OBJECTIVES: To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) METHODS: The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified RESULTS: When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor. CONCLUSIONS The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals
Amelogenin ; Dental Enamel Proteins ; Durapatite ; Humans

OBJECTIVERecombinant human leucine-rich amelogenin peptide (LRAP) was studied by cryogenic transmission electron microscopy (TEM); evaluation focused on its self-assembly and crystal growth in vitro.

METHODSHuman LRAP was recombined through prokaryotic expression vector pCold-SUMO and transformed into Escherichia coli BL21plys to acquire purified proteins. Cryogen TEM recorded assembly and self-assembling of LRAP from pH 3.5 to pH 8.0, and the hydroxyapatite crystal growth in the mixture of LRAP protein solution and artificial saliva was observed using TEM and selected area electron diffraction.

RESULTSMore than 90% purity LRAP was expressed, purified and identified as described in methods. LRAP linked into oligomers, nanospheres, nanochains, and microribbons, whereas pH value increased from 3.5 to 8.0. Mature hydroxyapatite crystal growth was guided in artificial saliva filled with calcium phosphate.

CONCLUSIONSLRAP is simplified amelogenin functional domain and conserved the basic characters of amelogenin such as self-assembling and inducing crystallization along c axis. In the area of acellular synthesis of hydroxyapatite using extracellular enamel matrix protein, LRAP is one of candidate repair materials for irregular hard tissue defection.
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Amelogenin ; Bone Density ; Calcium Phosphates ; Crystallization ; Dental Enamel ; Dental Enamel Proteins ; Durapatite ; Humans ; Microscopy, Electron, Transmission

Tooth enamel is a complex mineralized tissue consisting of long and parallel apatite crystals configured into decussating enamel rods. In recent years, multiple approaches have been introduced to generate or regenerate this highly attractive biomaterial characterized by great mechanical strength paired with relative resilience and tissue compatibility. In the present review, we discuss five pathways toward enamel tissue engineering, (i) enamel synthesis using physico-chemical means, (ii) protein matrix-guided enamel crystal growth, (iii) enamel surface remineralization, (iv) cell-based enamel engineering, and (v) biological enamel regeneration based on de novo induction of tooth morphogenesis. So far, physical synthesis approaches using extreme environmental conditions such as pH, heat and pressure have resulted in the formation of enamel-like crystal assemblies. Biochemical methods relying on enamel proteins as templating matrices have aided the growth of elongated calcium phosphate crystals. To illustrate the validity of this biochemical approach we have successfully grown enamel-like apatite crystals organized into decussating enamel rods using an organic enamel protein matrix. Other studies reviewed here have employed amelogenin-derived peptides or self-assembling dendrimers to re-mineralize mineral-depleted white lesions on tooth surfaces. So far, cell-based enamel tissue engineering has been hampered by the limitations of presently existing ameloblast cell lines. Going forward, these limitations may be overcome by new cell culture technologies. Finally, whole-tooth regeneration through reactivation of the signaling pathways triggered during natural enamel development represents a biological avenue toward faithful enamel regeneration. In the present review we have summarized the state of the art in enamel tissue engineering and provided novel insights into future opportunities to regenerate this arguably most fascinating of all dental tissues.
Acid Etching, Dental ; Amelogenin ; Biomimetics ; trends ; Dental Enamel ; metabolism ; Dental Enamel Proteins ; Dentistry ; trends ; Tissue Engineering ; methods ; Tooth Remineralization

OBJECTIVETo prepare the polyclonal antibody to amelogenin.

METHODSThe fetal porcine dental enamel was collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7.4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund's adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test.

RESULTSThe Gel filtration showed that amelogenin components is at molecular weights of 15 kD and 13 kD apparently, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1:1000.

CONCLUSIONThe antibody prepared in this study can be used for the detection of amelogenin.

Amelogenin ; Animals ; Animals, Newborn ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Dental Enamel ; chemistry ; Dental Enamel Proteins ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; immunology ; Immunoglobulin G ; analysis ; biosynthesis ; Rabbits ; Swine ; Tooth Germ ; chemistry

OBJECTIVETo invesitgate the expression patterns of amelogenin and enamelin in the developing tooth germs.

METHODSMandible sections of postnatal day 1, 3, 7 and 14 mouse were prepared, immunohistochemical analysis and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect the expression patterns of amelogenin and enamelin in mandibular first molars.

RESULTSAmelogenin was observed in the cytoplasm of secretory ameloblasts and the whole enamel matrix layer. It was also transiently expressed in the odontoblasts of postnatal day 1 molars. Enamelin proteins were observed in the enamel layer deposited by secretory ameloblasts, especially intense beneath the ameloblast process and dentino-enamel junction. The mRNA levels of both amelogenin and enamelin were highest on postnatal day 7 (the ratio to glyceraldehyde phosphate dehydrogenase of amelogenin and enamelin: 0.813 ± 0.085 and 0.799 ± 0.064, respectively, P < 0.05).

CONCLUSIONSAmelogenin and enamelin were enamel matrix proteins predominately expressed by secretory ameloblasts. The temporal-spatial expression patterns of amelogenin and enamelin indicate the important roles they played in amelogenesis and biomineralization.

Ameloblasts ; metabolism ; Amelogenesis ; Amelogenin ; genetics ; metabolism ; Animals ; Dental Enamel ; metabolism ; Dental Enamel Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Molar ; metabolism ; Odontoblasts ; metabolism ; RNA, Messenger ; metabolism ; Time Factors ; Tooth Germ ; growth & development ; metabolism

OBJECTIVEUsing nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis.

METHODSOne (or two) lymphocyte and blastomere (n=50/group) were collected and prepared under the following conditions: (1) water only (H(2)O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65 degree centigrade, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene.

RESULTSThe amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method.

CONCLUSIONThe KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.

Amelogenin ; Blastocyst ; cytology ; metabolism ; Blastomeres ; cytology ; metabolism ; DNA ; genetics ; Dental Enamel Proteins ; genetics ; Female ; Genotype ; Humans ; Lymphocytes ; cytology ; metabolism ; Male ; Polymerase Chain Reaction ; methods ; Sex Determination Analysis ; methods

OBJECTIVEThe purpose of this study was to construct a eukaryotic expression vector for human amelogenin (AMG).

METHODSPCR was performed to amplify the AMG encoding region. Amplified fragments for human AMG were recovered and inserted into eukaryotic expression vectors PsecTaq2A. The recombinant plasmid PsecTaq2A-AMG was constructed and their positive clones were identified.

RESULTS1. Amplified products were checked by electrophoresis and the results were satisfactory. 2. The recombinant plasmid PsecTaq2A-AMG was analyzed by restriction endonuclease mapping and DNA sequencing. The results of sequencing were consistent with those from GenBank.

CONCLUSIONThe recombinant plasmid PsecTaq2A-AMG was successfully constructed with properly inserted DNA sequence encoding mature amelogenin.

Amelogenin ; Clone Cells ; metabolism ; Cloning, Molecular ; DNA, Recombinant ; biosynthesis ; genetics ; Dental Enamel Proteins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Nine short tandem repeat (STR) markers (D3S1358, VWA, FGA, THO1, TPOX, CSFIPO, D5S818, D13S317, and D7S820) and a sex-identification marker (Amelogenin locus) were amplified with multiplex PCR and were genotyped with a four-color fluorescence method in samples from 174 unrelated Han individuals in North China. The allele frequencies, genotype frequencies, heterozygosity, probability of discrimination powers, probability of paternity exclusion and Hardy-Weinberg equilibrium expectations were determined. The results demonstrated that the genotypes at all these STR loci in Han population conform to Hardy-Weinberg equilibrium expectations. The combined discrimination power (DP) was 1.05 x 10(-10) within nine STR loci analyzed and the probability of paternity exclusion (EPP) was 0.9998. The results indicate that these nine STR loci and the Amelogenin locus are useful markers for human identification, paternity and maternity testing and sex determination in forensic sciences.
Amelogenin ; China ; Dental Enamel Proteins ; genetics ; Electrophoresis ; Ethnic Groups ; genetics ; Forensic Medicine ; methods ; Gene Frequency ; Genetics, Population ; Genotype ; Heterozygote ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Sex Determination Analysis ; methods ; Tandem Repeat Sequences ; genetics

OBJECTIVETo observe the effect of overdose fluoride on the expression of enamelin in rat mandibular incisor.

METHODSTwenty Wistar rats were divided randomly into two groups. Animals were maintained in standard environment with free access to food and distilled water (control group) or water added with 100 mg/L F-(experimental group). The rats were killed in the eighth week. HE staining was used to observe the morphology of ameloblasts. Immunohistochemical staining was adopted to study the expressions of enamelin in rat incisor.

RESULTSThe ameloblasts of the treated rat were arranged in multi-layer. The ameloblasts in group II were thinner than those in group I. The structure of enamel matrix was in disorder. The expressions of enamelin in ameloblasts and odontoblasts were obviously inhibited in group II (P < 0.01).

CONCLUSIONThe overdose fluoride inhibits the secretion of enamelin and leads to the abnormal development of enamel matrix.

Ameloblasts ; Animals ; Dental Enamel ; Dental Enamel Proteins ; Fluorides ; Incisor ; Mandible ; Odontoblasts ; Phosphates ; Rats ; Rats, Wistar

OBJECTIVETo localize the gene (s) responsible for autosomal dominant hypocalcified amelogenesis imperfecta in a Chinese family.

METHODSA Chinese family which was diagnosed as autosomal dominant hypocalcified amelogenesis imperfecta (AD) was studied. Venous blood from nineteen family members was collected and genomic DNA was extracted from the blood. Eight short tandem repeats (STRs) spanning five hereditary AI candidate genes were selected and linkage analysis between the genetic markers and the disease loci was performed.

RESULTSGenotype of the eight STRs were acquired, the linkage analysis result can not support that the gene for AI pedigrees was linked to ENAM, AMBN, TUF1, KLK4 or MMP-20.

CONCLUSIONThe results can not support all proposed candidate gene regions as causal for autosomal dominant hypocalcified AI in this family. These linkage findings provide further evidence for genetic heterogeneity among families with autosomal dominant AI and indicate that, at least, some forms of autosomal dominant AI are not caused by a gene in the five most commonly reported AI candidate genes.

Amelogenesis Imperfecta ; Dental Enamel Proteins ; Genotype ; Humans ; Pedigree