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The effects of fabrication method on the microstructure and sinterability of W-1 wt% Y2O3 were analyzed. W composite powders dispersed with Y2O3 particles were synthesized by the ultrasonic spray pyrolysis process or the ultrasonic spray pyrolysis/polymer solution process. A dense composite was fabricated by a combination of spark plasma sintering and final hot isostatic pressing. The powder synthesized by the ultrasonic spray pyrolysis had fine dispersed particles on the surface of the cubic powder and was composed of W, Y2O3 and W-oxides. On the other hand, in the case of the ultrasonic spray pyrolysis/polymer solution process, the nano-sized particles formed agglomerates and existed only as pure W and Y2O3 phases. All the sintered compacts treated with HIP showed an increase in relative density, and the sintered compacts of the powder synthesized by the ultrasonic spray pyrolysis/polymer solution process showed a maximum relative density of 97~99% and a fine grain size. The change in microstructure with different powder processing was explained by the presence of W-oxide and the size and distribution of Y2O3 particles. The Vickers hardness of the sintered compact reached the largest value of about 5 GPa in the powder synthesized by the ultrasonic spray pyrolysis/polymer solution process, which was interpreted to be a result of the relatively high density and decreased grain size.

This study summarizes the recent cutting-edge approaches for dentin regeneration that still do not offer adequate solutions. Tertiary dentin is formed when odontoblasts are directly affected by various stimuli. Recent preclinical studies have reported that stimulation of the Wnt/β-catenin signaling pathway could facilitate the formation of reparative dentin and thereby aid in the structural and functional development of the tertiary dentin. A range of signaling pathways, including the Wnt/β-catenin pathway, is activated when dental tissues are damaged and the pulp is exposed. The application of small molecules for dentin regeneration has been suggested as a drug repositioning approach. This study reviews the role of Wnt signaling in tooth formation, particularly dentin formation and dentin regeneration. In addition, the application of the drug repositioning strategy to facilitate the development of new drugs for dentin regeneration has been discussed in this study.

Several factors, including genetic and environmental insults, impede protein folding and secretion in the endoplasmic reticulum (ER). Accumulation of unfolded or mis-folded protein in the ER manifests as ER stress. To cope with this morbid condition of the ER, recent data has suggested that the intracellular event of an unfolded protein response plays a critical role in managing the secretory load and maintaining proteostasis in the ER. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone and hydrophilic bile acid that is known to inhibit apoptosis by attenuating ER stress. Numerous studies have revealed that TUDCA affects hepatic diseases, obesity, and inflammatory illnesses. Recently, molecular regulation of ER stress in tooth development, especially during the secretory stage, has been studied. Therefore, in this study, we examined the developmental role of ER stress regulation in tooth morphogenesis using in vitro organ cultivation methods with a chemical chaperone treatment, TUDCA. Altered cellular events including proliferation, apoptosis, and dentinogenesis were examined using immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, altered localization patterns of the formation of hard tissue matrices related to molecules, including amelogenin and nestin, were examined to assess their morphological changes. Based on our findings, modulating the role of the chemical chaperone TUDCA in tooth morphogenesis, especially through the modulation of cellular proliferation and apoptosis, could be applied as a supporting data for tooth regeneration for future studies.

Enamel knot (EK)—a signaling center—refers to a transient morphological structure comprising epithelial tissue. EK is believed to regulate tooth development in early organogenesis without its own cellular alterations, including proliferation and differentiation. EKs show a very simple but conserved structure and share functions with teeth of recently evolved vertebrates, suggesting conserved signaling in certain organs, such as functional teeth, through the course of evolution. In this study, we examined the expression patterns of key EK-specific genes including Dusp26 , Fat4, Meis2, Sln , and Zpld1 during mice embryogenesis. Expression patterns of these genes may reveal putative differentiation mechanisms underlying tooth morphogenesis.

miRNAs are conserved short non-coding RNAs that play a role in the modulation of various biological pathways during tissue and organ morphogenesis. In this study, the function of miRNA-221-3p in tooth development, through its loss or gain in function was evaluated. A variety of techniques were utilized to evaluate detailed functional roles of miRNA-221-3p during odontogenesis, including in vitro tooth cultivation, renal capsule transplantation, in situ hybridization, real-time PCR, and immunohistochemistry. Two-day in vitro tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of miRNA-221-3p. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including β-catenin, Bmp2, Bmp4, Fgf4, Ptch1, and Shh, when inhibited with miRNA-221-3p and mimic. Also, the inhibition of miRNA-221-3p demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of Shh and Fgf4 within inner enamel epithelium (IEE) in E13 + 2 days in vitro cultivated teeth. Moreover, 1-week renal transplantation of in vitro cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and Shh expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of miRNA-221-3p did not affect tooth phenotype, while the loss of function resulted in long and slender teeth with short mesiodistal length. This study provides evidence that a suitable level of miRNA-221-3p is required for the modulation of major signaling pathways, including Wnt, Bmp, and Shh, during tooth morphogenesis.

For hard tissue formation, cellular mechanisms, involved in protein folding, processing, and secretion play important roles in the endoplasmic reticulum (ER). In pathological and regeneration conditions, ER stress hinders proper formation and secretion of proteins, and tissue regeneration by unfolded protein synthesis. 4-Phenylbutyric acid (4PBA) is a chemical chaperone that alleviates ER stress through modulation in proteins folding and protein trafficking. However, previous studies about 4PBA only focused on the metabolic diseases rather than on hard tissue formation and regeneration. Herein, we evaluated the function of 4PBA in dentin regeneration using an exposed pulp animal model system via a local delivery method as a drug repositioning strategy. Altered morphological changes and cellular physiology were examined with histology and immunohistochemistry. The 4PBA treatment modulated the inflammation reaction and resolved ER stress in the early stage of pulp exposure. In addition, 4PBA treatment activated blood vessel formation and TGF-𝛽1 expression in the dentin-pulp complex. Micro-computed tomography and histological examinations confirmed the facilitated formation of the dentin bridge in the 4PBA-treated specimens. These results suggest that proper modulation of ER stress would be an important factor for secretion and patterned formation in dentin regeneration.

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson&rsquo, s trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including &alpha, SMA, CK14, IL-1&beta, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

Circumvallate papilla (CVP) is a distinctively structured with dome-shaped apex, and the surrounding trench which contains over two hundred taste buds on the lateral walls. Although CVP was extensively studied to determine the regulatory mechanisms during organogenesis, it still remains to be elucidated the principle mechanisms of signaling regulations on morphogenesis including taste buds formation. The key role of Yes-associated protein (YAP) in the regulation of organ size and cell proliferation in vertebrates is well understood, but little is known about the role of this signaling pathway in CVP development. We aimed to determine the putative roles of YAP signaling in the epithelial patterning during CVP morphogenesis. To evaluate the precise localization patterns of YAP and other related signaling molecules, including β-catenin, Ki67, cytokeratins, and PGP9.5, in CVP tissue, histology and immunohistochemistry were employed at E16 and adult mice. Our results suggested that there are specific localization patterns of YAP and Wnt signaling molecules in developing and adult CVP. These concrete localization patterns would provide putative involvements of YAP and Wnt signaling for proper epithelial cell differentiation including the formation and maintenance of taste buds.

The endoplasmic reticulum (ER) is a site where protein folding and posttranslational modifications occur, but when unfolded or misfolded proteins accumulate in the ER lumen, an unfolded protein response (UPR) occurs. A UPR activates ER-stress signalling genes, including inositol-requiring enzyme-1 (Ire1), activating transcription factor 6 (Atf6), and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (Perk), to maintain homeostasis. The involvement of ER stress molecules in metabolic disease and hard tissue matrix formation has been established; however, an understanding of the role of ER-stress signalling molecules in tooth development is lacking. The aims of this study are to define the stage-specific expression patterns of ER stress-related molecules and to elucidate their putative functions in the organogenesis of teeth. This study leverages knowledge of the tissue morphology and expression patterns of a range of signalling molecules during tooth development. RT-qPCR, in situ hybridization, and immunohistochemistry analyses were performed to determine the stage-specific expression patterns of ER-stress-related signalling molecules at important stages of tooth development. RT-qPCR analyses showed that Atf6 and Perk have similar expression levels during all stages of tooth development; however, the expression levels of Ire1 and its downstream target X-box binding protein (Xbp1) increased significantly from the cap to the secretory stage of tooth development. In situ hybridization results revealed that Atf6 and Xbp1 were expressed in cells that form the enamel knot at cap stage and ameloblasts and odontoblasts at secretory stage in stage-specific patterns. In addition, Atf6, Ire1, and Xbp1 expression exhibited distinct localization patterns in secretory odontoblasts and ameloblasts of PN0 molars. Overall, our results strongly suggest that ER-stress molecules are involved in tooth development in response to protein overload that occurs during signaling modulations from enamel knots at cap stage and extracellular matrix secretion at secretory stage.