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Three dimensional (3D) printing materials are widely used in dental applications, but their biocompatibility and interactions with human cells require evaluation. This study aimed to identify materials meeting biocompatibility, mechanical strength, and tissue-forming requirements for safe dental applications. We assessed the cytotoxicity of resins and thermoplastic filaments in human HaCaT keratinocytes, gingival fibroblasts (hGFs), and stem cells from human exfoliated deciduous teeth (SHED) using PrestoBlue assays. Three resins, including two types of surgical guide resins, exhibited strong cytotoxicity after 4–72 h, while 2 h exposure to an FDA-approved surgical guide resin did not affect SHED cell viability. In contrast, six thermoplastic filaments showed no significant cytotoxicity even after 72 h. Among these, polycarbonate–acrylonitrile butadiene styrene (PC-ABS) demonstrated excellent toughness, heat resistance, and surface quality at a low cost. SHED cells cultured on PC-ABS dishes and micro bone structures showed strong proliferation and osteogenic potential. Culture inserts made of PC-ABS also supported the growth of HaCaT keratinocytes and the hGFs formed gingival tissue, which was superior to that formed on commercially available PET inserts. In conclusion, PC-ABS is a promising 3D printing material for dental applications due to its biocompatibility, ability to promote osteogenesis, and support for gingival tissue formation, with no observed cytotoxicity.

Abstract Background Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs. Methods We selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells). Results Our results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1α-dependent production and uniform distribution of collagen type I and its interactions with integrins α2β1 and α11β1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization. Conclusion The use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.

The aim of this study is to investigate the repressive effects of enzyme-digested edible bird’s nest (EBND) on the combination of arid environment and UV-induced intracellular oxidative stress, cell death, DNA double-strand breaks (DSBs) and inflammatory responses in human HaCaT keratinocytes and three-dimensional (3D) epithelium equivalents. An oxygen radical antioxidant capacity assay showed that EBND exhibited excellent peroxyl radical scavenging activity and significantly increased cellular antioxidant capacity in HaCaT cells. When EBND was administered to HaCaT cells and 3D epitheliums, it exhibited significant preventive effects on air-drying and UVA (Dry-UVA)-induced cell death and apoptosis. Dry-UVA markedly induced intracellular reactive oxygen species (ROS) generation in HaCaT cells and 3D epitheliums as quantified by CellROX® Green/Orange reagents. Once HaCaT cells and 3D epitheliums were pretreated with EBND, Dry-UVA-induced intracellular ROS were significantly reduced. The results from anti-γ-H2A.X antibody-based immunostaining showed that EBND significantly inhibited Dry-UVA-induced DSBs in HaCaT keratinocytes. Compared with sialic acid, EBND showed significantly better protection for both keratinocytes and 3D epitheliums against Dry-UVA-induced injuries. ELISA showed that EBND significantly suppressed UVB-induced IL-6 and TNF-α secretion. In conclusion, EBND could decrease arid environments and UV-induced harmful effects and inflammatory responses in human keratinocytes and 3D epithelium equivalents partially through its antioxidant capacity.

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.

As human-origin cells, human dental pulp stem cells (hDPSCs) are thought to be potentially useful for biological and medical experiments. They are easily obtained from lost primary teeth or extracted wisdom teeth, and they are mesenchymal stem cells that are known to differentiate into osteoblasts, chondrocytes, and adipocytes. Although hDPSCs originate from neural crest cells, it is difficult to induce hDPSCs to differentiate into neuron-like cells. To facilitate their differentiation into neuron-like cells, we evaluated various differentiation conditions. Activation of K+ channels is thought to regulate the intracellular Ca2+ concentration, allowing for manipulation of the cell cycle to induce the differentiation of hDPSCs. Therefore, in addition to a conventional neural cell differentiation protocol, we activated K+ channels in hDPSCs. Immunocyto­chemistry and real-time PCR revealed that applying a combination of 3 stimuli (high K+ solution, epigenetic reprogramming solution, and neural differentiation solution) to hDPSCs increased their expression of neuronal markers, such as β3-tubulin, postsynaptic density protein 95, and nestin within 5 days, which led to their rapid differentiation into neuron-like cells. Our findings indicate that epigenetic reprogramming along with cell cycle regulation by stimulation with high K+ accelerated the differentiation of hDPSCs into neuron-like cells. Therefore, hDPSCs can be used in various ways as neuron-like cells by manipulating their cell cycle.

Abstract Background Currently, ex-vivo handling of stem cells, including transport after harvest and therapeutic preparation, is generally done in culture media containing fetal bovine serum (FBS), which promotes cell attachment, proliferation, and differentiation. However, because of safety concerns associated with the use of FBS, including potential transmission of zoonotic agents and transplant rejection because of the incorporation of foreign proteins into the stem cells, there is a need for xenogeneic serum-free culture media for clinical handling of stem cells. Methods Dental pulp stem cells were derived from wisdom teeth donated by eight healthy volunteers and cultured in xenogeneic serum-free culture medium (XFM) or xenogeneic serum-containing culture medium (SCM). Cells were subjected to morphological, proliferation, karyotype, differentiation, marker expression, cryopreservation, and cytotoxic susceptibility analyses in vitro, as well as transplantation in vivo. Results In primary culture, XFM cells showed lower adhesion and slightly different morphology, although the single-cell size was similar to that of SCM cells. XFM cells exhibited typical mesenchymal stem cell (MSC) characteristics in vitro and in vivo, including marker gene/protein expression, trilineage differentiation potential, and hard, osteo-dentin tissue formation. Additionally, XFM cells maintained a normal karyotype in vitro and nontumorigenic potential in vivo; however, XFM cells were more susceptible to H2O2 and ultraviolet cytotoxic stimuli. XFM cells formed a multilayered structure showing excessive cell death/division in contrast to the monolayered structure of SCM cells when reaching overconfluence. Proliferation was disrupted in overconfluent XFM cells, and these cells could not be subcultured. Dimethyl sulfoxide-free cryopreserved XFM cells yielded similar results in all of the experiments. Conclusions This study is the first reporting successful isolation and expansion of an MSC population from donor-derived tissue (dental pulp) under xenogeneic serum-free culture conditions, as well as the application of cryopreservation, using a research strategy based on clinically oriented in-vitro and in-vivo experiments.

Ultraviolet-A (UVA) irradiation induces harmful effects on skin cells and accelerates skin aging through oxidative stress. In this study, the effects of a hydrogen-generating silica material named ULH-002 against UVA injuries in human cells and 3D skin equivalents were investigated. The oxygen radical absorption capacity (ORAC) assay showed that both freshly prepared ULH-002 solutions and 7-day-old solutions exhibited equal peroxyl radical (ROO·) scavenging activities concentration-dependently. CellROX® green/orange staining showed that ULH-002 could reduce UVA-induced oxidative stress in human keratinocytes HaCaT and human gingival fibroblasts (HGFs). ULH-002 significantly prevented UVA-induced apoptotic/necrotic cell death and cell-viability decline in HGFs and keratinocytes, as shown by Annexin V/PI apoptosis assay and PrestoBlue assay, respectively. Immunostaining showed that ULH-002 prevented the UVA-induced deterioration of expression of both type IV and I collagens in the 3D skin equivalents, and similarly in monolayer HGFs. UVA-enhanced melanogenesis was observed in human melanocytes HMV-II and HMV-II cell-containing 3D skin equivalents, but markedly prevented by ULH-002 as demonstrated by Fontana–Masson’s staining. In conclusion, our data suggested that ULH-002 could protect human keratinocytes and fibroblasts from UVA-induced injuries, prevent the loss of type IV and I collagens, as well as reduce melanogenesis. ULH-002 might be developed as a skin care reagent in the cosmetic industry.

Introduction: The diagnostic tools of nucleic acid amplification tests and antigen tests have been extensively employed for the detection of Coronavirus disease 2019 (COVID-19). Although the reverse-transcriptase polymerase chain reaction (RT)-PCR test has high sensitivity and specificity, it is a time-consuming and labor-intensive process. On the other hand, antigen tests are simple and prompt, however, their low sensitivity and potential for false positives have been identified as limitations. In light of these factors, the development of novel tests that combine speed and clinical dependability is a promising prospect., Methods: Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) excites chromophores by means of an enhanced electromagnetic field induced on a gold film surface. It enables the highly sensitive measurement of biomarkers in a short and simple 20-min window. In this study, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) SPFS-based antigen test targeting the SARS-CoV-2 nucleocapsid protein was performed and evaluated in 25 patients with COVID-19 and 10 non-infected controls., Results: A positive correlation was observed between antigen levels determined by SPFS and RNA levels determined via RT-PCR. The sensitivity values were 100 %, 92 %, and 62.5 %; and the specificity values were 100 %, 90 %, and 100 %; for nasopharyngeal swabs, nasal swabs, and saliva specimens when the cutoff values were set to 65.1, 0.2, and 1.5 pg/mL, respectively. No clinically problematic cross-reactivity with analogous coronaviruses was observed., Conclusions: The SARS-CoV-2 SPFS antigen test showed excellent clinical diagnostic accuracy for nasopharyngeal and nasal swabs, with a rapid turnaround., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Silicon carbide fibers have superior flexural properties and chemical stability compared to glass fibers. We investigated the flexural strength and modulus of an experimental, short silicon carbide fiber-reinforced resin. Short silicon carbide fibers with lengths of ~0.5, 1, 2, and 3 mm were prepared and silanized. Urethane dimethacrylate and triethylene glycol dimethacrylate were mixed at a 70:30 wt% ratio and used as the matrix resins. Each length of short silicon carbide fibers and the matrix resin were combined using a mixing machine and then used for specimen preparation. The three-point bending test conditions were in accordance with ISO 4049:2009. The fracture surfaces of the specimens after the three-point bending test were observed using secondary electron images. The data were statistically analyzed with a one-way analysis of variance and Tukey's HSD test (α = 0.05). The flexural strength and modulus of the specimens containing 2 mm or 3 mm silicon carbide fibers were significantly higher than the other specimens. The river pattern was observed more clearly in specimens containing shorter silicon carbide fibers, although this pattern was observed in all specimens.

Takotsubo cardiomyopathy (TC), an acute cardiac event is often associated with acute emotional stress, usually in the setting of cardiovascular risk factors. This case report attempts to review one of the triggers of TC beer potomania-induce hyponatremia with imaging findings that shows the link between severe hyponatremia and TC., Competing Interests: The authors declare that they have no competing interest to disclose., (© 2022 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.)

5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD ( R 2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng μL -1 . In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.

Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several cancers; therefore, global methylation levels have been considered as biomarkers for cancer diagnosis. Bisulfite conversion analysis has been widely used as the gold standard method for quantification of DNA methylation levels. However, this method requires cumbersome and time-consuming steps. To quantify global DNA methylation levels in homogeneous solutions, we exemplify a sensing system based on bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (MBD-FLuc) and unmethyl-CpG binding domain (CXXC)-fused firefly luciferase (CXXC-FLuc). MBD-FLuc and CXXC-FLuc bind to methylated and unmethylated CpGs, respectively, in the genomic DNA to excite BOBO-3, an intercalating dye on genomic DNA. These BOBO-3 emission intensities depend on the methylated and unmethylated CpG content. The global DNA methylation levels can be quantified from the BOBO-3 emission intensities. Moreover, we introduce a multicolor BRET assay using MBD-FLuc and CXXC-fused Oplophorus luciferase (CXXC-OLuc) for the simultaneous quantification of methylated and unmethylated CpG content in genomic DNA. CXXC-OLuc excites the BOBO-1 DNA-intercalating dye depending on the unmethylated CpG content. Thus, the emission intensities of BOBO-1 and BOBO-3 excited by CXXC-OLuc and MBD-FLuc, respectively, can be simultaneously measured, thereby enabling the determination of global DNA methylation level in a single step. Here, we describe the detailed protocols for the expression of MBD-FLuc, CXXC-FLuc, and CXXC-OLuc in Escherichia coli and determine the global DNA methylation levels using these BRET assays., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Zirconia has been used as a prosthesis material for over a decade because of its excellent mechanical properties and esthetics. The surface treatment for zirconia generally involves sandblasting and the application of primers for favorable bond strength between the surface and resin. However, sandblasting causes the microcracking and chipping of the zirconia surface. To overcome these challenges, the metallization of the zirconia surface was performed. Ti and Au were sputtered on yttria stabilized zirconia (YSZ) disks and heated to 800 °C for 15 min in air. These disks were bonded to stainless-steel rods using resin cement. Then, shear bond strength tests were performed using an Instron-type testing machine. The shear bond strength of the Ti sputtering group was significantly higher than that of the other groups. According to the results of X-ray photoelectron spectroscopy and electron probe microanalysis, the Ti-sputtered YSZ surface contained both sub-titanium oxide and titanium oxide before heating. Sub-titanium oxide was converted to titanium oxide by heating. These results suggest that metallization using Ti is effective for zirconia surface treatment to improve the shear bond strength between YSZ and resin cement. This metallization technique for YSZ has potential in clinical applications.

DNA methylation is one of the best studied epigenetic modifications. Alteration of the global DNA methylation level occurs in abnormal cells, such as those associated with cancers and Alzheimer's disease. Several assays are used to determine the global DNA methylation level, including the bisulfite-based assay, high-performance liquid chromatography (HPLC)-based assay, enzyme-linked immunosorbent assay (ELISA), and methyl acceptance assay. However, these assays require several cumbersome steps to detect methylation levels. We developed a simpler enzymatic assay for the quantification of the global DNA methylation level using the Ten-eleven translocation (TET) protein. TET proteins mediate DNA demethylation through the oxidation of 5-methylcytosine (5mC) in CpG in mammalian cells. Succinate is produced during this oxidation reaction, and the amount of succinate produced correlates to the global DNA methylation level. The catalytic domain of the TET2 was expressed in Escherichia coli (E. coli), and the purified TET2 catalytic domain was reacted with human genomic DNA. The reaction solution was used for enzymatic succinate quantification with no purification step. The results showed that the succinate produced through TET-mediated oxidation increased with increasing global DNA methylation levels in human genomic DNA, which was determined using the bisulfite method. These results show that the global DNA methylation level is quantifiable by measuring the amount of succinate produced by the TET2-mediated 5mC oxidation reaction. Graphical abstract.

We investigated the effect of helium atmospheric-pressure plasma (PL) and deep-ultraviolet (UV) light treatments on the adhesive properties of fiber-reinforced poly(ether-ether-ketone) polymer (PEEK). PEEK disks reinforced with carbon (CPEEK) or glass (GPEEK) fibers were polished, modified with PL and UV for 60 s, and the surface energy was calculated by measuring the contact angles. The disk surfaces were analyzed by X-ray photoemission spectroscopy. Shear bond strength testing was performed using a universal testing machine, and the fracture surfaces were observed by electron probe microanalyzer. Data were analyzed with one and two-way ANOVA and Tukey's post-hoc test ( p < 0.05). The surface energies were increased by the modifications, which created OH functional groups on the surfaces. The bond strengths of CPEEK were increased by PL, and those of GPEEK were increased by PL and UV, owing to chemical bonding at the interface.

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases regulates multiple biological processes, such as cell proliferation, migration, apoptosis, and differentiation. Various genetic alterations that drive activation of the receptors and the pathway are associated with tumor growth and survival; therefore, the FGFR family represents an attractive therapeutic target for treating cancer. Here, we report the discovery and the pharmacological profiles of 8 (CH5183284/Debio 1347), an orally available and selective inhibitor of FGFR1, FGFR2, and FGFR3. The chemical modifications, which were guided by 3D-modeling analyses of the inhibitor and FGFRs, led to identifying an inhibitor that is selective to FGFR1, FGFR2, and FGFR3. In in vitro studies and xenograft models in mice, 8 shows antitumor activity against cancer cell lines that harbor genetically altered FGFRs. These results support the potential therapeutic use of 8 as a new anticancer agent.

The 2013 American College of Cardiology/American Heart Association guidelines recommend statins for adults age ≤75 years who have clinical atherosclerotic cardiovascular disease (IA) and adults age 40 to 75 years with diabetes mellitus and LDL-C 70-189 mg/dl (IA). Our aim was to estimate the prevalence and likelihood of statin use among selected statin benefit groups. Using data from the National Health and Nutrition Examination Survey (NHANES) 2011-2012, we examined 5319 adults age ≥20 years. We estimated weighted frequencies and prevalence of statin use for adults with diabetes mellitus and dyslipidemia (or low-density lipoprotein cholesterol ≥70 mg/dL), defined as statin benefit group 1 (SBG1); and for adults with atherosclerotic cardiovascular disease, defined as statin benefit group 2 (SBG2). We constructed a logistic regression model to estimate odds of statin use in SBG1. Overall, an estimated 38.6 million Americans are on a statin. In adjusted models, uninsured and Hispanic adults were less likely to be on a statin compared with white adults; 59.5% (95% confidence interval [CI]: 53.0-66.1) of all adults in SBG1, 58.8% (95% CI: 51.5-66.1) of adults age 40 to 75 in SBG1, and 63.5% (95% CI: 55.6-71.4) of all adults in SBG2 were on a statin. Although the prevalence of statin use has increased over time, Hispanic ethnicity and lack of insurance remain barriers to statin use. Black-white racial disparities were not significant. Our study provides a baseline estimate of statin use in the noninstitutionalized population just prior to introduction of the new guidelines and provides a reference for evaluating the impact of the new guidelines on statin utilization., (© 2016 Wiley Periodicals, Inc.)

Introduction: Facing the rapid scale-up of antiretroviral treatment (ART) programs in resource-limited settings, monitoring of treatment outcome is essential in order to timely detect and tackle drawbacks [1]., Methods: In a prospective cohort study, 300 consecutive patients starting first-line ART were enrolled between 2009 and2010 in a large HIV treatment centre in rural Cameroon. Patients were followed up for 12 months. Virologic failure was defined as a VL >1000 cop/mL at month 12. Besides CD4 and viral load (VL) analysis, HIV-1 drug resistance testing was performed in patients with VL>1000 copies (c)/mL plasma. In those patients and controls, minority HIV-1 drug resistance mutations at baseline, and plasma drug levels were analyzed in order to identify the risk factors for virologic failure., Results: Most enrolled patients (71%) were female. At baseline median CD4 cell count was 162/µL (IQR 59-259), median log10 VL was 5.4 (IQR 5.0-5.8) c/mL, and one-third of patients had World Health Organisation (WHO) stage 3 or 4; 30 patients died during follow-up. Among all patients who completed follow-up 38/238 had virologic failure. These patients were younger, had lower CD4 cell counts and more often had WHO stage 3 or 4 at baseline compared to patients with VL<1000c/mL. Sixty-three percent of failing patients (24/38) had at least one mutation associated with high-level drug resistance. The M184V mutation was the most frequently detected nucleoside reverse transcriptase inhibitor (NRTI) mutation (n=18) followed by TAMs (n=5) and multi-NRTI resistance mutations (n=4). The most commonly observed non-nucleoside reverse-transcriptase inhibitor (NNRTI) resistance mutations were K103N (n=10), Y181C (n=7), and G190A (n=6). Drug resistance mutations at baseline were detected in 12/65 (18%) patients, in 6 patients with and 6 patients without virological failure (p=0.77). Subtherapeutic NNRTI levels (OR 6.67, 95% CI 1.98-22.43, p<0.002) and poorer adherence (OR 1.54, 95% CI 1.00-2.39, p=0.05) were each associated with higher risk of virologic failure in the matched pair analysis. Unavailability of ART at the treatment centre was the single most common cause (37%) for incomplete adherence in these patients., Conclusions: Virologic failure after one year of first-line ART in rural Cameroon was not associated with transmitted drug resistance, but with reduced drug plasma levels and incomplete adherence. Strategies to assure adherence and uninterrupted drug supply are important factors for therapy success.