Alleles ; Amelogenin/genetics* ; Chromosomes, Human, Y ; Humans ; Male ; Sex Determination Analysis

OBJECTIVE: To compare a new thermosensitive recombinant human amelogenin (rhAm) carrier and traditional propylene glycol alginate (PGA) carrier for their characteristics, antibacterial activity, and biocompatibility with human periodontal membrane fibroblasts. METHODS: PGA-rhAm was prepared by mixing 3.3% PGA and rhAm, and CS-βGP-rhAm was prepared by mixing 2% chitosan (CS) with rhAm and then with 60% β-sodium glycerophosphate solution (βGP) as the crosslinking agent. The biophysical properties of the prepared carriers were characterized, and their antibacterial activity was assessed by observing Staphylococcus aureus growth. The biocompatibility of the carriers was evaluated in human periodontal membrane fibroblasts (hPDLFs) using CCK8 assay and scratch test, and mRNA and protein expressions of osteogenic genes of the cells incubated with the carriers were detected using RT-qPCR and Western blotting; osteogenic differentiation of the cells was detected using alkaline phosphatase staining. RESULTS: PGA-rhAm had a viscosity value of 3.262±0.055 Pa.s. CS-βGP-rhAm had a solidification capacity of 6 min at 37 ℃ with a pH value close to that of the oral cavity and a swelling rate of about 90%. CS-β GP-rhAm maintained sustained release of rhAm for over 2 weeks with a self-degradation time over 3 weeks. CS-βGPrhAm more effectively inhibited the growth of S. aureus than rhAm-loaded PGA. While PGA did not obviously affect the proliferation of hPDLFs, both CS-βGP and CS-βGP-rhAm significantly promoted the cell proliferation(P < 0.001). Scratch test showed that after rhAm loading, both CS-βGP and PGA promoted cell migration (P < 0.01). CS-βGP-rhAm significantly enhanced the mRNA expressions of RUNX2 and OCN mRNA level and the protein expressions of Ki67, RUNX2, collagen I, and β-catenin (P < 0.05); PGA-rhAm only enhanced RUNX2 (P < 0.05) and OCN (P < 0.01) mRNA expressions without significant effects on the protein expressions. Alkaline phosphatase staining results showed that CS-βGP, but not PGA, promoted osteogenic differentiation of hPDLFs. CONCLUSION CS-βGP carrier is capable of sustained release of rhAm, inhibiting the growth of S. aureus, and improving the biological activity of hPDLFs without affecting the bioactivity of rhAm after drug loading.
Alginates ; Alkaline Phosphatase ; Amelogenin ; Anti-Bacterial Agents/pharmacology* ; Cell Differentiation ; Cells, Cultured ; Chitosan/pharmacology* ; Collagen ; Core Binding Factor Alpha 1 Subunit ; Delayed-Action Preparations ; Glycerophosphates ; Humans ; Ki-67 Antigen ; Osteogenesis ; Periodontal Ligament ; RNA, Messenger ; Staphylococcus aureus ; beta Catenin

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

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

BACKGROUND AND OBJECTIVES: Recombinant amelogenin protein (RAP) was reported to induce soft-tissue regeneration in canine infected endodontically treated permanent teeth with open apices. To characterize identities of the cells found in the RAP regenerated tissues compared to authentic pulp by identifying: 1) stem cells by their expression of Sox2; 2) nerve fibers by distribution of the axonal marker peripherin; 3) axons by their expression of calcitonin gene–related peptide (CGRP); 4) the presence of astrocytes expressing glial fibrillary acidic proteins (GFAP).METHODS: A total of 240 open-apex root canals in dogs were used. After establishment of oral contamination to the pulp, the canals were cleaned, irrigated, and 120 canals filled with RAP, and the other 120 with calcium hydroxide.RESULTS: After 1, 3, and 6 months, teeth were recovered for immune-detection of protein markers associated with native pulp tissues. Regenerated pulp and apical papilla of RAP group revealed an abundance of stem cells showing intense immunoreactivity to Sox2 antibody, immunoreactivity of peripherin mainly in the A-fibers of the odontoblast layer and immunoreactivity to CGRP fibers in the central pulp region indicative of C-fibres. GFAP immunoreactivity was observed near the odontoblastic, cell-rich regions and throughout the regenerated pulp.CONCLUSIONS: RAP induces pulp regeneration following regenerative endodontic procedures with cells identity by gene expression demonstrating a distribution pattern similar to the authentic pulp innervation. A- and C-fibers, as well as GFAP specific to astrocytic differentiation, are recognized. The origin of the regenerated neural networks may be derived from the Sox2 identified stem cells within the apical papilla.
Amelogenin ; Animals ; Astrocytes ; Axons ; Calcitonin ; Calcitonin Gene-Related Peptide ; Calcium Hydroxide ; Dental Pulp Cavity ; Dental Pulp Necrosis ; Dogs ; Gene Expression ; Glial Fibrillary Acidic Protein ; Nerve Fibers ; Odontoblasts ; Periapical Periodontitis ; Regeneration ; Stem Cells ; Tooth

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.
Alkaline Phosphatase ; metabolism ; Amelogenin ; physiology ; Animals ; Biomarkers ; metabolism ; Calcification, Physiologic ; Cell Adhesion Molecules ; metabolism ; Cell Proliferation ; Cementogenesis ; physiology ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Gene Expression Regulation ; In Vitro Techniques ; Integrin-Binding Sialoprotein ; metabolism ; Mice ; Microscopy, Confocal ; Osteocalcin ; metabolism ; Osteopontin ; metabolism ; Real-Time Polymerase Chain Reaction

The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary 2(nd) molar germs of rats. We used the maxillary 2(nd) molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary 2(nd) molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.
Ameloblasts ; Amelogenesis ; Amelogenin ; Animals ; Blotting, Western ; Dental Enamel ; Fluorescent Antibody Technique ; Molar ; Polymerase Chain Reaction ; Rats ; Reverse Transcription ; RNA, Messenger

OBJECTIVES: To establish multiplex system of 16 miniSTR loci, and explore its application value for the degraded materials in forensic medicine. METHODS: The multiplex system of 16 miniSTR loci was established using a six-dye fluorescence labeling technology and its application value in forensic medicine was assessed. RESULTS: A six-dye fluorescence labeling miniSTR amplification kit was developed, which enabled 15 autosomal STR loci, Amelogenin locus and DYS391 to be typed simultaneously. This method showed good specificity and could provide stable and accurate typing results with a sensitivity of 50 pg. This system also provided a good test result for the normal biological sample of actual cases. CONCLUSIONS The multiplex system of 16 miniSTR loci has application value for degraded and trace materials with the advantages of high sensitivity and database compatibility, which can be used for forensic casework.
Amelogenin ; DNA Fingerprinting ; DNA Primers ; Forensic Medicine/methods* ; Microsatellite Repeats/genetics* ; Polymerase Chain Reaction

OBJECTIVES: To observe and analyse the Amelogenin allelic loss in parent-child identification cases, and to explore the type and mechanism of Amelogenin allelic loss as well as its influence on gender identification and solutions. METHODS: After the detection by SiFaSTR™ 23plex DNA identification system, samples had the characteristics of the peak area of Amelogenin X was the same as the one of adjacent heterozygote or lower than one half of adjacent homozygote in females while Amelogenin X loss was observed in males were selected. X chromosome STR （X-STR） typing and Amelogenin X sequencing were performed. The samples with Amelogenin Y loss in males were confirmed by the detection of Y chromosome STR typing and sex-determining region of Y （SRY）. The type and rate of Amelogenin allelic loss were confirmed and calculated, and the mechanism and influence of this variation were also analysed. RESULTS: Amelogenin X allelic loss was observed in one male sample, the mutation in primer-binding region was confirmed by sequencing. The suspected Amelogenin X allelic loss was observed in four female samples, but the mutation in primer-binding region was confirmed by sequencing in only one sample. Amelogenin Y allelic loss was observed in seven male samples, SRY positive cases was detected in five of them, and two were SRY negative. Y-STR type was detected in four cases of the five SRY positive cases, which was not detected in the two SRY negative cases. The rate of Amelogenin allelic loss was about 0.029%. CONCLUSIONS Amelogenin X allelic loss does not affect the gender identification, but Amelogenin Y allelic loss may cause wrong gender identification. Thus, Y-STR or SRY should be detected for gender confirmation. When Y-STR genotypes are not detected in a "male" whose SRY detection is also negative, then the chromosome karyotype analysis and sex differentiation related genes test should be taken to further confirm the gender.
Amelogenin/genetics* ; DNA/genetics* ; Female ; Humans ; Loss of Heterozygosity/genetics* ; Male ; Sex Determination Analysis

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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