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Objective: The aim of the present study was to select the optimal concentration of TiF 4 solution to facilitate the remineralization of early dentine caries lesions., Design: Sixty human dentine specimens were cut and randomly divided into 6 groups (1%, 2%, 3%, 4% TiF 4 groups, 2.712% NaF group and distilled deionized water (DDW) control group). Artificial dentine caries-like lesions were created. After being subjected to fluoride treatment and immersed in remineralizing solution for 2weeks, the specimens were observed by microCT, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Data were analysed using linear regression analysis (P<0.05)., Results: The lesion depths of the specimens treated by 2% TiF 4 solution were statistically less than those of the other groups. Further, the greyscale values of these lesion areas were greater. The 3% and 4% TiF 4 solutions caused further lesion demineralization. The 2.712% NaF solution seemed to be detrimental to remineralization during the experimental time, as the subsurface area remained hypomineralized with a thick precipitation layer on the surface., Conclusions: The 2% TiF 4 solution demonstrated better remineralizing potency than did the other treatments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We argue in favor of representing living cells as automata and review demonstrations that autonomous cells can form patterns by responding to local variations in the strain fields that arise from their individual or collective motions. An autonomous cell's response to strain stimuli is assumed to be effected by internally-generated, internally-powered forces, which generally move the cell in directions other than those implied by external energy gradients. Evidence of cells acting as strain-cued automata have been inferred from patterns observed in nature and from experiments conducted in vitro. Simulations that mimic particular cases of pattern forming share the idealization that cells are assumed to pass information among themselves solely via mechanical boundary conditions, i.e., the tractions and displacements present at their membranes. This assumption opens three mechanisms for pattern formation in large cell populations: wavelike behavior, kinematic feedback in cell motility that can lead to sliding and rotational patterns, and directed migration during invasions. Wavelike behavior among ameloblast cells during amelogenesis (the formation of dental enamel) has been inferred from enamel microstructure, while strain waves in populations of epithelial cells have been observed in vitro. One hypothesized kinematic feedback mechanism, "enhanced shear motility", accounts successfully for the spontaneous formation of layered patterns during amelogenesis in the mouse incisor. Directed migration is exemplified by a theory of invader cells that sense and respond to the strains they themselves create in the host population as they invade it: analysis shows that the strain fields contain positional information that could aid the formation of cell network structures, stabilizing the slender geometry of branches and helping govern the frequency of branch bifurcation and branch coalescence (the formation of closed networks). In simulations of pattern formation in homogeneous populations and network formation by invaders, morphological outcomes are governed by the ratio of the rates of two competing time dependent processes, one a migration velocity and the other a relaxation velocity related to the propagation of strain information. Relaxation velocities are approximately constant for different species and organs, whereas cell migration rates vary by three orders of magnitude. We conjecture that developmental processes use rapid cell migration to achieve certain outcomes, and slow migration to achieve others. We infer from analysis of host relaxation during network formation that a transition exists in the mechanical response of a host cell from animate to inanimate behavior when its strain changes at a rate that exceeds 10 -4 -10 -3 s -1 . The transition has previously been observed in experiments conducted in vitro.

Endocytosis and phagocytosis are important physiologic activities occurring during ameloblast differentiation. We have previously found that excess fluoride inhibited ameloblasts endocytotic functions. Here, we hypothesized that increasing amounts of fluoride may affect ameloblast phagocytotic function during their differentiation. Using cell culture, we first induced maturation of the mouse ameloblast-like LS8 cells by treatment with exogenous retinoic acid (RA) and dexamethasone (DEX). We measured their phagocytotic activity by fluorescent microscopy using a live cell visualization station. We found that ameloblast-like LS8 cells matured with RA/DEX treatment and the increasing amounts of fluoride demonstrated the up-regulated expression of the phagocytotic marker proteins, LAMP1 and CD68. A connection between phagocytosis and apoptosis was confirmed by the increased number of phagocytotic vacuole-like structures and the heterochromatin margination phenomenon observed in the RA/DEX with NaF treatment group. The increase in albumin uptake by ameloblasts was confirmed using whole organ culture of incisor tooth germs. Here, in fluoride treated tooth germs, mature canonical ameloblasts showed greater amounts of albumin uptake, which was accompanied by decreased expression of the anti-apoptosis marker, Bcl-2 along with up-regulated expression of CD68. From these observations, we inferred that high doses of fluoride may cause apoptosis by increasing the phagocytosis of protein particles in mature-stage ameloblasts and loss of Bcl-2 signals might be involved in this process., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To investigate the effects of two different fluoride concentrations on the expression of enamel proteins, alkaline phosphatase (ALP), cytokines and interleukins by an ameloblast-derived cell line., Methods: Murine ameloblast-derived cells (LS-8), mouse odontogenic epithelia, were exposed to 1 or 5ppm sodium fluoride (NaF) (0.46 and 2.25ppm F, respectively) for 1, 3 and 7 days. The effect of NaF on the mRNA expression of enamel proteins was quantified; the secretion of cytokines, and interleukins, and the alkaline phosphatase (ALP) activity, into the cell culture medium was measured and compared to untreated controls. The effect on cell growth after 1- and 3-days in culture was measured using BrdU incorporation., Results: Fluoride at 2.25ppm reduced mRNA expression of the structural enamel matrix proteins amelogenin (amel), ameloblastin (ambn), enamelin (enam), and the enamel protease matrix metallopeptidase-20 (MMP-20). Similarly several vascularisation factors (vascular endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and interferon inducible protein 10 (IP-10), was also reduced by 2.25ppm fluoride. ALP activity and proliferation were stimulated by 0.46ppm fluoride but inhibited by 2.25ppm fluoride., Conclusions: These results indicate that fluoride may impact on the expression of structural enamel proteins and the protease responsible for processing these proteins during the secretory stage of amelogenesis and go some way to explaining the mineralization defect that characterises fluorotic enamel., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objectives: Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells., Design: Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 microg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern., Results: Maximum surface binding was achieved with 50 microg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1., Conclusions: The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Electroporation was used to introduce foreign genes into cells derived from the mouse enamel organ epithelia (EOE). Optimal conditions for this electroporation were established. The introduction of a plasmid construct bearing the coding region for the large T-antigen from polyoma virus into EOE cells permitted the establishment of a derivative cell line that has the following characteristics: (1) the cells could be passaged many times; (2) they expressed a keratin-containing cytoskeleton; and (3) approx. 60% of the cells expressed amelogenin, a tissue-specific gene product unique to ameloblasts. Potential uses for such a cell line include analysis of: (1) the upstream regulatory regions required for temporally and spatially restricted expression of amelogenin; (2) the post-translational modification of amelogenin in synchronized cells and (3) the organization and biomineralization of enamel extracellular matrix in monolayer culture.

Human dentine contains relatively large amounts of transforming growth factor-beta (TGF-beta), which might originate from odontoblasts. The expression of the TGF-beta 1 message in developing teeth was examined by in situ hybridization. The analysis was made on 5-microns serial sections of mandibular third molars of neonatal sheep cut from tissues that had been fixed in glutaraldehyde and paraffin-embedded. A 35S-labelled cRNA probe, complementary to TGF-beta 1 mRNA, was constructed from human TGF-beta 1 cDNA. Northern analysis of total RNA from sheep placenta and neonatal third molars demonstrated hybridization to a single 2.4 kb TGF-beta 1 transcript from both tissues, indicating cross-reactivity of the human probe in the sheep. In the neonatal molars, in situ hybridization was observed in cells of the inner enamel epithelium, mature ameloblasts and mature odontoblasts, but not within preodontoblasts before dentine matrix formation. TGF-beta 1 mRNA expression was also evident in the cells of the dental papilla but scarcely so in the stellate reticulum. The most striking feature was the appearance of hybridization signal in the cells of the stratum intermedium before hybridization was evident in the inner enamel epithelium. Control sections incubated with RNAase before incubation with probe did not show evidence of hybridization. These findings suggest that TGF-beta 1 may have an important regulatory role in the differentiation of ameloblasts and odontoblasts, perhaps by modulating matrix formation during amelogenesis or odontogenesis. They also suggest a potential novel regulatory role for the cells of the stratum intermedium.

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

To better characterize the molecular mechanism responsible for the bromodeoxyuridine (BrdU)-mediated arrest of mammalian odontogenesis in vitro, the nature of nuclear DNA-analogue interactions was determined. Bioactive doses of the radiolabelled analogue were added to tissue culture medium of 16-day old embryonic rat incisor primordia. Control rudiments were similarly exposed to equimolar, radiolabelled thymidine. After 16-18 h, DNA was isolated and purified from the labelled organ cultures. Following sedimentation to equilibrium through neutral CsCl density gradients, [3H]-BrdU-labelled DNA revealed a buoyant density indicative of a 12-15 per cent level of substitution in place of thymidine. Furthermore, similar centrifugation of DNA through alkaline density gradients suggested that the substitution was localized predominantly within a single-strand. DNA-DNA reassociation kinetics subsequently revealed that disproportionately more radiolabelled BrdU was concentrated within repetitive DNA nucleotide sequences in contrast to the more random distribution of [3H]-thymidine moieties. Thus it is likely that BrdU exerts its inhibitory effects on odontogenic differentiation through a relatively small proportion of rat embryo nuclear DNA.