation. There are a number of murine models for human AI and several ENU-induced mutations in Enam have been shown to result in hypoplastic AI in the mouse (Masuya et al, Hum Mol Genet 14:275, 2005). Objectives: To compare incisor enamel thickness and mineral density in mice with each of two distinct ENU-induced Enam mutations: a G to T substitution at position 6489 resulting in an amino acid change at residue 55 of enamelin protein (ENAMRgsc395) and a T to A substitution at the splicing donor site in intron 4, resulting in a frameshift giving rise to a premature stop codon (ENAMRgsc521). Methods: Lower incisors from wild-type (WT) mice and those heterozygous (+/-) and homozygous (-/-) for the mutant alleles were embedded in methylmethacrylate and sectioned trans...
Amelogenin is essential for amelogenesis and amelogenin mutations lead to X-linked amelogenesis i... more Amelogenin is essential for amelogenesis and amelogenin mutations lead to X-linked amelogenesis imperfecta (AI) in humans. Previously we showed that the Y64H amelogenin mutation causes AI by a mechanism involving increased ameloblast apoptosis when compared to wild type mice(Barron et al (2010), Hum. Mol. Genet. doi:10.1093/hmg/ddq001). Objective: To investigate the role of cellular stress in the molecular pathogenesis of AI in mice expressing the Y64H amelogenin mutation. Methods: The mandibular incisor teeth of mice expressing the Y64H amelogenin mutation were investigated by immunohistochemistry, electron microscopy and in situ hybridisation. Results: Immunohistochemistry of affected secretory ameloblasts revealed the accumulation of vesicles containing both amelogenin and ameloblastin. Vesicles were positive for the endoplasmic reticulum (ER) markers calnexin and protein disulphide isomerase but also contained the Golgi markers GM130 and COPI. Ultrastructurally, the vesicles app...
A number of murine models have appeared for human AD hypoplastic AI either by knockout or by indu... more A number of murine models have appeared for human AD hypoplastic AI either by knockout or by induced mutation in the enamelin gene. One such model involves a G to T substitution at position 6489 resulting in an amino acid change at position 55 of the translated protein (Masuya et al, Hum Mol Genet 14:275, 2005). Objectives: To correlate mineral density, microhardness, and composition with enamel architecture in mice carrying this mutation, designated ENAMRgsc395. This will then allow direct comparison between this data and that obtained for human teeth for which this is a potential model. Methods: Lower incisors from wild-type mice and those heterozygous for the mutant allele were embedded in methylmethacrylate and sectioned transverse to their long axis with a peripheral diamond disc. Sections from mature enamel were polished plano-parallel to a thickness of 100 m, etched for 15 sec with 35 % phosphoric acid and thoroughly washed. Mineral density across the full enamel thickness wa...
The 32kDa post-secretory breakdown product of enamelin (a minor component of the developing ename... more The 32kDa post-secretory breakdown product of enamelin (a minor component of the developing enamel matrix) has been predicted to have an important function in enamel biomineralisation. This view is supported by the fact that mutations in the ENAM gene are associated with mineralisation defects, resulting in amelogenesis imperfecta. Objectives: To determine the mineral nucleating potential of a recombinant 32 kDa enamelin (rEnamelin) in vitro. Methods: rEnamelin was expressed in an eukaryotic expression system and purified for use in the steady state model described by Hunter and Goldburg. rEnamelin (1 ug/mL 10 ug/mL) was added to agarose gels and incubated under steady state conditions for 7d at 37C. Agarose alone and poly-glutamate (PGA, 10 g/mL) served as negative and positive controls respectively. Mineral deposition was determined by measurement of phosphate in the gels at the end of each run. Mineral deposits were characterised using SEM. Results: rEnamelin nucleated mineral at...
Objectives: Understanding the formation of enamel is of particular interest in the study of biomi... more Objectives: Understanding the formation of enamel is of particular interest in the study of biomineralisation processes and the development of biomaterials. Amelogenin is the most abundant component of the extracellular matrix associated with enamel formation. To distinguish the role of full-length amelogenin (P173) and its processing product lacking the hydrophilic C-terminal (P148), we have studied amelogenin adsorption onto a model crystal surface. In both the outer and inner regions of developing enamel the interface between protein matrix and crystal surface is the critical juncture, therefore we have used the novel technique of neutron reflectometry (NR) to probe this interface directly. Methods: Adsorption of nascent full-length P173 amelogenin protein and its 148 residue processing product onto silica substrates was studied using NR. For each experiment, the bare substrate was first characterised, then amelogenin P173 and P148 were introduced at a concentration of 0.1 mg/ml ...
Amelogenins are self-assembling proteins comprising 90% of the developing enamel matrix. AMELX ge... more Amelogenins are self-assembling proteins comprising 90% of the developing enamel matrix. AMELX gene mutations result in X-linked amelogenesis imperfecta (AI). We recently reported an amelogenin tyrosine to histidine mutation (Y64H) in mice treated with N-ethyl-N-nitrosurea (M100888, RIKEN, Japan) and displaying an AI phenotype. Western blotting of the developing affected enamel indicated an absence of full length amelogenin from the tissue. Objectives: To express recombinant wild type (WT) and Y64H amelogenins and compare their self assembly properties and degradation by enamel proteases in vitro. Methods: Amelogenin genes were inserted into a pET 28 vector modified with the addition of a human retrovirus 3C protease site for expression in E. coli. Recombinant proteins were purified using affinity chromatography and preparative electrophoresis and characterised by SDS-PAGE, Western blotting and mass spectroscopy. Functional analysis included in vitro proteolytic degradation with ena...
Objective: Amelogenesis Imperfecta (AI) is a collective term for a heterogeneous group of enamel ... more Objective: Amelogenesis Imperfecta (AI) is a collective term for a heterogeneous group of enamel biomineralisation abnormalities. Our research group aims to identify AI-causing gene mutations that give insight into the network of genes critical to human enamel formation. This study aims to describe the genetic cause of generalised hypoplastic AI observed in three siblings from a second cousin consanguineous family. Method: Family members were assessed through clinical examination and supporting investigations. DNA and other samples were collected following informed consent and where appropriate ethics committee approval. SNP chip genotyping identified a locus consistent with inheritance identical by descent that differed from those in unaffected members. DNA from one affected individual was whole exome sequenced on an Illumina HiSeq 2500 sequencer. The sequence data was aligned to human reference genome (GRCh37) and analysed for potential relevant mutations within the homozygous int...
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research
Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early ... more Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during matur...
Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase ... more Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography-electrospray ionization-tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.
Objective: Therapeutic effects of enamel matrix derivatives have been extensively documented. But... more Objective: Therapeutic effects of enamel matrix derivatives have been extensively documented. But, the physiological expression and functions of enamel matrix peptides (EMPs) are a matter of debate. The present study explores endogenous amelogenin (AMELX) and ameloblastin (AMBN)mRNA/proteins in bones. Method: Samples. of mandibular alveolar (AB) and basal (BB) bone of growing (7 PN days) and adult (8 weeks) mice were microdissected and RNAs and proteins, extracted and analyzed. Laser-captured tissue extracts were also studied at 3 PN days. Results: RT-qPCR and Western-blotting evidenced AMELX and AMBN expression in AB and BB (about 10x less relatively to incisor enamel organ at 7PN days). These levels decreased with age and varied according to the anatomical sites (AB ≥BB). In situ studies were monitored using (1) enamel and ameloblasts as positive controls and (2) RT-qPCR/Western-blot data on their relative levels and (3) a set of negative controls (notably AMEL-KO mice –NIDCR, NIH...
ABSTRACT Enamel maturation is characterized by massive crystal growth in both width and thickness... more ABSTRACT Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.
Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomi... more Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In s...
International Journal of Paediatric Dentistry, 2006
ABSTRACT Objectives: To compare the effect of brushing versus dipping using three different conce... more ABSTRACT Objectives: To compare the effect of brushing versus dipping using three different concentrations of fluoridated toothpastes on enamel erosion in vitro using surface profilometry.Methods: In a randomised, blinded experiment six groups of seven enamel slabs each were cut and mounted into resin blocks, ground and checked for surface flatness using a scanning profilometer (Scantron Proscan 2000). Each slabs’ surface was covered with nail varnish except for a small window (1 x 2 mm). Each group was immersed under static conditions for 2 minutes, five times daily in fresh 200 ml aliquots of citric acid 0.3% (pH = 3.6). In addition, three groups were immersed in three different fluoridated toothpastes (0 ppm F, 1100 ppm F, or 1450 ppm F) twice daily morning and evening for 2 minutes each time. The other three groups were brushed using the same toothpastes twice daily for 2 minutes each time. The total cycling period lasted 16 days during which the slabs were incubated overnight and between erosive challenges in artificial saliva at 37oC. A 60-minute gap was left between daytime immersions. Before and after dipping in the erosive solutions the slabs were rinsed with de-ionised water. After the cycling period, the slabs were analysed with the scanning profilometer to measure the amount of surface loss at day 4, 8, 12, and 16.Results: Surface loss ± SD of enamel at day 16 caused by citric acid combined with dipping using non-fluoridated toothpaste was 61.19 ± 8.50 μm, 1100 ppm F was 43.44 ± 10.94 μm or 1450 ppm F was 34.98 ± 4.29 μm. Surface Loss ± SD of enamel at day 16 caused by citric acid combined with brushing using 0 ppm F, 1100 ppm F, or 1450 ppm F toothpastes was 75.62 ±10.64, 63.51 ± 5.27 and 48.94 ± 13.67 μm, respectively.Conclusion: Enamel erosion was increased significantly (CI 95%) using brushing with toothpastes compared to dipping. In addition, enamel erosion showed a dose-response to fluoridated toothpastes.Acknowledgment: This project was supported by GlaxoSmithKline.
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Papers by Steven Brookes