Effective Differentiation of Induced Pluripotent Stem Cells Into Dental Cells.

BACKGROUND: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. RESULTS: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. CONCLUSIONS: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129-139, 2019. © 2018 Wiley Periodicals, Inc.

Inhibition of the Zeb family prevents murine palatogenesis through regulation of apoptosis and the cell cycle.

Mammalian palate separates the oral and nasal cavities for normal feeding, breathing and speech. The palatal shelves are a pair of maxillary prominences that consist of the neural crest-derived mesenchyme and surrounding epithelium. Palatogenesis is completed by the fusion of the midline epithelial seam (MES) after the medial edge epithelium (MEE) cells make contact between the palatal shelves. Various cellular and molecular events, such as apoptosis, cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT), are involved in palatogenesis. The Zeb family of transcription factors is an essential player during normal embryonic development. The distinct role of the Zeb family has not been thoroughly elucidated to date. In mouse palate, the Zeb family factors are expressed in the palatal mesenchyme until MEE contact. Interestingly, the expression of the Zeb family has also been observed in MES, which is already fused with the mesenchymal region. The regulatory roles of the Zeb family in palatogenesis have not been elucidated to date. The purpose of this study is to determine the Zeb family effects on the cellular events. To investigate the functions of the Zeb family, siRNA targeting Zeb family was used to treat in vitro organ culture for temporary inhibition of the Zeb family during palatogenesis. In the cultured palate containing siRNA, MES was clearly observed, and E-cadherin, an epithelial marker, was still expressed. Inhibition of the Zeb family results in the suppression of apoptosis, increased cell proliferation, and defective cell migration in the developing palate. Our data suggest that the Zeb family plays multiple roles in the stimulation and inhibition of apoptosis and cell proliferation and efficient mesenchymal cell migration during palatogenesis.

Uniform-sized neurosphere-mediated motoneuron differentiation in microwell arrays.

We developed the photocrosslinkable hydrogel microwell arrays for uniform-sized neurosphere-mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform-sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform-sized neurospheres harvested from microwell arrays were replated into laminin-coated substrate. In parallel, uniform-sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three-dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150 µm in diameter) on motoneuron differentiation, showing that the largest uniform-sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform-sized neurosphere-mediated motoneuron differentiation.

Hydrogel microfluidic co-culture device for photothermal therapy and cancer migration.

We developed the photo-crosslinkable hydrogel microfluidic co-culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi-permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co-culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)-treated GelMA hydrogel-embedding microchannels. The main advantage of this hydrogel microfluidic co-culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod-mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co-culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.

Novel insights into a retinoic-acid-induced cleft palate based on Rac1 regulation of the fibronectin arrangement.

Retinoic acid (RA)-induced cleft palate results from both extrinsic obstructions by the tongue and internal factors within the palatal shelves. Our previous study showed that the spatiotemporal expression of Rac1 regulates the fibronectin (FN) arrangement through cell density alterations that play an important role in palate development. In this study, we investigate the involvement of the Rac1 regulation of the FN arrangement in RA-induced cleft palate. Our results demonstrate that RA-induced intrinsic alterations in palatal shelves, including a delayed progress of cell condensation, delay palate development, even after the removal of the tongue. Further analysis shows that RA treatment diminishes the region-distinctive expression of Rac1 within the palatal shelves, which reversely alters the fibrillar arrangement of FN. Furthermore, RA treatment disrupts the formation of lamellipodia, which are indicative structures of cell migration that are regulated by Rac1. These results suggest that the Rac1 regulation of the FN arrangement is involved in RA-induced cleft palate through the regulation of cell migration, which delays the progress of cell condensation and subsequently influences the FN arrangement, inducing a delay in palate development. Our study provides new insights into the RA-induced impairment of palatal shelf elevation based on cell migration dynamics.

Gastrin-releasing peptide expression and its effect on the calcification of developing mouse incisor.

Gastrin-releasing peptide (GRP) is considered to be one of the cancer growth factors. This peptide's receptor (GRPR) is known as a G protein-coupled receptor, regulating intracellular calcium storage and releasing signals. This study is the first to investigate the function of GRP during mouse incisor development. We hypothesized that GRP is one of the factors that affects the regulation of calcification during tooth development. To verify the expression pattern of GRP, in situ hybridization was processed during incisor development. GRP was expressed at the late bell stage and hard tissue formation stage in the epithelial tissue. To identify the genuine function of GRP during incisor development, a gain-of-function analysis was performed. After GRP overexpression in culture, the phenotype of ameloblasts, odontoblasts and predentin was altered compared to control group. Moreover, enamel and dentin thickness was increased after renal capsule transplantation of GRP-overexpressed incisors. With these results, we suggest that GRP plays a significant role in the formation of enamel and dentin by regulating ameloblasts and predentin formation, respectively. Thus, GRP signaling is strongly related to calcium acquisition and secretion during mouse incisor development.

Role of region-distinctive expression of Rac1 in regulating fibronectin arrangement during palatal shelf elevation.

Palatal shelf elevation is a crucial process in palate development, with the contribution of various factors. Disturbances in any factor during this process result in cleft palate. Prior to palatal shelf elevation starting from embryonic day 12.5, the Rac1 expression level in the bend region of the mid-palatal shelf progressively increases and the cell densities in the bend and groove regions gradually become higher than those in the middle region. The comparative decrease of cell density in the middle region is correlated with a gradual alteration of the arrangement of fibronectin (FN) fibers, whereas the bend and groove regions with higher cell densities maintain ring-like FN arrangements. Rac1 overexpression alters the fibrillar FN arrangement in the middle region to the ring-like arrangement by increasing cell density. This alteration is sufficient to induce the failure of palatal shelf elevation, ultimately leading to cleft palate. Furthermore, the inhibition of FN delays palatal shelf elevation. Thus, the spatiotemporal expression of Rac1 plays an impressive role in palatal shelf elevation by regulating FN arrangement within the palatal shelf.

Molecular and engineering approaches to regenerate and repair teeth in mammals.

Continuous replacement of teeth throughout the lifespan of an individual is possibly basal for most of the vertebrates including fish and reptiles; however, mammals generally have a limited capacity of tooth renewal. The ability to induce cellular differentiation in adults to replace lost or damaged cells in mammals, or to tissue-engineer organs in vitro, has hence become one of the major goals of regenerative medicine. In this article, we will revisit some of the important signals and tissue interactions that regulate mammalian tooth development, and will offer a synopsis of the latest progress in tooth regeneration and repair via molecular and engineering approaches. It is hoped that this article will not only offer an overview of recent technologies in tooth regeneration and repair but will also stimulate more interdisciplinary research in this field to turn the pursuit of tooth regeneration and repair into practical reality.

Morphological and molecular changes associated with Pitchfork during mouse palate development.

Mammalian palate development is regulated by complex processes. Many cellular and molecular events, such as cell proliferation, apoptosis, cell migration and the epithelial mesenchymal transition, regulate proper palate development and some abnormalities in palate development lead to cleft palate. Various developmental disorders, such as cleft palate and disorders of the lung, kidney and heart, are known to be associated with ciliary defects. Pitchfork, a mouse embryonic node gene, is associated with ciliary targeting complexes located at the basal body during primary cilia disassembly. To determine the function of Pitchfork during palate development, we examine Pitchfork expression patterns and morphological changes in the developing secondary palate after Pitchfork over-expression. From embryonic day 12.5 (E12.5) to E13.5 in mice, Pitchfork was highly expressed in the developing mouse secondary palate. Morphological differences were observed in vitro in cultured palates in the Pitchfork over-expression group compared with the control group. Pitchfork over-expression induced primary cilia disassembly during palate development. Sonic hedgehog and Patched1 expression levels and palatine rugae morphology were altered in the over-expressed Pitchfork group during palate development. Thus, the proper expression levels of Pitchfork might play a pivotal role in normal secondary palate morphogenesis.

Effects of icariin on dental pulp stem cells and its potential applications in dentin repair.

OBJECTIVES: As dental pulp therapy evolves towards regenerative approaches, biomolecules such as icariin, derived from Epimedium flowers, are being evaluated for their therapeutic potential. This study investigates icariin's effectiveness in promoting odontogenic differentiation in human dental pulp stem cells (hDPSCs) in vitro and as a pulp-capping agent in vivo. DESIGN: The study explored the effects of icariin on hDPSCs at concentrations of 10, 20, and 40 µM. Cell viability and migration assays were conducted to evaluate cytotoxicity and chemotaxis. Odontogenic differentiation was assessed using alkaline phosphatase staining and alizarin red S (ARS) staining, complemented by real-time PCR and Western blot analyses of key markers such as RUNX family transcription factor 2 (RUNX2), collagen type I alpha 1 chain (COL1A1), alkaline phosphatase (ALPL), and dentin sialophosphoprotein (DSPP). Additionally, the in vivo effects of icariin were tested in a rat maxillary molar model, where icariin-treated collagen sponges were used for direct pulp capping to evaluate its potential to induce reparative dentin formation. RESULTS: Icariin showed no cytotoxic effects on hDPSCs at any tested concentration, enhanced migratory activity in a dose-dependent manner, and significantly increased alkaline phosphatase activity and calcium deposition. Gene and protein expression analyses revealed a dose-dependent increase in odontogenic differentiation markers in icariin-treated hDPSCs. In vivo, icariin effectively promoted reparative dentin formation in exposed rat pulp. CONCLUSIONS: Icariin enhances odontogenic differentiation of hDPSCs and has promising potential as a pulp-capping agent for vital pulp therapy.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

miR-200b regulates cell migration via Zeb family during mouse palate development.

Palate development requires coordinating proper cellular and molecular events in palatogenesis, including the epithelial-mesenchymal transition (EMT), apoptosis, cell proliferation, and cell migration. Zeb1 and Zeb2 regulate epithelial cadherin (E-cadherin) and EMT during organogenesis. While microRNA 200b (miR-200b) is known to be a negative regulator of Zeb1 and Zeb2 in cancer progression, its regulatory effects on Zeb1 and Zeb2 in palatogenesis have not yet been clarified. The aim of this study is to investigate the relationship between the regulators of palatal development, specifically, miR-200b and the Zeb family. Expression of both Zeb1 and Zeb2 was detected in the mesenchyme of the mouse palate, while miR-200b was expressed in the medial edge epithelium. After contact with the palatal shelves, miR-200b was expressed in the palatal epithelial lining and epithelial island around the fusion region but not in the palatal mesenchyme. The function of miR-200b was examined by overexpression via a lentiviral vector in the palatal shelves. Ectopic expression of miR-200b resulted in suppression of the Zeb family, upregulation of E-cadherin, and changes in cell migration and palatal fusion. These results suggest that miR-200b plays crucial roles in cell migration and palatal fusion by regulating Zeb1 and Zeb2 as a noncoding RNA during palate development.

MiR-200b is involved in Tgf-ß signaling to regulate mammalian palate development.

Various cellular and molecular events are involved in palatogenesis, including apoptosis, epithelial-mesenchymal transition (EMT), cell proliferation, and cell migration. Smad2 and Snail, which are well-known key mediators of the transforming growth factor beta (Tgf-ß) pathway, play a crucial role in the regulation of palate development. Regulatory effects of microRNA 200b (miR-200b) on Smad2 and Snail in palatogenesis have not yet been elucidated. The aim of this study is to determine the relationship between palate development regulators miR-200b and Tgf-ß-mediated genes. Expression of miR-200b, E-cadherin, Smad2, and Snail was detected in the mesenchyme of the mouse palate, while miR-200b was expressed in the medial edge epithelium (MEE) and palatal mesenchyme. After the contact of palatal shelves, miR-200b was no longer expressed in the mesenchyme around the fusion region. The binding activity of miR-200b to both Smad2 and Snail was examined using a luciferase assay. MiR-200b directly targeted Smad2 and Snail at both cellular and molecular levels. The function of miR-200b was determined by overexpression via a lentiviral vector in the palatal shelves. Ectopic expression of miR-200b resulted in suppression of these Tgf-ß-mediated regulators and changes of apoptosis and cell proliferation in the palatal fusion region. These results suggest that miR-200b plays a crucial role in regulating the Smad2, Snail, and in apoptosis during palatogenesis by acting as a direct non-coding, influencing factor. Furthermore, the molecular interactions between miR-200b and Tgf-ß signaling are important for proper palatogenesis and especially for palate fusion. Elucidating the mechanism of palatogenesis may aid the design of effective gene-based therapies for the treatment of congenital cleft palate.

Activated recovery of PVC from contaminated waste extension cord-cable using a weak acid.

Waste electronic and electrical equipment are complex mixtures of valuable and/or toxic materials, which pose serious challenges in their recycling or disposal, for example, electrical transmission wires insulated in polyvinyl chloride materials. These materials are frequently found contaminated with toxic chemical elements, such as Pb, Hg, Cr, or Cd, and are discarded without decontamination. To resolve this problem, we developed a microwave-assisted extraction process to remove toxic metals from plastic e-waste. We processed diluted (30 wt%) citric acid at 210 °C for 1 h inside a pressurized vessel heated by microwave, and found it was suitable not only for the extraction of the toxic metals (∼100%) but also for a significant plastic recovery (>50 wt%). To predict an optimized process window, the support vector regression machine learning algorithm was applied, which reduced the amount of experimentation required while still giving accurate results. Conditions optimized for the reference sample also led to maximum extraction of toxic metals from real-life extension cord waste. We also report that the recovered plastic's properties remained intact after the extraction.

Machine learning-assisted optimization of TBBPA-bis-(2,3-dibromopropyl ether) extraction process from ABS polymer.

The increasing amount of e-waste plastics needs to be disposed of properly, and removing the brominated flame retardants contained in them can effectively reduce their negative impact on the environment. In the present work, TBBPA-bis-(2,3-dibromopropyl ether) (TBBPA-DBP), a novel brominated flame retardant, was extracted by ultrasonic-assisted solvothermal extraction process. Response Surface Methodology (RSM) achieved by machine learning (support vector regression, SVR) was employed to estimate the optimum extraction conditions (extraction time, extraction temperature, liquid to solid ratio) in methanol or ethanol solvent. The predicted optimum conditions of TBBPA-DBP were 96 min, 131 mL g-1, 65 °C, in MeOH, and 120 min, 152 mL g-1, 67 °C in EtOH. And the validity of predicted conditions was verified.

Direct reuse of electronic plastic scraps from computer monitor and keyboard to direct stem cell growth and differentiation.

Reuse of electronic wastes is a critical aspect for a more sustainable circular economy as it provides the simplest and most direct route to extend the lifespan of non-renewable resources. Herein, the distinctive surface and micro topographical features of computer electronic-plastic (E-plastic) scraps were unconventionally repurposed as a substrate material to guide the growth and differentiation of human adipose-derived mesenchymal stem cells (ADSCs). Specifically, the E-plastics were scavenged from discarded computer components such as light diffuser plate (polyacrylates), prismatic sheet (polyethylene terephthalate), and keyboards (acrylonitrile butadiene styrene) were cleaned, sterilized, and systematically characterized to determine the identity of the plastics, chemical constituents, surface features, and leaching characteristics. Multiparametric analysis revealed that all the E-plastics could preserve stem-cell phenotype and maintain cell growth over 2 weeks, rivalling the performance of commercial tissue-culture treated plates as cell culture plastics. Interestingly, compared to commercial tissue-culture treated plastics and in a competitive adipogenic and osteogenic differentiation environment, ADSCs cultured on the keyboard and light diffuser plastics favoured bone cells formation while the grating-like microstructures of the prismatic sheet promoted fat cells differentiation via the process of contact guidance. Our findings point to the real possibility of utilizing discarded computer plastics as a "waste-to-resource" material to programme stem cell fate without further processing nor biochemical modification, thus providing an innovative second-life option for E-plastics from personal computers.

Shh signaling is essential for rugae morphogenesis in mice.

Palatal ridges, or rugae palatinae, are corrugated structures observed in the hard palate region. They are found in most mammalian species, but their number and arrangement are species-specific. Nine palatal rugae are found in the mouse secondary palate. Previous studies have shown that epithelial Shh signaling in the palatal ridge plays an important role during rugae development. Moreover, Wnt family members, including LEF1, play a functional role in orofacial morphogenesis. To explore the function of Shh during rugae development, we utilized the maternal transfer of 5E1 (anti-Shh antibody) to mouse embryos. 5E1 induced abnormal rugae patterning characterized by a spotted shape of palatal ridge rather than a stripe. The expression patterns of Shh and Shh-related genes, Sostdc1, Lef1 and Ptch1, were disrupted following 5E1 injection. Moreover, rugae-specific cell proliferation and inter-rugae-specific apoptosis were affected by inhibition of Shh signaling. We hypothesize that the altered gene expression patterns and the change in molecular events caused by the inhibition of Shh signaling may have induced abnormal rugae patterning. Furthermore, we propose a reaction-diffusion model generated by Wnt, Shh and Sostdc1 signaling. In this study, we show that Sostdc1, a secreted inhibitor of the Wnt pathway, is a downstream target of Shh and hypothesize that the interaction of Wnt, Shh and Sostdc1 is a pivotal mechanism controlling the spatial patterning of palatal rugae.

Highly porous core-shell polymeric fiber network.

Core-shell nanofibers are of great interest in the field of tissue engineering and cell biology. We fabricated porous core-shell fiber networks using an electrospinning system with a water-immersed collector. We hypothesized that the phase separation and solvent evaporation process would enable the control of the pore formation on the core-shell fiber networks. To synthesize porous core-shell fiber networks, we used polycaprolactone (PCL) and gelatin. Quantitative analysis showed that the sizes of gelatin-PCL core-shell nanofibers increased with PCL concentrations. We also observed that the shapes of the pores created on the PCL fiber networks were elongated, whereas the gelatin-PCL core-shell fiber networks had circular pores. The surface areas of porous nanofibers were larger than those of the nonporous nanofibers due to the highly volatile solvent and phase separation process. The porous core-shell fiber network was also used as a matrix to culture various cell types, such as embryonic stem cells, breast cancer cells, and fibroblast cells. Therefore, this porous core-shell polymeric fiber network could be a potentially powerful tool for tissue engineering and biological applications.

Surface morphology changes of graphene on flexible PET substrate upon thermal annealing.

The performance of a polymer photovoltaic device using multilayered graphene on an amorphous PET substrate as the electrode was studied. The changes in surface morphology of graphene coated polyethylene terephthalate (PETG) substrate upon thermal annealing were investigated by atomic force microscopy (AFM), field emission scanning electron microscope (FE-SEM) and current-voltage characteristics. The root mean square (RMS) roughness of PETG substrate before annealing was 36.5 nm that decreased to 11.5 nm after 10 min thermal annealing at 110 degrees C. The mean grain size of the substrate decreased from 2301 nm2 to 848 nm2. The PETG surface became smooth when thermally annealed as the voids created by the bubbles in the graphene layer were filled up with thermal expansion of the PET substrate. However, cracks present initially on the graphene due to surface stress between the graphene and PET layer grew further upon annealing that deteriorated the device performance. This study on the graphene surface morphology change upon annealing and the consequent drop in device performance vis-à-vis an ITO glass electrode shows potential drawback of solar cell device fabrication on such flexible substrates.

Synthesis of double-layered rotavirus-like particles using internal ribosome entry site vector system in stably-transformed Drosophila melanogaster.

We established a bicistronic expression system using an encephalomyocarditis virus (EMCV)-derived internal ribosomal entry site (IRES) element to generate stably transformed Drosophila melanogaster Schneider 2 (S2) cells expressing human rotavirus Wa capsid proteins, VP2 and VP6, for the synthesis of VP2/6 double-layered virus-like particle (DVLP). The EMCV-derived IRES permitted bicistronic translation of recombinant VP6. Recombinant VP2 and VP6 were detected in extracellular fractions of stably transformed S2 cells. A wheel-like DVLP (diam ~ 50-55 nm) with short spikes was produced from the extracellular fraction of stably transformed S2 cells. A bicistronic expression system using an EMCV-derived IRES element can thus be used to express two proteins of interest in stably transformed S2 cells. The bi-or tri-cistronic expression of recombinant VP2/6/7 using stably transformed S2 cells can also be used to produce rotavirus VLPs.