Molecular studies on the roles of Runx2 and Twist1 in regulating FGF signaling.

BACKGROUND: Supernumerary teeth are often observed in patients suffering from cleidocranial dysplasia due to a mutation in Runx2 that results in haploinsufficiency. However, the underlying molecular mechanisms are poorly defined. In this study, we assessed the roles of Runx2 and its functional antagonist Twist1 in regulating fibroblast growth factor (FGF) signaling using in vitro biochemical approaches. RESULTS: We showed that Twist1 stimulated Fgfr2 and Fgf10 expression in a mesenchymal cell line and that it formed heterodimers with ubiquitously expressed E12 (together with E47 encoded by E2A gene) and upregulated Fgfr2 and Fgf10 promoter activities in a dental mesenchyme-derived cell line. We further demonstrated that the bHLH domain of Twist1 was essential for its synergistic activation of Fgfr2 promoter with E12 and that the binding of E12 stabilized Twist1 by preventing it from undergoing lysosomal degradation. Although Runx2 had no apparent effects on Fgfr2 and Fgf10 promoter activities, it inhibited the stimulatory activity of Twist1 on Fgfr2 promoter. CONCLUSIONS: These findings suggest that Runx2 haploinsufficiency might result in excessive unbound Twist1 that can freely bind to E12 and enhance FGF signaling, thereby promoting the formation of extra teeth.

Co-production of 2,5-dihydroxymethylfuran and furfuralcohol from sugarcane bagasse via chemobiocatalytic approach in a sustainable system.

2,5-Dihydroxymethylfuran and furfuryl alcohol serve as versatile building-blocks in pharmaceuticals, polymers, and value-added intermediates. To develop an efficient and sustainable method for their production from biomass, a combined approach using deep eutectic solvent Citric acid:Betaine (CTA:BT) for bagasse catalysis and recombinant E. coli SCFD23 for bioreduction of bagasse-derived 5-hydroxymethylfurfural and furfural was devised. Bagasse was effectively transformed into 5-hydroxymethylfurfural (48 mM) and furfural (14 mM) in CTA:BT (8 wt%)-water at 170 °C for 30 min. Bioreduction of 5-hydroxymethylfurfural and furfural by SCFD23 cell co-expressing formate dehydrogenase and NAD(P)H-dependent aldehyde reductase (SsCR) yielded 2,5-dihydroxymethylfuran (90.0 % yield) and furfuryl alcohol (99.0 % yield) in 6 h, using biomass-derived formic acid, xylose and glucose as co-substrates. Molecular docking confirmed the stable binding and reductase activity of SsCR with the biomass-derived 5-hydroxymethylfurfural and furfural. An efficient and eco-friendly chemobiological approach was applied for co-production of 2,5-dihydroxymethylfuran and furfuryl alcohol from biomass in one-pot two-step reaction.

Improved biotransformation of lignin-valorized vanillin into vanillylamine in a sustainable bioreaction medium.

Lignin is a critical biopolymer for creating a large number of highly valuable biobased compounds. Vanillin, one of lignin-derived aromatics, can be used to synthesize vanillylamine that is a key fine chemical and pharmaceutical intermediate. To produce vanillylamine, a productive whole-cell-catalyzed biotransformation of vanillin was developed in deep eutectic solvent - surfactant - H2O media. One newly created recombinant E. coli 30CA cells expressing ω-transaminase and L-alanine dehydrogenase was employed to transform 50 mM and 60 mM vanillin into vanillylamine in the yield of 82.2% and 8.5% under 40 °C, respectively. The biotransamination efficiency was enhanced by introducing surfactant PEG-2000 (40 mM) and deep eutectic solvent ChCl:LA (5.0 wt%, pH 8.0), and the highest vanillylamine yield reached 90.0% from 60 mM vanillin. Building an effective bioprocess was utilized for transamination of lignin-derived vanillin to vanillylamine with newly created bacteria in an eco-friendly medium, which had potential application for valorization of lignin to value-added compounds.

Enhanced proton conductivity of Nafion membrane induced by incorporation of MOF-anchored 3D microspheres: a superior and promising membrane for fuel cell applications.

A novel 3D core-shell material composed of polyacrylate carboxyl microspheres (PCMs) and ZIF-8 nanoparticles was used to promote proton conduction in the Nafion matrix. The Nafion/ZIF-8@PCM membranes displayed excellent proton conductivity (0.24 S cm-1) and physicochemical properties due to special structural characteristics. More importantly, this new concept has a strong practical guiding significance for the fabrication of novel PEMs.

Binding of Sudan II and IV to lecithin liposomes and E. coli membranes: insights into the toxicity of hydrophobic azo dyes.

BACKGROUND: Sudan red compounds are hydrophobic azo dyes, still used as food additives in some countries. However, they have been shown to be unsafe, causing tumors in the liver and urinary bladder in rats. They have been classified as category 3 human carcinogens by the International Agency for Research on Cancer. A number of hypotheses that could explain the mechanism of carcinogenesis have been proposed for dyes similar to the Sudan red compounds. Traditionally, investigations of the membrane toxicity of organic substances have focused on hydrocarbons, e.g. polycyclic aromatic hydrocarbons (PAHs), and DDT. In contrast to hydrocarbons, Sudan red compounds contain azo and hydroxy groups, which can form hydrogen bonds with the polar head groups of membrane phospholipids. Thus, entry may be impeded. They could have different toxicities from other lipophilic hydrocarbons. The available data show that because these compounds are lipophilic, interactions with hydrophobic parts of the cell are important for their toxicity. Lipophilic compounds accumulate in the membrane, causing expansion of the membrane surface area, inhibition of primary ion pumps and increased proton permeability. RESULTS: This work investigated the interactions of the amphiphilic compounds Sudan II and IV with lecithin liposomes and live Escherichia coli (E. coli). Sudan II and IV binding to lecithin liposomes and live E. coli corresponds to the Langmuir adsorption isotherm. In the Sudan red compounds--lecithin liposome solutions, the binding ratio of Sudan II to lecithin is 1/31 and that of Sudan IV to 1/314. The binding constant of the Sudan II-lecithin complex is 1.75 x 104 and that of the Sudan IV-lecithin complex 2.92 x 105. Besides, the influences of pH, electrolyte and temperature were investigated and analyzed quantitatively. In the Sudan red compounds--E.coli mixture, the binding ratios of Sudan II and Sudan IV to E.coli membrane phospholipid are 1/29 and 1/114. The binding constants of the Sudan II--and Sudan IV- E.coli membrane phospholipid complexes are 1.86 x 104 and 6.02 x 104. Over 60% of Sudan II and 75% of Sudan IV penetrated into E.coli, in which 90% of them remained in the E.coli membrane. CONCLUSION: Experiments of Sudan II and IV binding to lecithin liposomes and live E. coli indicates that amphiphilic compounds may be sequestered in the lecithin liposomes and membrane phospholipid bilayer according to the Langmuir adsorption law. Penetration into the cytosol was impeded and inhibited for Sudan red compounds. It is possible for such compounds themselves (excluding their metabolites and by-products)not result directly in terminal toxicity. Therefore, membrane toxicity could be manifested as membrane blocking and membrane expansion. The method established here may be useful for evaluating the interaction of toxins with membranes.

Extrinsic stain removal efficacy of a dual-phase dentifrice.

PURPOSE: To evaluate the extrinsic stain removal benefit of a sodium hexametaphosphate containing dual-phase dentifrice (Crest Vivid White) relative to a marketed negative control (Colgate Cavity Protection). METHODS: This was a parallel groups, examiner-blind, randomized and controlled clinical trial. A total of 200 healthy adults with natural stain on their anterior teeth were enrolled into the study. Following baseline examination, subjects were randomly assigned to one of the two treatment groups based on baseline Lobene composite scores, smoking status (yes/no), tea/coffee consumption (yes/no), and gender. Subjects brushed twice daily, at least 1 minute every time over 6 weeks. Clinical examinations including extrinsic stain evaluation and oral soft tissue examination were conducted at BL, Weeks 3 and 6. Extrinsic stain removal was evaluated on the anterior teeth by a dental examiner using Lobene stain index. RESULTS: Of the 200 subjects who were randomized to treatment, 195 were available for the 3-week examination and 193 subjects completed the study. The dual-phase dentifrice exhibited a statistically significant (P< 0.01) reduction in Lobene stain composite scores when compared to the negative control dentifrice at Week 6. Adjusted mean Lobene composite reduction for the dual-phase dentifrice group (0.32) was twice as big as the negative control group (0.16). Change in Lobene stain extent (area) contributed primarily to the overall composite score reduction. All test products were well tolerated over the 6-week treatment period.

Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium.

Investigations of the odontoblast phenotype are hindered by obstacles such as the limited number of odontoblasts within the dental pulp and the difficulty in purification of these cells. Therefore, it is necessary to develop a cell culture system in which the local environment is inductive and can promote dental pulp stem cells (DPSCs) to differentiate into odontoblast lineage. In this study, we investigated the effect of conditioned medium from developing tooth germ cells (TGCs) on the differentiation and dentinogenesis of DPSCs both in vitro and in vivo. DPSCs were enzymatically isolated from the lower incisors of 4-week-old Sprague-Dawley rats and co-cultured with TGC conditioned medium (TGC-CM). The cell phenotype of induced DPSCs presents many features of odontoblasts, as assessed by the morphologic appearance, cell cycle modification, increased alkaline phosphatase level, synthesis of dentin sialoprotein, type I collagen and several other noncollagenous proteins, expression of the dentin sialophosphoprotein and dentin matrix protein 1 genes, and the formation of mineralized nodules in vitro. The induced DPSC pellets in vivo generated a regular-shaped dentin-pulp complex containing distinct dentinal tubules and predentin, while untreated pellets spontaneously differentiated into bone-like tissues. To our knowledge, this is the first study to mimic the dentinogenic microenvironment from TGCs in vitro, and our data suggest that TGC-CM creates the most odontogenic microenvironment, a feature essential and effective for the regular dentinogenesis mediated by DPSCs.

Odontoblast-like cell differentiation and dentin formation induced with TGF-ß1.

OBJECTIVE: To investigate the inductive potential of scaffold material combing with transforming growth factor-ß1 (TGF-ß1), and to induce odontoblast differentiation and dentin formation from dental pulp cells both in vitro and in vivo. METHODS: Primarily cultured dental pulp cells were used for MTT, ALP activity assay and Alizarin red staining in the presence of TGF-ß1. Pelleted cells were put on the filters combining with or not with TGF-ß1 and cultured in vitro or in vivo. The in vitro and in vivo cell response and tissue formation were analysed with Haematoxylin-Eosin (HE), transmission electron microscopy (TEM) and immunohistochemical staining. RESULTS: TGF-ß1 increased the mineralization and ALP activity of dental pulp cells as revealed by Alizarin red staining and ALP activity assay. After in vitro culture for 7 days, cells polarized in the TGF-ß1 group and expressed dentin sialoprotein (DSP), osteopontin (OPN) and type I collagen (Col I). After in vivo transplantation for 7 days, columnar odontoblast formed on the surface of filter in experimental group, and tubular dentin expressing DSP formed after 3 months transplantation. CONCLUSION: It was concluded that TGF-ß1 combining with transfilter could induce odontoblast differentiation and dentin formation. Our results implied that suitable substrate for the progenitors of odontoblast to anchor on and inductive signals to initiate the differentiation of odontoblast should be taken into consideration when designing scaffold material for inducing dentin tissue engineering.

[The use of dental papillae cells and millipore filter for bioengineered dentin].

OBJECTIVE: To observe the ability of SD rat dental papillae cells forming dentin-like structure induced by millipore filter combined with transforming growth factor-ß(1) (TGF-ß(1)). METHODS: The first passage SD rat dental papillae cells were enzymatically dissociated and centrifuged to obtain a cell mass. The cell mass was seeded on the millipore filter combined with TGF-ß(1). The complex was incubated for 6 d in vitro or transplanted under the renal capsule for 2 weeks. Then the differentiation of dental papillae cells on the filter and the formation of mineral tissue on the implant were analyzed. RESULTS: A layer of polarized columnar cells were observed along the surface of the millipore filter, with cell processes extending into the porous media. Dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP-1) were positive in these cells. After 2 weeks, tubular dentin matrix was deposited on the surface of the aligned cells. Scanning electron microscopy showed that the thickness of newly formed tubular dentin was consistent. DSP and DMP-1 were expressed in columnar cells, tubular matrix and the dental papillae cells adjacent to the filter. CONCLUSIONS: The millipore filter combined with TGF-ß(1) could effectively recruit progenitors onto its surface and induce odontoblast differentiation, secrete matrix in a homogenous manner, leading to dentinogenesis.

[Effects of nimodipine on human dentinogenesis].

OBJECTIVE: Studies have showed that L type calcium channel plays an important role in dentin calcification and affects tooth development and tooth reparation after injury. The objective of this article is to study the effects of nimodipine, blocking agent of L type calcium channel, on human dentinogenesis using human tooth slice organ culture in vitro. METHODS: Young healthy human premolars were collected, and cut into 2 mm-thick transverse slices by low speed diamond saw. Agarose beads dipped in nimodipine solution and PBS weresy minetrically placed on tooth slices, and the slices were then embedded in a semisolid agarose-based medium and cultured with organ culture method for 1 week. Fluorescent band of tetracycline, Von-Kossa staining, immunohistochemical staining of the slices and transmission electron microscopy (TEM) of odontoblasts were observed to evaluate dentinogenesis changes of the slices. RESULTS: Tooth slices were successfully cultured in vitro for 1 week and the odontoblasts could maintain their original morphology. After treatment with nimodipine, the fluorescent band of tetracycline was narrow and weak, and globular calcification in predentine was decreased compared with the control. TEM showed that secretory vesicles in odontoblast were somewhat increased, hut iminunohistochemical staining for collagen I showed no difference between the two groups. CONCLUSION: Nimodipine can influence the calcification of dentine, but has no obvious influence on the synthesis and secretion of dentine matrix. The results show that L type calcium channel is important in dentin calcification.

Cementum and periodontal ligament-like tissue formation induced using bioengineered dentin.

Stem cell-mediated root regeneration offers opportunities to regenerate a bio-root and its associated periodontal tissues to restore tooth loss. Periodontal ligament (PDL) and cementum complex and dentin pulp complex have been tissue engineered using human dental pulp stem cells and PDL stem cells, respectively. The aim of this study was to explore whether dentin formation could be induced using an inductive substrate and whether bioengineered dentin could induce cementum and PDL formation. First, dentin was bioengineered from tooth papillae of Sprague-Dawley (SD) rats with an inductive substrate, and its phenotype was characterized; then primarily cultured human PDL cells were seeded on the surface of dentin and transplanted under the skin of immunocompromised mice. Histological, immunohistochemical, and scanning electronic microscopy examinations results showed that bioengineered dentin could induce cementogenesis and PDL formation, and condense PDL arranged perpendicularly on the dentin surface via a layer of cementum-like tissue. The results indicated that tissue-engineered dentin could be induced using an inductive substrate and could be used as a further substrate for cementum and PDL tissue engineering.