Ameloblastin Research Articles

Co-exposure to fluoride and sulfur dioxide induces abnormal enamel mineralization in rats via the FGF9-mediated MAPK signaling pathway

Fluoride (F) and sulfur dioxide (SO2) contamination is recognized as a public health concern worldwide. Our previous research has shown that Co-exposure to F and SO2 can cause abnormal enamel mineralization. Ameloblastin (AMBN) plays a crucial role in the process of enamel mineralization. However, the process by which simultaneous exposure to F and SO2 influences enamel formation by regulating AMBN expression still needs to be understood. This study aimed to establish in vivo and in vitro models of F–SO2 Co-exposure and investigate the relationship between AMBN and abnormal enamel mineralization. By overexpressing/knocking out the Fibroblast Growth Factor 9 (FGF9) gene, we investigated the impact of FGF9-mediated Mitogen-Activated Protein Kinase (MAPK) signaling on AMBN synthesis to elucidate the mechanism underlying the induction of abnormal enamel mineralization by F–SO2 Co-exposure in rats. The results showed that F–SO2 exposure damaged the structure of rat enamel and ameloblasts. When exposed to F or SO2, gradual increases in the protein expression of FGF9 and phosphorylated p38 mitogen-activated protein kinase (p-P38) were observed. Conversely, the protein levels of AMBN, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) were decreased. AMBN expression was significantly correlated with FGF9, p-ERK, and p-JNK expression in ameloblasts. Interestingly, FGF9 overexpression reduced the levels of p-ERK and p-JNK, worsening the inhibitory effect of F–SO2 on AMBN. Conversely, FGF9 knockout increased the phosphorylation of ERK and JNK, partially reversing the F–SO2-induced downregulation of AMBN. Taken together, these findings strongly demonstrate that FGF9 plays a critical role in F–SO2-induced abnormal enamel mineralization by regulating AMBN synthesis through the JNK and ERK pathways.

Proteolytic profiles of two isoforms of human AMBN expressed in E. coli by MMP-20 and KLK-4 proteases

Ameloblastin is a protein in biomineralization of tooth enamel. However recent results indicate that this is probably not its only role in an organism. Enamel matrix formation represents a complex process enabled via specific crosslinking of two proteins – the most abundant amelogenin and the ameloblastin (AMBN). The human AMBN (hAMBN) gene possesses 13 protein coding exons with alternatively spliced transcripts and the longest isoform about 447 amino acid residues. It has been described that AMBN molecules in vitro assemble into oligomers via a sequence encoded by exon 5. Enamel is formed by the processing of enamel proteins by two specific proteases - enamelysin (MMP-20) and kallikrein 4 (KLK-4). The scaffold made of AMEL and non-amelogenin proteins is cleaved and removed from the developed tooth enamel. The hAMBN is expressed in two isoforms (ISO I and II), which could lead to their different utilization determined by distinct proteolytic profiles. In this study, we compared proteolytic profiles of both isoforms of hAMBN expressed in E. coli after proteolysis by MMP-20, KLK-4, and their 1:2 mixture. Proteolysis products were analysed and cleavage sites were identified by mass spectrometry. The proteolytic profiles of two AMBN isoforms showed different results, although we have to determine that the analysed AMBN was not post-translationally modified as expressed in prokaryotic cells. These results may lead to the suggestion of potentially divergent roles of AMBN isoforms cleavage products in various cell signalling pathways such as calcium buffering or signalling cascades.

Novel Ameloblastin Variants, Contrasting Amelogenesis Imperfecta Phenotypes

Amelogenesis imperfecta (AI) comprises a group of rare, inherited disorders with abnormal enamel formation. Ameloblastin (AMBN), the second most abundant enamel matrix protein (EMP), plays a critical role in amelogenesis. Pathogenic biallelic loss-of-function AMBN variants are known to cause recessive hypoplastic AI. A report of a family with dominant hypoplastic AI attributed to AMBN missense change p.Pro357Ser, together with data from animal models, suggests that the consequences of AMBN variants in human AI remain incompletely characterized. Here we describe 5 new pathogenic AMBN variants in 11 individuals with AI. These fall within 3 groups by phenotype. Group 1, consisting of 6 families biallelic for combinations of 4 different variants, have yellow hypoplastic AI with poor-quality enamel, consistent with previous reports. Group 2, with 2 families, appears monoallelic for a variant shared with group 1 and has hypomaturation AI of near-normal enamel volume with pitting. Group 3 includes 3 families, all monoallelic for a fifth variant, which are affected by white hypoplastic AI with a thin intact enamel layer. Three variants, c.209C>G; p.(Ser70*) (groups 1 and 2), c.295T>C; p.(Tyr99His) (group 1), and c.76G>A; p.(Ala26Thr) (group 3) were identified in multiple families. Long-read AMBN locus sequencing revealed these variants are on the same conserved haplotype, implying they originate from a common ancestor. Data presented therefore provide further support for possible dominant as well as recessive inheritance for AMBN-related AI and for multiple contrasting phenotypes. In conclusion, our findings suggest pathogenic AMBN variants have a more complex impact on human AI than previously reported.

Revisiting ameloblastin; addressing the EMT-ECM axis above and beyond oral biology.

Ameloblastin (AMBN) is best characterized for its role in dental enamel formation, regulating cell differentiation and mineralization, and cell matrix adhesion. However, AMBN has also been detected in mesenchymal stem cells in addition to bone, blood, and adipose tissue. Using immunofluorescence in a pilot scheme, we identified that AMBN is expressed in different parts of the gastrointestinal (GI) tract. AMBN mRNA and protein detection in several tissues along the length of the GI tract suggests a role for AMBN in the structure and tissue integrity of the extracellular matrix (ECM). Intracellular AMBN expression in subsets of cells indicates a potential alternative role in signaling processes. Of note, our previous functional AMBN promoter analyses had shown that it contains epithelial-mesenchymal transition (EMT) regulatory elements. ΑΜΒΝ is herein presented as a paradigm shift of the possible associations and the spatiotemporal regulation of the ECM regulating the EMT and vice versa, using the example of AMBN expression beyond oral biology.

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

The influence of IGF-I and Emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model

Aim: Tooth decay and associated periodontal disease remain the most common chronic diseases in current society. In the future, individually tailored, more effective therapeutic treatment options could be provided through the application of tissue engineering and regenerative medicine and dentistry (TERMD) approaches. A relevant finding is the potential of both dental mesenchymal and dental epithelial cells to regenerate mineralized dentin and enamel tissues, respectively.Materials and Methods: In the current study, a multi-layered, bioengineered tooth bud model was assembled by combining human dental mesenchymal (hDM) and porcine dental epithelial cells (pDE). The hDM cells were seeded onto poly(epsilon-caprolactone)/ poly (lactide-co-glycolide) (PCL/PLGA) wet electro-spun scaffolds overlayed with confluent pDE cell sheet harvested from thermo-reversible tissue culture plates. The so-formed multi-layered bioengineered tooth bud was then used to study the mineralization potential of the dental cells in in vitro culture. It was hypothesized that the addition of the in media soluble factors Insulin-like growth factor 1 (IGF-I) and the extracellular matrix derivate Emdogain (EMD) would result in enhanced differentiation and mineralized dental tissue formation. Scanning Electron morphological observation was used to characterize scaffolds porosity. Histological and immunofluorescent analyses confirmed the localization of hDM cells inside the scaffold, an intact pDE cell sheet, and the presence of beta-integrin 1-positive cell-cell junctions connecting the two.Results: Scanning Electron Microscopy showed that EMD, in particular, enhanced the mineralization potential of pDE cells. qRT-PCR analyses showed that both EMD and IGF-1 significantly enhanced the expression of Ameloblastin (AMBN), reflecting pDE cell differentiation.Conclusion: In conclusion, these results proved the hypothesis that both EMD and IGF-1 should be considered for their utility in preclinical dental tissue engineering approaches.

Production of recombinant human ameloblastin by a fully native purification pathway

Ameloblastin (Ambn) is an intrinsically disordered protein (IDP) with a specific function of forming heterogenous homooligomers. The oligomeric function is led through a specific sequence encoded by exon 5 of Ambn. Due to the IDP character of Ambn to form oligomers, protein purification is subject to many challenges. Human ameloblastin (AMBN) and its two isoforms, I and II have already been purified as a recombinant protein in a bacterial expression system and functionally characterized in vitro. However, here we present a new purification protocol for the production of native AMBN in its original formation as a homooligomeric heterogeneous IDP. The purification process consists of three chromatographic steps utilizing His-tag and Twin Strep-tag affinity chromatography, along with size exclusion and reverse affinity chromatography. The presented workflow offers the production of AMBN in sufficient yield for in vitro protein characterizations and can be used to produce both AMBN isoforms I and II.

Comprehensive Analysis Identifies Ameloblastin-Related Competitive Endogenous RNA as a Prognostic Biomarker for Testicular Germ Cell Tumour.

Simple SummaryTesticular germ cell tumour is a common tumour in young males, and although it is one of the most curable cancers, many patients still experience recurrence after the chemotherapy. Tumour recurrence is not detected with high sensitivity by established blood tumour markers. Ameloblastin is identified as an extracellular matrix protein and has shown to be associated with tumour progression. We validated ameloblastin’s expression in testicular tissue, and used comprehensive bioinformatics analysis of 156 patients with testicular germ cell tumour to show that the level of ameloblastin was associated with the time of tumour recurrence after the first cure. In the analysis of ameloblastin differential genes in the tumour, a ceRNA (competing endogenous RNA) regulatory network associated with tumour diagnosis and an independent prognostic factor for the tumour, PELATON (Plaque Enriched LncRNA In Atherosclerotic And Inflammatory Bowel Macrophage Regulation), were identified, which could provide evidence for prediction of tumour prognosis.Testicular Germ Cell Tumour (TGCT) is one of the most common tumours in young men. Increasing evidence shows that the extracellular matrix has a key role in the prognosis and metastasis of various human cancers. This study analysed the relationship between the matrix protein ameloblastin (AMBN) and potential biological markers associated with TGCT diagnosis and prognosis. The relationship between AMBN and TGCT prognosis was determined by bioinformatic analysis using the expression profiles of three RNAs (long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs) from The Cancer Genome Atlas (TCGA) database, and available clinical information of the corresponding patients. Prediction and validation of competitive endogenous RNA (ceRNA) regulatory networks related to AMBN was performed. AMBN and its associated ceRNA regulatory network were found to be related to the recurrence of TGCT, and LINC02701 may be used as a diagnostic factor in TGCT. Furthermore, we identified PELATON (Plaque Enriched LncRNA In Atherosclerotic And Inflammatory Bowel Macrophage Regulation) as an independent prognostic factor for TGCT progression-free interval.

How prenatal environmental factors affect rat molar enamel formation?

Amelogenin (AMELX) and ameloblastin (AMBN) are crucial for enamel formation, and interruptions in the production of these proteins may cause enamel defects. We investigated how prenatal environmental factors (chronic stress, bisphenol A (BPA), amoxicillin, and lipopolysaccharide (LPS)) affect AMELX and AMBN production of ameloblasts. Fifteen pregnant Sprague-Dawley rats were divided into four experimental groups and a control group. Chronic-stress group rats were exposed to a 12:12 light/light cycle (LL) from day E18 until delivery. BPA group rats were orally administered 5μg/kg BPA daily from day E1 until delivery. Amoxicillin group rats were injected 100mg/kg amoxicillin daily from day E18 until delivery. LPS-infection group rats were injected 125μg/kg bacterial LPS once on day E18. Seven pups from the control group and ten pups from the experimental groups were euthanized on P10. Sections were stained with hematoxylin and eosin (H&E) and Gomori's one-step trichrome staining (GT) and incubated with rabbit polyclonal antibodies to AMELX and AMBN, to evaluate staining intensity at ameloblast stages. The surface morphology was evaluated with a stereomicroscope. AMELX (p = 0.008, p = 0.0001, p = 0.009) and AMBN (p = 0.002, p = 0.001, p = 0.0001) staining of all groups were significantly lower than that of the control group in the secretory, transitional, and maturation stages. Abnormal enamel matrix formation was observed in the H&E and GT staining sections of all experimental groups. Yellowish coloration of the amoxicillin group was observed in morphologic evaluation.

Ameloblastin promotes polarization of ameloblast cell lines in a 3-D cell culture system

Studies on animal models with mutations in ameloblastin gene have suggested that the extracellular matrix protein ameloblastin (AMBN) plays important roles in controlling cell-matrix adhesion and ameloblast polarization during amelogenesis. In order to examine the function of AMBN in cell polarization and morphology, we developed an in vitro 3D cell culture model to examine the effect of AMBN and amelogenin (AMEL) addition on ameloblast cell lines. We further used high resolution confocal microscopy to detect expression of polarization markers in response to AMBN addition. Addition of AMBN to the 3D culture matrix resulted in the clustering and elongation (higher aspect ratio) of ALC in a dose dependent manner. The molar concentration of AMEL required to exact this response from ALC was 2.75- times greater than that of AMBN. This polarization effect of ameloblastin was attributable directly to an evolutionary conserved domain within its exon 5-encoded region. The lack of exon 6-encoded region also influenced AMBN-cell interactions but to a lesser extent. The clusters formed with AMBN were polarized with expression of E-cadherin, Par3 and Cldn1 assembly at the nascent cell-cell junctions. The elongation effect was specific to epithelial cells of ameloblastic lineage ALC and LS8 cells. Our data suggest that AMBN may play critical signaling roles in the initiation of cell polarity by acting as a communicator between cell-cell and cell-matrix interactions. Our investigation has important implications for understanding the function of ameloblastin in enamel-cell matrix adhesion and the outcomes may contribute to efforts to develop strategies for enamel tissue regeneration.

A systematic review and meta-analysis of CA VI, AMBN, and TUFT1 polymorphisms and dental caries risk

Dental caries is a multifactorial disease and the cause of caries has been studied for many years. Herein, a meta-analysis purposed to estimate the association between polymorphism of Carbonic anhydrase VI (CA VI), Ameloblastin (AMBN), and Toftelin (TUFT1) genes and dental caries susceptibility. Four databases of Cochrane Library, Web of Science, PubMed/Medline, and Scopus databases were searched comprehensively until February 13, 2020. The pooled analyses were implemented by Review Manager 5.3 software and publication bias analyzed by the Comprehensive Meta-Analysis version 2.0 software. Eight studies were included and analyzed in the meta-analysis. The pooled analyses illustrated that the CA VI rs2274328, CA VI rs2274333, CA VI rs2274327, AMBN rs4694075, AMBN rs34538475, TUFT1 rs4970957, and TUFT1 rs3790506 polymorphisms were not associated with the susceptibility to dental caries. The age group and ethnicity were not effective factors on the results of the polymorphisms. The sensitivity analyses confirmed the stability of the pooled results. In addition, there was no publication bias across the studies. The results of this meta-analysis suggested a lack of association between AMBN, TUFT1, and CA VI polymorphisms and susceptibility to dental caries.

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties.

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel—the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN—its oligomerization ability—is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.

Ablation of Fam20c causes amelogenesis imperfecta via inhibiting Smad dependent BMP signaling pathway

BackgroundAmelogenesis imperfecta (AI) is a type of hereditary diseases that manifest defects in the formation or mineralization of enamel. Recently, it is reported that inactivation of FAM20C, a well-known Golgi casein kinase, caused AI. However, the mechanism of it is still unknown. The aim of this study was to explore the molecular mechanism of AI, which caused by ablation of FAM20C.ResultsIn the Sox2-Cre;Fam20Cfl/fl (cKO) mouse, we found abnormal differentiation of ameloblasts, improper formation and mineralization of enamel, and downregulation of both mRNA and protein level of enamel matrix proteins, including amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). The levels of BMP2, BMP4 and BMP7, the ligands of BMP signaling pathway, and phosphorylation of Smad1/5/8, the key regulators of BMP signaling pathway, were all decreased in the enamel matrix and the ameloblast of the cKO mice, respectively. The expression of cyclin-dependent kinase inhibitor (P21), muscle segment homeobox genes 2 (Msx2), which are the target genes of the BMP signaling pathway, and laminin 3, the downstream factor of Msx2, were all significantly decreased in the ameloblasts of the cKO mice compared to the control mice.Conclusionthe results of our study suggest that ablation of FAM20C leads to AI through inhibiting the Smad dependent BMP signaling pathway in the process of amelogenesis.

Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns.

Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

Reptile enamel matrix proteins: Selection, divergence, and functional constraint.

The three major enamel matrix proteins (EMPs): amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM), are intrinsically linked to tooth development in tetrapods. However, reptiles and mammals exhibit significant differences in dental patterning and development, potentially affecting how EMPs evolve in each group. In most reptiles, teeth are replaced continuously throughout life, while mammals have reduced replacement to only one or two generations. Reptiles also form structurally simple, aprismatic enamel while mammalian enamel is composed of highly organized hydroxyapatite prisms. These differences, combined with reported low sequence homology in reptiles, led us to predict that reptiles may experience lower selection pressure on their EMPs as compared with mammals. However, we found that like mammals, reptile EMPs are under moderate purifying selection, with some differences evident between AMEL, AMBN, and ENAM. We also demonstrate that sequence homology in reptile EMPs is closely associated with divergence times, with more recently diverged lineages exhibiting high homology, along with strong phylogenetic signal. Lastly, despite sequence divergence, none of the reptile species in our study exhibited mutations consistent with diseases that cause degeneration of enamel (e.g. amelogenesis imperfecta). Despite short tooth retention time and simplicity in enamel structure, reptile EMPs still exhibit purifying selection required to form durable enamel.

An ameloblastin C-terminus variant is present in human adipose tissue

ObjectiveTranscriptional regulatory elements in the ameloblastin (AMBN) promoter indicate that adipogenesis may influence its expression. The objective here was to investigate if AMBN is expressed in adipose tissue, and have a role during differentiation of adipocytes. DesignAMBN expression was examined in adipose tissue and adipocytes by real-time PCR and ELISA. Distribution of ameloblastin was investigated by immunofluorescence in sections of human subcutaneous adipose tissue. The effect of recombinant proteins resembling AMBN and its processed products on proliferation of primary human pre-adipocytes and murine 3T3-L1 cell lines was measured by [3H]-thymidine incorporation. The effect on adipocyte differentiation was evaluated by the expression profile of the adipogenic markers PPARγ and leptin, and the content of lipids droplets (Oil-Red-O staining). ResultsAMBN was found to be expressed in human adipose tissue, human primary adipocytes, and in 3T3-L1 cells. The C-terminus of the AMBN protein and a 45 bp shorter splice variant was identified in human subcutaneous adipose tissue. The expression of AMBN was found to increase four-fold during differentiation of 3T3-L1 cells. Administration of recombinant AMBN reduced the proliferation, and enhanced the expression of PPARγ and leptin in 3T3-L1 and human pre-adipocytes, respectively. ConclusionsThe AMBN C-terminus variant was identified in adipocytes. This variant may be encoded from a short splice variant. Increased expression of AMBN during adipogenesis and its effect on adipogenic factors suggests that AMBN also has a role in adipocyte development.

GuttaFlow Bioseal promotes spontaneous differentiation of human periodontal ligament stem cells into cementoblast-like cells

ObjectivesTo evaluate in vitro the cementogenic potential and the biological effects of GuttaFlow Bioseal, GuttaFlow 2, MTA Fillapex and AH Plus on human periodontal ligament stem cells (hPDLSCs). MethodsCell viability, cell migration and cell morphology assays were performed using eluates of each material. To evaluate cell attachment, hPDLSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy (SEM). The effects of endodontic sealers on cementum protein 1 (CEMP1), cementum-derived attachment protein (CAP), bone sialoprotein (BSP), ameloblastin (AMBN), amelogenin (AMELX) and alkaline phosphatase (ALP) gene expression on hPDLSCs were investigated by qPCR and immunofluorescence (IF). Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α<0.05). ResultsMore than 90% of viable cells were obtained using extracts of GuttaFlow Bioseal and GuttaFlow2 after 72h of culture. By contrast, AH Plus and MTA Fillapex induced significantly lower levels of cell viability. GuttaFlow2 and GuttaFlow Bioseal promoted wound closure in a concentration-dependent manner, comparable to that observed with control extracts (*p<0.05). However, with AH Plus and MTA Fillapex, cell migration was significantly lower than in the control (***p<0.0001). SEM analysis pointed to an organized stress fiber assembly and high degree of cell adhesion on GuttaFlow Bioseal disks but low rates on GuttaFlow2, MTA Fillapex and AH Plus. When hPDLSCs were cultured with GuttaFlow Bioseal-conditioned media, qPCR assays and IF showed a higher level of AMELX, AMBN, CEMP1 and CAP expression than the control (*p<0.05)), whereas no such expression was observed in the other sealers. SignificanceOur results showed that GuttaFlow sealers were more cytocompatible than AH Plus and MTA Fillapex, while GuttaFlow Bioseal favored cementoblast differentiation of hPDLSCs in the absence of any growth factors.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Ablation of Runx2 in Ameloblasts Suppresses Enamel Maturation in Tooth Development

Runt-related transcription factor 2 (Runx2) is involved in the early stage of tooth development. However, only few studies have reported the role of Runx2 in enamel development, which may be attributed to that Runx2 full knockout mice cannot survive after birth. In the present study, we successfully established a Runx2-deficient mouse model using a conditional knockout (cKO) method. We observed a significant reduction in the degree of mineralization and the decreased size of enamel rods in cKO mice. Histological analysis showed the retained enamel proteins in enamel layer at maturation stage in cKO molars. Further analysis by qRT-PCR revealed that the expressions of genes encoding enamel structure proteins, such as amelogenin (AMELX), ameloblastin (AMBN) and enamelin (ENAM), were increased in cKO enamel organs. On the other hand, the expression of kallikrein-related peptidase-4 (KLK4) at the mRNA and protein levels was dramatically decreased from late secretory stage to maturation stage in cKO enamel organs, while the expression of matrix metalloproteinase-20 (MMP-20) was not significantly altered. Finally, immunohistochemistry indicated that the uptake of amelogenins by ameloblasts was significantly decreased in cKO mice. Taken together, Runx2 played critical roles in controlling enamel maturation by increasing synthesis of KLK4 and decreasing synthesis of AMELX, AMBN and ENAM.

Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells

This study aimed to construct the expression of bone morphogenetic protein-4 (BMP4) lentiviral vector gene and explore its influence on the biological activity of mouse induced pluripotent stem (iPS) cells. iPS cell lines stably overexpressing BMP4 were constructed by lentivirus transfection (BMP4-overexpressing group). Cells without transfection served as the blank group, and cells with only vector transfection served as the empty-vector group. Cell proliferation was detected by CCK8, and the expression levels of ameloblastin (AMBN), cytokeratin (CK) 14, dentin sialophospho-protein (DSPP), bone sialoprotein (BSP), and Runx2 mRNA were detected by quantitative polymerase chain reaction. Alkaline phosphatase (ALP) activity was used to detect the degree of cell differentiation. Compared with blank and empty-vector groups, proliferation activity and ALP activity of BMP4-overexpressing group obvious increased (P<0.05), BMP4, AMBN, CK14, DSPP, BSP, Runx2 mRNA expression also increased (P<0.05). BMP4 can significantly promote the odontogenic differentiation of iPS.