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Calcium phosphate-based materials (CaP) are introduced as potential dental pulp capping materials for deciduous teeth. The present study investigated the influence of inorganic phosphate (P i ) on regulating stem cells isolated from human exfoliated deciduous teeth (SHED). SHEDs were treated with P i . Cell cycle progression and apoptosis were examined using flow cytometry analysis. Osteo/odontogenic and adipogenic differentiation were analyzed using alizarin red S and oil red O staining, respectively. The mRNA expression profile was investigated using a high-throughput RNA sequencing technique. P i increased the late apoptotic cell population while cell cycle progression was not altered. P i upregulated osteo/odontoblastic gene expression and enhanced calcium deposition. P i -induced mineralization was reversed by pretreatment of cells with Foscarnet, or p38 inhibitor. P i treatment inhibited adipogenic differentiation as determined by decreased PPARγ expression and reduced intracellular lipid accumulation. Bioinformatic analysis of gene expression profiles demonstrated several involved pathways, including PI3K/AKT, MAPK, EGFR, and VEGF signaling. In conclusion, P i enhanced osteo/odontogenic but inhibited adipogenic differentiation in SHED., (© 2024. The Author(s).)

Nerve-derived factors have attracted attention in bone regeneration therapy due to their ability to promote bone regeneration and nerve innervation. Mesenchymal stem cells transported to target sites promote osteogenesis. However, there are few reports on the effects of neural stem cells on bone regeneration. Therefore, the aim of this study was to investigate the role of neural stem cells in osteogenesis. Here, embryoid bodies (EB) or primary neurospheres (1NS) were generated using mouse induced pluripotent stem cells (iPS cells), which were then seeded onto gelatin (Gel) sponges. The seeded Gel sponges were then transplanted into mouse calvarial bone defects. We noted that 1NS-seeded Gel promoted bone regeneration and the presence of tartrate-resistant acid phosphatase (TRAP)-positive cells, whereas the EB-seeded Gel did not. RNA-sequencing of the 1NS-seeded and EB seeded Gels showed an upregulation of the transforming growth factor (TGF)-β signaling pathway in the 1NS-seeded Gel group. Immunostaining confirmed the presence of Id3 positive cells in mice with bone defects treated with the 1NS-seeded Gel. These findings suggest that the transplantation of neural stem cells may contribute to the promotion of bone regeneration. STATEMENT OF SIGNIFICANCE: This study aimed to investigate whether neural stem cells, when seeded in Gel sponges, promoted bone regeneration. It has been well documented that bone is tightly linked with the nervous systems. Bioscaffolds comprising factors that promote innervation and bone regeneration have been investigated for use in bone therapy. However, there is limited research on the use of neural stem cells for promoting bone formation. To assess this relationship, we conducted both in vivo and in vitro assays to determine whether neural stem cells promoted bone formation. We noted that 1NS-seeded Gel sponges promoted bone formation significantly in mice with calvarial defects after 4 weeks. This study provides a novel approach of neural stem cells for bone therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: There have been only a few cases showing the efficacy of pembrolizumab on granulocyte-colony - stimulating factor (G-CSF)-producing non-small-cell lung cancer (NSCLC) with high programmed cell death ligand 1 (PD-L1) expression. Herein, we report the first case showing the efficacy of pembrolizumab for G-CSF-producing NSCLC with high PD-L1 expression, although the patient had factors indicative of poor pembrolizumab efficacy, such as poor performance status (PS) due to the tumor-induced inflammation and corticosteroids administration., Case Presentation: A 77-year-old woman was diagnosed with G-CSF-producing NSCLC-not otherwise specified, classified as clinical stage IVB, T2N3M1c. She had fever and her PS was 3, and her C-reactive protein (CRP) was 6.47 mg/dL due to inflammation by a G-CSF-producing tumor. Thus, we initiated the administration of dexamethasone (3.3 mg/day). Her fever abated the next day, and CRP dropped to 3.22 mg/dL after 4 days. Driver mutations were negative, and PD-L1, tumor proportion score, was highly expressed at 100%. Thus, pembrolizumab was started. Subsequently, the white blood cell count decreased, and the tumor shrank, indicating a partial response. After three cycles of pembrolizumab therapy, the anorexia improved, and she was discharged. The patient developed sclerosing cholangitis after discharge. Therefore, the pembrolizumab treatment was discontinued. The primary lesion was enlarged, indicating progressive disease. However, the patient and her family did not want additional treatment. Finally, her progression-free survival and overall survival were 6 and 7 months, respectively., Conclusion: Pembrolizumab may be effective against G-CSF-producing NSCLC with high PD-L1 expression. Corticosteroids seemed to inhibit inflammation induced by the tumor, and exert the efficacy of pembrolizumab., Competing Interests: Takeshi Masuda reports personal fee from Daiichi-Sankyo Co., Ltd., Taiho Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., Kyowa Kirin Co., Ltd., Eli Lilly Japan K.K., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., and AstraZeneca K.K. outside the submitted work. Kakuhiro Yamaguchi reports honoraria from Ono Pharmaceutical Co., Ltd., outside the submitted work. Shinjiro Sakamoto reports honoraria from AstraZeneca K.K., Nippon Boehringer Ingelheim Co., Ltd., Chugai Pharmaceutical Co., Ltd., Kyowa Kirin Co., Ltd., and Novartis Pharma K.K. outside the submitted work. Yasushi Horimasu reports honoraria from Nippon Boehringer Ingelheim Co., Ltd., outside the submitted work. Taku Nakashima reports honoraria from AstraZeneca K.K. and Chugai Pharmaceutical Co., Ltd., outside the submitted work. Noboru Hattori reports honoraria from AstraZeneca K.K., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., and Ono Pharmaceutical Co., Ltd., outside the submitted work. No other disclosures are reported., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Introduction: Type 2 diabetes (T2D) is the predominant form of diabetes mellitus and is among the leading causes of death with an increasing prevalence worldwide. However, the pathological mechanism underlying T2D remains complex and unclear. An increasing number of studies have suggested an association between circadian clock disruption and high T2D prevalence., Method: This review explores the physiological and genetic evidence underlying T2D symptoms associated with circadian clock disturbances, including insulin secretion and glucose metabolism., Results and Discussion: Notably, circadian clock disruption reduces insulin secretion and insulin sensitivity and negatively affects glucose homeostasis. The circadian clock regulates the hypothalamic-pituitary-adrenal axis, an important factor that regulates glucose metabolism and influences T2D progression. Therefore, circadian clock regulation is an attractive, novel therapeutic approach for T2D, and various circadian clock stabilizers play therapeutic roles in T2D. Lastly, this review suggests novel therapeutic and preventive approaches using circadian clock regulators for T2D., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tran, Kondo, Ashry, Fu, Okawa, Sawangmake and Egusa.)

Background: Appropriate load distribution among the supporting elements is essential for the long-term success of implant-assisted removable partial dentures; however, there is little information available on load distribution., Purpose: This study aimed to evaluate the effect of implant location on load distribution in implant-assisted removable partial dentures by reviewing in vitro models and finite-element analysis studies., Materials and Methods: English-language studies which examined the load distribution of implant-assisted removable partial dentures and were published between January 2001 and October 2022 were extracted from PubMed, ScienceDirect, and Scopus online databases, and manual searching. Two reviewers selected the articles based on the predetermined inclusion and exclusion criteria, followed by data extraction and analysis., Results: Forty-seven studies were selected after evaluating the titles and abstracts of 264 articles; two were identified manually. After screening the text, 12 studies were included: six in vitro model experiments and six finite-element analysis studies. All included studies used a mandibular free-end missing model (Kennedy Class I or II). The influence of implant location on load distribution to the abutment tooth, implant, and mucosa under the denture base was summarized in three cases: implant at the premolar, first molar, and second molar region. Due to differences in the measurement method of load distribution and loading condition to the denture, the results differed among the studies., Conclusions: The implant location in implant-assisted removable partial dentures can affect load distribution to the supporting elements, such as the abutment tooth, implant, and mucosa under the denture base., (© 2023. Deutsche Gesellschaft für Implantologie im Zahn‐, Mund‐ und Kieferbereich e.V., Japanese Society of Oral Implantology.)

Objective: Adenosine triphosphate (ATP) is an essential nucleotide that is normally present in both intracellular and extracellular compartments. Extracellular ATP (eATP) has a pivotal role in both physiological and pathological processes of periodontal ligament tissues. Here, this review aimed to explore the various functions of eATP that are involved in the control of behaviours and functions of periodontal ligament cells., Methods: To identify the included publications for review, the articles were searched in PubMed (MEDLINE) and SCOPUS with the keywords of adenosine triphosphate and periodontal ligament cells. Thirteen publications were used as the main publications for discussion in the present review., Results: eATP has been implicated as a potent stimulator for inflammation initiation in periodontal tissues. It also plays a role in proliferation, differentiation, remodelling, and immunosuppressive functions of periodontal ligament cells. Yet, eATP has diverse functions in regulating periodontal tissue homeostasis and regeneration., Conclusion: eATP may provide a new prospect for periodontal tissue healing as well as treatment of periodontal disease especially periodontitis. It may be utilized as a useful therapeutic tool for future periodontal regeneration therapy., (© 2023. The Author(s).)

Zirconia restorations are becoming increasingly common. However, zirconia reduces the polymerization of dual-cured resin cement owing to light attenuation, resulting in residual resin monomers. This study investigated the effects of dual-cured resin cement, with incomplete polymerization owing to attenuated light through zirconia, on the inflammatory response in vitro. The dual-cured resin cement (SA Luting Multi, Kuraray) was light-irradiated through zirconia with three thickness diameters (1.0, 1.5, and 2.0 mm). The light transmittance and the degree of conversion (DC) of the resin cement significantly decreased with increasing zirconia thickness. The dual-cured resin cement in 1.5 mm and 2.0 mm zirconia and no-irradiation groups showed significantly higher amounts of hydroxyethylmethacrylate and triethyleneglycol dimethacrylate elution and upregulated gene expression of proinflammatory cytokines IL-1β and IL-6 from human gingival fibroblasts (hGFs) and TNFα from human monocytic cells, compared with that of the 0 mm group. Dual-cured resin cement with lower DC enhanced intracellular reactive oxygen species (ROS) levels and activated mitogen-activated protein (MAP) kinases in hGFs and monocytic cells. This study suggests that dual-cured resin cement with incomplete polymerization induces inflammatory responses in hGFs and monocytic cells by intracellular ROS generation and MAP kinase activation.

A micro-physiological system is generally fabricated using soft materials, such as polydimethylsiloxane silicone (PDMS), and seeks an inflammatory osteolysis model for osteoimmunological research as one of the development needs. Microenvironmental stiffness regulates various cellular functions via mechanotransduction. Controlling culture substrate stiffness may help spatially coordinate the supply of osteoclastogenesis-inducing factors from immortalized cell lines, such as mouse fibrosarcoma L929 cells, within the system. Herein, we aimed to determine the effects of substrate stiffness on the osteoclastogenesis-inducing potential of L929 cells via cellular mechanotransduction. L929 cells showed increased expression of osteoclastogenesis-inducing factors when cultured on type I collagen-coated PDMS substrates with soft stiffness, approximating that of soft tissue sarcomas, regardless of the addition of lipopolysaccharide to augment proinflammatory reactions. Supernatants of L929 cells cultured on soft PDMS substrates promoted osteoclast differentiation of the mouse osteoclast precursor RAW 264.7 by stimulating the expression of osteoclastogenesis-related gene markers and tartrate-resistant acid phosphatase activity. The soft PDMS substrate inhibited the nuclear translocation of YES-associated proteins in L929 cells without reducing cell attachment. However, the hard PDMS substrate hardly affected the cellular response of the L929 cells. Our results showed that PDMS substrate stiffness tuned the osteoclastogenesis-inducing potential of L929 cells via cellular mechanotransduction.

Background: Needlestick and sharps injuries (NSIs) are serious problems for dental health care workers (DHCWs) because they are at risk for occupational blood-borne infections. In this study, risk factors for NSIs in DHCWs at Tohoku University Hospital (TUH) in Japan over 19 years were analysed., Methods: NSI data of DHCWs at TUH from April 2002 to March 2020 were collected from the Exposure Prevention Information Network (EPINet) and statistically analysed., Results: A total of 195 NSIs occurred during the 19-year study period. Approximately 58.5% of NSIs occurred in DHCWs with less than 5 years of experience. Injection needles were the most frequent cause of NSIs (19.0%) followed by suture needles (13.3%) and ultrasonic scaler chips (12.8%). Needle injuries occurred mainly on the left hand, whereas ultrasonic scaler chip and bur injuries occurred on the right hand and other body parts whilst DHCWs were placing the instruments back on the dental unit hanging holder without removing the sharps. NSIs from other instruments primarily occurred on both hands and foot insteps during cleanup. No case of occupational blood-borne infection caused by NSIs was observed during the study period at TUH., Conclusions: NSIs occurred in DHCWs with less experience, and there were associations between the instruments, timing of use, and NSI site. EPINet was considered a valuable tool for monitoring NSIs in order to develop future strategies for minimising NSIs., Competing Interests: Conflict of interest None disclosed., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Soft gingiva is often compromised in gingival health; however, the underlying biological mechanisms remain unknown. Extracellular matrix (ECM) stiffness is involved in the progression of various fibroblast-related inflammatory disorders via cellular mechanotransduction. Gingival stiffness might regulate cellular mechanotransduction-mediated proinflammatory responses in gingival fibroblasts. This in vitro study aims to investigate the effects of substrate stiffness on proinflammatory responses in human gingival fibroblasts (hGFs). The hGFs isolated from two healthy donors cultured on type I collagen-coated polydimethylsiloxane substrates with different stiffnesses, representing soft (5 kPa) or hard (25 kPa) gingiva. Expression levels of proinflammatory mediators, prostaglandin E2 or interleukin-1β, in hGFs were significantly higher with the soft substrate than with the hard substrate, even without and with lipopolysaccharide (LPS) to induce inflammation. Expression levels of gingival ECM and collagen cross-linking agents in hGFs were downregulated more with the soft substrate than with the hard substrate through 14 days of culture. The soft substrate suppressed the expression of mechanotransduction-related transcriptional factors and activated the expression of inflammation-related factors, whereas the hard substrate demonstrated the opposite effects. Soft substrate induced proinflammatory responses and inhibition of ECM synthesis in hGFs by inactivating cellular mechanotransduction. This supports the importance of ECM stiffness in gingival health., (© 2023. The Author(s).)

Background/purpose: Mechanical force differentially regulates periodontal ligament functions depending on types, magnitudes, and duration of stimulation. Intermittent compressive force (ICF) promotes an in vitro mineralization in human periodontal ligament cells. The present study investigated the effect of ICF on dentin matrix protein-1 (DMP1) expression in human periodontal ligament stem cells (hPDLSCs)., Materials and Methods: Cells were treated with ICF in a serum-free culture medium for 24 h The mRNA and protein expression were examined using real-time polymerase chain reaction, immunofluorescence staining and Western blot analysis, respectively., Results: The exposure to ICF in a serum-free condition significantly induced DMP1 expression in both mRNA and protein levels. The effect of ICF-induced DMP1 expression was inhibited by pretreatment with cycloheximide, indicating the requirement of the intermediated molecule(s). Pretreatment with transforming growth factor β (TGF-β) receptor inhibitor (SB431542) or neutralized antibody against TGF-β1 prior to ICF application abolished the effect of ICF-induced DMP1 expression. Further, recombinant TGF-β1 treatment stimulated DMP1 expression., Conclusion: The present study illustrated that ICF induces DMP1 expression in hPDLSCs via the regulation of TGF-β signaling pathway., (© 2022 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

Background: For biomechanical consideration of dental implants, an understanding of the three-dimensional (3D) load exerted on the implant is essential, but little information is available on the in vivo load, including the measuring devices., Purpose: This review aimed to evaluate studies that used specific load-measuring devices that could be mounted on an implant to measure the functional load in vivo., Materials and Methods: An electronic search utilizing the internet research databases PubMed, Google Scholar, and Scopus was performed. The articles were chosen by two authors based on the inclusion and exclusion criteria., Results: In all, 132 studies were selected from the database search, and 16 were selected from a manual search. Twenty-three studies were finally included in this review after a complete full-text evaluation. Eleven studies were related to the force measurements using the strain gauges, and 12 were related to the piezoelectric force transducer. The principles of the two types of devices were completely different, but the devices produced comparable outcomes. The dynamics of the load magnitude and direction on the implant during function were clarified, although the number of participants in each study was small., Conclusions: The load exerted on the implant during function was precisely measured in vivo using specific measuring devices, such as strain gauges or piezoelectric force transducers. The in vivo load data enable us to determine the actual biomechanical status in more detail, which might be useful for optimization of the implant prosthetic design and development of related materials. Due to the limited data and difficulty of in vivo measurements, the development of a new, simpler force measurement device and method might be necessary., (© 2022. The Author(s).)

The self-organizing potential of induced pluripotent stem cells (iPSCs) represents a promising tool for bone tissue engineering. Shear stress promotes the osteogenic differentiation of mesenchymal stem cells, leading us to hypothesize that specific shear stress could enhance the osteogenic differentiation of iPSCs. For osteogenesis, embryoid bodies were formed for two days and then maintained in medium supplemented with retinoic acid for three days, followed by adherent culture in osteogenic induction medium for one day. The cells were then subjected to shear loading (0.15, 0.5, or 1.5 Pa) for two days. Among different magnitudes tested, 0.5 Pa induced the highest levels of osteogenic gene expression and greatest mineral deposition, corresponding to upregulated connexin 43 (Cx43) and phosphorylated Erk1/2 expression. Erk1/2 inhibition during shear loading resulted in decreased osteogenic gene expression and the suppression of mineral deposition. These results suggest that shear stress (0.5 Pa) enhances the osteogenic differentiation of iPSCs, partly through Cx43 and Erk1/2 signaling. Our findings shed light on the application of shear-stress technology to improve iPSC-based tissue-engineered bone for regenerative bone therapy., (© 2022. The Author(s).)

Osteocyte network architecture is closely associated with bone turnover. The cellular mechanosensing system regulates osteocyte dendrite formation by enhancing focal adhesion. Therefore, titanium surface nanotopography might affect osteocyte network architecture and improve the peri-implant bone tissue quality, leading to strengthened osseointegration of bone-anchored implants. We aimed to investigate the effects of titanium nanosurfaces on the development of osteocyte lacunar-canalicular networks and osseointegration of dental implants. Alkaline etching created titanium nanosurfaces with anisotropically patterned dense nanospikes, superhydrophilicity, and hydroxyl groups. MLO-Y4 mouse osteocyte-like cells cultured on titanium nanosurfaces developed neuron-like dendrites with increased focal adhesion assembly and gap junctions. Maturation was promoted in osteocytes cultured on titanium nanosurfaces compared to cells cultured on machined or acid-etched micro-roughened titanium surfaces. Osteocytes cultured in type I three-dimensional collagen gels for seven days on nano-roughened titanium surfaces displayed well-developed interconnectivity with highly developed dendrites and gap junctions compared to the poor interconnectivity observed on the other titanium surfaces. Even if superhydrophilicity and hydroxyl groups were maintained, the loss of anisotropy-patterned nanospikes reduced expression of gap junction in osteocytes cultured on alkaline-etched titanium nanosurfaces. Four weeks after placing the titanium nanosurface implants in the upper jawbone of wild-type rats, osteocytes with numerous dendrites were found directly attached to the implant surface, forming well-developed lacunar-canalicular networks around the nano-roughened titanium implants. The osseointegration strength of the nano-roughened titanium implants was significantly higher than that of the micro-roughened titanium implants. These data indicate that titanium nanosurfaces promote osteocyte lacunar-canalicular network development via nanotopographical cues and strengthen osseointegration. STATEMENT OF SIGNIFICANCE: The clinical stability of bone-anchoring implant devices is influenced by the bone quality. The osteocyte network potentially affects bone quality and is established by the three-dimensional (3D) connection of neuron-like dendrites of well-matured osteocytes within the bone matrix. No biomaterials are known to regulate formation of the osteocyte network. The present study provides the first demonstration that titanium nanosurfaces with nanospikes created by alkali-etching treatment enhance the 3D formation of osteocyte networks by promoting osteocyte dendrite formation and maturation by nanotopographic cues, leading to strengthened osseointegration of titanium implants. Osteocytes attached to the titanium nanosurfaces via numerous cellular projections. The success of osteocyte regulation by nanotechnology paves the way for development of epoch-making technologies to control bone quality., Competing Interests: Declaration of Competing Interest All authors have no conflict of interest in this research. Those authors have no conflict of interest., (Copyright © 2022. Published by Elsevier Ltd.)

The exact profiles of the clinical symptoms related to the SARS-CoV-2 Omicron variant (B.1.1.529) remain largely uncertain. Therefore, this study aimed to clarify the clinical manifestations of infection with this variant. We enrolled individuals who were tested by quantitative nasopharyngeal swab reverse transcription-polymerase chain reaction (RT-PCR) test at a large screening center in a city of Japan during the B.1.1.529 Omicron variant wave between January and May 2022, after contact with COVID-19 patients. Swab tests were planned to be performed approximately 4-5 days after contact. The presence of COVID-19-related symptoms was assessed at the swab test site. Among the 2,507 enrolled individuals, 943 (37.6%) were RT-PCR test-positive and 1,564 (62.4%) were test-negative. Among the 943 PCR test-positive participants, the prevalence of the symptoms was as follows: 47.3% with cough, 32.9% with sore throat, 18.4% with fatigability, 12.7% with fever of ≥ 37.5℃, 9.9% with dyspnea, 2.1% with dysosmia, and 1.4% with dysgeusia. The prevalence of cough, sore throat, dyspnea, and fatigability was higher among adults aged ≥ 18 years than among children and adolescents. The prevalence of dysosmia and dysgeusia remarkably decreased during the Omicron wave (1-3%) compared to during the pre-Omicron variant waves (15-25%). In summary, common COVID-19-related symptoms during the Omicron variant wave included cough and sore throat, followed by fatigability, fever, and dyspnea. The prevalence of most of these symptoms was higher in adults than in non-adults. The prevalence of dysosmia and dysgeusia remarkably decreased with the Omicron variant than with pre-Omicron variants.

Bone tissue engineering has been developed using a combination of mesenchymal stem cells (MSCs) and calcium phosphate-based scaffolds. However, these complexes cannot regenerate large jawbone defects. To overcome this limitation of MSCs and ceramic scaffolds, a novel bone regeneration technology must be developed using cells possessing high bone forming ability and a scaffold that provides space for vertical bone augmentation. To approach this problem in our study, we developed alveolar bone-derived immature osteoblast-like cells (HAOBs), which have the bone regenerative capacity to correct a large bone defect when used as a grafting material in combination with polylactic acid fibers that organize the 3D structure and increase the strength of the scaffold material (3DPL). HAOB-3DPL constructs could not regenerate bone via xenogeneic transplantation in a micromini pig alveolar bone defect model. However, the autogenic transplantation of mouse calvaria-derived immature osteoblast-like cells (MCOBs) isolated using the identical protocol for HAOBs and mixed with 3DPL scaffolds successfully regenerated the bone in a large jawbone defect mouse model, compared to the 3DPL scaffold alone. Nanoindentation analysis indicated that the regenerated bone had a similar micromechanical strength to native bone. In addition, this MCOB-3DPL regenerated bone possesses osseointegration ability wherein a direct structural connection is established with the titanium implant surface. Hence, a complex formed between a 3DPL scaffold and immature osteoblast-like cells such as MCOBs represents a novel bone tissue engineering approach that enables the formation of vertical bone with the micromechanical properties required to treat large bone defects., (© The Author(s) 2022. Published by Oxford University Press on behalf of the National Academy of Sciences.)

The administration of a third booster dose of messenger ribonucleic acid (mRNA) vaccines against coronavirus disease 2019 (COVID-19) has progressed worldwide. Since January 2022, Japan has faced a nationwide outbreak caused by the Omicron variant, which occurred simultaneously with the progression of mass vaccination with the third booster dose. Therefore, this study evaluated the effectiveness of the third dose of vaccine by reverse transcription-polymerase chain reaction (RT-PCR) test using nasopharyngeal swab samples from adults aged ≥ 18 years tested after having close contact with COVID-19 cases between January and May 2022. Participants who completed only one dose were excluded from the study. Among the 928 enrolled participants, 139 had never been vaccinated, 609 had completed two doses, 180 had completed three doses before the swab test, and the overall RT-PCR test positivity rate in each group was 48.9%, 46.0%, and 32.2%, respectively. The vaccine effectiveness of the third dose to prevent infection after close contact was approximately 40% (95% confidence interval: 20-60%), which was the highest at 10-70 days after receiving the third dose. In conclusion, the effectiveness of the three-dose mRNA COVID-19 vaccine after close contact during the Omicron outbreak is approximately 40%., (© 2022. The Author(s).)

Induced pluripotent stem cells (iPSCs) offer an unlimited source for cartilage regeneration as they can generate a wide spectrum of cell types. Here, we established a tetracycline (tet) controlled bone morphogenetic protein-4 ( BMP-4 ) expressing iPSC (iPSC- Tet/BMP-4 ) line in which transcriptional activation of BMP-4 was associated with enhanced chondrogenesis. Moreover, we developed an efficient and simple approach for directly guiding iPSC- Tet/BMP-4 differentiation into chondrocytes in scaffold-free cartilaginous pellets using a combination of transcriptional activation of BMP-4 and a 3D shaking suspension culture system. In chondrogenic induction medium, shaking culture alone significantly upregulated the chondrogenic markers Sox9, Col2a1 , and Aggrecan in iPSCs- Tet/BMP-4 by day 21. Of note, transcriptional activation of BMP-4 by addition of tet (doxycycline) greatly enhanced the expression of these genes. The cartilaginous pellets derived from iPSCs- Tet/BMP-4 showed an oval morphology and white smooth appearance by day 21. After day 21, the cells presented a typical round morphology and the extracellular matrix was stained intensively with Safranin O, alcian blue, and type II collagen. In addition, the homogenous cartilaginous pellets derived from iPSCs- Tet/BMP-4 with 28 days of induction repaired joint osteochondral defects in immunosuppressed rats and integrated well with the adjacent host cartilage. The regenerated cartilage expressed the neomycin resistance gene, indicating that the newly formed cartilage was generated by the transplanted iPSCs- Tet/BMP-4 . Thus, our culture system could be a useful tool for further investigation of the mechanism of BMP-4 in regulating iPSC differentiation toward the chondrogenic lineage, and should facilitate research in cartilage development, repair, and osteoarthritis., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Macrophage phagocytosis is an important research target to combat various inflammatory or autoimmune diseases; however, the phenomenon has never been controlled by artificial means. Titania nanospikes created by alkaline etching treatment can tune macrophage polarization toward a M1-like type and might regulate macrophage phagocytosis. This in vitro study aimed to determine whether the two-dimensional titania nanosurfaces created by alkaline etching treatment activated the macrophage phagocytosis by nanospike-mediated contact stimulation. On two-dimensional pure titanium sheets, alkaline etching treatments with different protocols created superhydrophilic nanosurfaces with hydroxyl function groups and moderate or dense nanospikes. Both types of titania nanosurfaces promoted the phagocytic activity of the mouse macrophage-like cell line, J774A.1, through upregulation of M1 polarization markers and phagocytosis-related receptors, such as toll-like receptors (TLR2 and 4). In contrast, the hydrophobic smooth or micro-roughened titanium surfaces did not activate macrophage phagocytosis or the expression of related receptors. These phenomena remained unchanged even under the antibody blockade of macrophage TLR2 but were either suppressed or augmented for each surface excited by ultraviolet irradiation. Titania nanospikes induced paxillin expression and provided physical stimuli to macrophages, the extent of which was positively correlated with TLR expression levels. Ligand stimulation with lipopolysaccharide did not upregulate macrophage TLR expression but further enhanced M1 marker expression by titania nanosurfaces. These results showed that the two-dimensional titania nanosurfaces activated macrophage phagocytosis by enhancing expression of phagocytosis-related receptors through nanospike-mediated contact stimulation, in assistance with physical surface properties, in a ligand-independent manner., (© 2022. The Author(s).)

The transcriptional regulation of induced pluripotent stem cells (iPSCs) holds promise for their directed differentiation into ameloblasts, which are usually lost after tooth eruption. Ameloblast differentiation is regulated by multiple signaling molecules, including bone morphogenetic proteins (BMPs). Epiprofin (Epfn), a transcription factor, is expressed in the dental epithelium, and epithelial Epfn overexpression results in ectopic ameloblast differentiation and enamel formation in mouse incisor, a striking phenotype resembling that of mice with deletion of follistatin (a BMP inhibitor). However, it remains unknown whether and how Epfn transcriptional activation promotes ameloblast induction from mouse iPSCs. Here, we generated doxycycline-inducible Epfn -expressing mouse iPSCs (Epfn-iPSCs). Ameloblasts, which are characterized by positive staining for keratin 14 and amelogenin and alizarin red S staining, were successfully derived from Epfn-iPSCs based on a stage-specific induction protocol, which involved the induction of the surface ectoderm, dental epithelial cells, and ameloblasts at stages 1, 2, and 3, respectively. Epfn activation by doxycycline at stages 2 and/or 3 decreased cell proliferation and promoted ameloblast differentiation, along with the upregulation of p-Smad1/5/8, a key regulator of the BMP-Smad signaling pathway. Gene analysis of the BMP-Smad signaling pathway-associated molecules revealed that Epfn activation decreased follistatin expression at stage 2, but increased BMP2/4/7 expression at stage 3. Perturbations in the ameloblast differentiation process were observed when the BMP-Smad signaling pathway was inhibited by a BMP receptor inhibitor (LDN-193189). Simultaneous LDN-193189 treatment and Epfn activation largely reversed the perturbations in ameloblast induction, with partial recovery of p-Smad1/5/8 expression, suggesting that Epfn activation promotes ameloblast induction from mouse iPSCs partially by upregulating BMP-Smad activity. These results reveal the potential regulatory networks between Epfn and the BMP-Smad pathway and suggest that Epfn is a promising target for inducing the differentiation of ameloblasts, which can be used in enamel and tooth regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Miao, Niibe, Fu, Zhang, Nattasit, Ohori-Morita, Nakamura, Jiang and Egusa.)

Mesenchymal stem cells (MSCs) exhibit self-renewal, multi-lineage differentiation potential and immunomodulatory properties, and are promising candidates for cellular therapy of various tissues. Despite the effective function of MSCs, the gradual loss of stem cell characteristics that occurs with repeated passages may significantly limit their therapeutic potential. A novel 3D shaking method was previously established to generate MSC spheroids in growth medium (GM-spheroids) and successfully maintain the multipotency of expanded MSCs, yet the expression of MSC-related genes was still low. In this study, we used a neurosphere culture technique to optimize the shaking culture method using human bone marrow-derived MSCs (BM-MSCs). MSC spheroids generated in neurosphere medium (NM-spheroids) maintained high expression of MSC-related genes during 3 weeks of prolonged shaking culture. Moreover, NM-spheroids generated from expanded MSCs showed high viability, upregulation of MSC-related and immune-related genes, and recovery of differentiation potential in vitro. Expanded adherent MSCs, GM-spheroids, and NM-spheroids were transplanted into a rat femur bone defect model to investigate their therapeutic potential in bone repair. Adherent MSCs and GM-spheroids showed delayed bone healing. In contrast, NM-spheroids showed high transplantation efficiency and enhanced bone regeneration. These data suggest that NM-spheroids generated using modified neurosphere culture conditions under continuous shaking recovered their stem cell characteristics in vitro and enhanced bone regeneration in vivo. Therefore, NM-spheroids should have great clinical potential for bone and tissue regenerative therapies as a stem cell-based biomaterial therapy., (© The Author(s) 2022. Published by Oxford University Press.)

Purpose: Evaluation of the clinical performance of computer-aided design/computer-aided manufacturing-produced resin composite crowns (CAD/CAM composite crowns) on molars with a particular focus on placement location., Methods: A retrospective cohort study was performed based on the clinical records of patients with CAD/CAM composite crowns on molars (June 2016 to March 2021). The hazard ratios (HRs) and 95% confidence intervals (95% CIs) were estimated based the Cox proportional hazard model to evaluate the effect of tooth location on complication type and occurrence. Covariates included crown location (maxilla/mandible, distalmost tooth/not distalmost tooth, and first molar/second or third molar) and endodontically treated (nonvital) or untreated (vital) tooth., Results: Overall, 362 crowns were evaluated (mean follow-up: 378 days, median: 286 days), and 106 crowns (29.3%) showed complications, most frequently crown debonding. The cumulative success and survival rates were 70.9% and 93.7%, respectively, after 1 year and 49.5% and 86.5%, respectively, after 3 years. There was no significant difference in the HRs and log-rank tests in the Kaplan-Meier curves based on crown location parameters (P > 0.05). However, placement on vital teeth was associated with higher risks than on nonvital teeth (HR, 1.55; 95% CI, 1.03-2.23). In addition, the cement as a covariate yielded a high HR., Conclusions: The location of CAD/CAM composite molar crowns is unlikely a risk factor for complications; therefore, these crowns can be clinically applied to all molars. However, the application of such molar crowns to vital teeth and the use of a cement other than adhesive resin cement present risks., Competing Interests: The authors have declared that no competing interests exist.

Background: Severe-early childhood caries (S-ECC) a global problem of significant concern, commonly manifest on the occlusal, and proximal surfaces of affected teeth. Despite the major ecological differences between these two niches the compositional differences, if any, in the microbiota of such lesions is unknown., Methods: Deep-dentine caries samples from asymptomatic primary molars of children with S-ECC (n 19) belonging to caries-code 5/6, (ICDAS classification) were evaluated. Employing two primer pools, we amplified and compared the bacterial 16S rRNA gene sequences of the seven hypervariable regions (V2-V4 and V6-V9) using NGS-based assay., Results: Bray-Curtisevaluation indicated that occlusal lesions (OL) had a more homogeneous community than the proximal lesions (PL) with significant compositional differences at the species level ( p = 0.01; R- 0.513). Together, the occlusal and proximal niches harbored 263 species, of which 202 (76.8%) species were common to both , while 49 (18.6%) and 12 (4.6%) disparate species were exclusively isolated from the proximal and occlusal niches, respectively. The most commonl genera at both niches included Streptococcus, Prevotella , and Lactobacillus. S. mutans was predominant in PL ( p ≤ 0.05), and Atopobium parvulum ( p = 0.01) was predominant in OL., Conclusions: Distinct differences exist between the caries microbiota of occlusal and proximal caries in S-ECC., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Small-molecule compounds strongly affecting osteogenesis can form the basis of effective therapeutic strategies in bone regenerative medicine. A cell-based high-throughput screening system might be a powerful tool for identifying osteoblast-targeting candidates; however, this approach is generally limited with using only one molecule as a cell-based sensor that does not always reflect the activation of the osteogenic phenotype. In the present study, we used the MC3T3-E1 cell line stably transfected with the green fluorescent protein (GFP) reporter gene driven by a fragment of type I collagen promoter (Col-1a1GFP-MC3T3-E1) to evaluate a double-screening system to identify osteogenic inducible compounds using a combination of a cell-based reporter assay and detection of alkaline phosphatase (ALP) activity. Col-1a1GFP-MC3T3-E1 cells were cultured in an osteogenic induction medium after library screening of 1280 pharmacologically active compounds (Lopack 1280 ). After 7 days, GFP fluorescence was measured using a microplate reader. After 14 days of osteogenic induction, the cells were stained with ALP. Library screening using the Col-1a1/GFP reporter and ALP staining assay detected three candidates with significant osteogenic induction ability. Furthermore, leflunomide, one of the three detected candidates, significantly promoted new bone formation in vivo. Therefore, this double-screening method could identify candidates for osteogenesis-targeting compounds more reliably than conventional methods.

In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication., (© 2021. The Author(s).)

Definitive prevention of inflammatory osteolysis around peri-implant bone tissue remains unestablished. M1 macrophages play a key role in the host defense against inflammatory osteolysis, and their polarization depends on cell shape. Macrophage polarization is controlled by environmental stimuli, particularly physicochemical cues and hence titanium nanosurface might tune macrophage polarization and function. This study determined whether titanium nanosurfaces with anisotropically patterned nanospikes regulates macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors. Alkaline-etching treatment with different protocols created two types of titanium nanosurfaces that had anisotropically patterned nanospikes with high or low distribution density, together with superhydrophilicity and the presence of hydroxyl groups. J774A.1 cells (mouse macrophage-like cell line), cultured on both titanium nanosurfaces, exhibited truly circulated shapes and highly expressed M1, but less M2, markers, without loss of viability. M1-like polarization of macrophages on both titanium nanosurfaces was independent of protein-mediated ligand stimulation or titanium surface hydrophilic or chemical status. In contrast, other smooth or micro-roughened titanium surfaces with little or no nanospikes did not activate macrophages under any culture conditions. Macrophage culture supernatants on both titanium nanosurfaces inhibited osteoclast differentiation of RAW264.7 cells (mouse osteoclast precursor cell line), even when co-incubated with osteoclast differentiation factors. The inhibitory effects on osteoclast differentiation tended to be higher in macrophages cultured on titanium nanosurfaces with denser nanospikes. These results showed that titanium nanosurfaces with anisotropically patterned nanospikes tune macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors, with nanotopographic cues rather than other physicochemical properties. STATEMENT OF SIGNIFICANCE: Peri-implant inflammatory osteolysis is one of the serious issues for dental and orthopedic implants. Macrophage polarization and function are key for prevention of peri-implant inflammatory osteolysis. Macrophage polarization can be regulated by the biomaterial's surface physicochemical properties such as hydrophilicity or topography. However, there was no titanium surface modification to prevent inflammatory osteolysis through immunomodulation. The present study showed for the first time that the titanium nanosurfaces with anisotropically patterned nanospikes, created by the simple alkali-etching treatment polarized macrophages into M1-like type producing the inhibitory factor on osteoclast differentiation. This phenomenon attributed to nanotopographic cues, but not hydrophilicity on the titanium nanosurfaces. This nanotechnology might pave the way to develop the smart implant surface preventing peri-implant inflammatory osteolysis through immunomodulation., Competing Interests: Declaration of Competing Interest All authors have no conflict of interest in this research., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Introduction: The retentive niches of deep caries lesions have a distinct biome. Methods: We evaluated the site-specific (occlusal and proximal) Candida -biome of Severe-Early Childhood Caries (S-ECC) in 66- children (132 lesions). Asymptomatic primary molars fitting the definition of the International Caries Detection and Assessment-(ICDAS)-caries-code 5/6 were analyzed. Deep-dentinal sampling and simultaneous assessment of pH were performed. Clinical isolates were speciated using multiplex-PCR and evaluated for their acidogenic and aciduric potential. Results: Surprisingly, a high prevalence of Candida species (72.7%), either singly or in combination, was noted from both the proximal and occlusal cavities. C. tropicalis was the most prevalent species (47%; 34/72), followed by C. krusei (43.1%; 31/72) and C. albicans (40.3%; 29/72), with C. glabrata being the least (9.7%; 7/72). Over 45% low-pH niches (pH <7) of both sites yielded either dual or triple species of Candida . Genotyping revealed three distinct C. albicans genotypes (A, B, and C) with (14/29; 48.3%) of strains belonging to Genotype A. All four evaluated Candida species exhibited acidogenic and aciduric potential, C. tropicalis being the most potent. Conclusion: This, the first report of the high-density, multispecies, yeast colonization of deep-dentinal lesions in S-ECC, suggests that the Candida -biome plays a significant etiologic role in the condition, possibly due to their profound acidogenicity in milieus rich in dietary carbohydrates., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Amelogenin comprises ~90% of enamel proteins; however, the involvement of Amelx transcriptional activation in regulating ameloblast differentiation from induced pluripotent stem cells (iPSCs) remains unknown. In this study, we generated doxycycline-inducible Amelx -expressing mouse iPSCs (Amelx-iPSCs). We then established a three-stage ameloblast induction strategy from Amelx-iPSCs, including induction of surface ectoderm (stage 1), dental epithelial cells (DECs; stage 2), and ameloblast lineage (stage 3) in sequence, by manipulating several signaling molecules. We found that adjunctive use of lithium chloride (LiCl) in addition to bone morphogenetic protein 4 and retinoic acid promoted concentration-dependent differentiation of DECs. The resulting cells had a cobblestone appearance and keratin14 positivity. Attenuation of LiCl at stage 3 together with transforming growth factor β1 and epidermal growth factor resulted in an ameloblast lineage with elongated cell morphology, positivity for ameloblast markers, and calcium deposition. Although stage-specific activation of Amelx did not produce noticeable phenotypic changes in ameloblast differentiation, Amelx activation at stage 3 significantly enhanced cell adhesion as well as decreased proliferation and migration. These results suggest that the combination of inducible Amelx transcription and stage-specific ameloblast induction for iPSCs represents a powerful tool to highlight underlying mechanisms in ameloblast differentiation and function in association with Amelx expression.

Lipin2 is a phosphatidate phosphatase that plays critical roles in fat homeostasis. Alterations in Lpin2 , which encodes lipin2, cause the autoinflammatory bone disorder Majeed syndrome. Lipin2 limits lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. However, little is known about the precise molecular mechanisms underlying its anti-inflammatory function. In this study, we attempted to elucidate the molecular link between the loss of lipin2 function and autoinflammatory bone disorder. Using a Lpin2 knockout murine macrophage cell line, we showed that lipin2 deficiency enhances innate immune responses to LPS stimulation through excessive activation of the NF-κB signaling pathway, partly because of TAK1 signaling upregulation. Lipin2 depletion also enhanced RANKL-mediated osteoclastogenesis and osteoclastic resorption activity accompanied by NFATc1 dephosphorylation and increased nuclear accumulation. These results suggest that lipin2 suppresses the development of autoinflammatory bone disorder by fine-tuning proinflammatory responses and osteoclastogenesis in macrophages. Therefore, this study provides insights into the molecular pathogenesis of monogenic autoinflammatory bone disorders and presents a potential therapeutic intervention.

Protamine-coated multi-shell calcium phosphate (CaP) was developed as a non-viral vector for tissue regeneration therapy. CaP nanoparticles loaded with different amounts of plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) were used to treat MC3T3E1 cells, and the yield of the released BMP-2 or IGF-1 was measured using ELISA 3 days later. Collagen scaffolds containing CaP nanoparticles were implanted into rat cranial bone defects, and BMP-2 and IGF-1 yields, bone formation, and bone mineral density enhancement were evaluated 28 days after gene transfer. The antibacterial effects of CaP nanoparticles against Streptococcus mutans and Aggregatibacter actinomycetemcomitans increased with an increase in the protamine dose, while they were lower for Staphylococcus aureus and Porphyromonas gingivalis. In the combination treatment with BMP-2 and IGF-1, the concentration ratio of BMP-2 and IGF-1 is an important factor affecting bone formation activity. The calcification activity and OCN mRNA of MC3T3E1 cells subjected to a BMP-2:IGF-1 concentration ratio of 1:4 was higher at 14 days. During gene transfection treatment, BMP-2 and IGF-1 were released simultaneously after gene transfer; the loaded dose of the plasmid DNA encoding IGF-1 did not impact the BMP-2 or IGF-1 yield or new bone formation ratio in vitro and in vivo. In conclusion, two growth factor-releasing systems were developed using an antibacterial gene transfer vector, and the relationship between the loaded plasmid DNA dose and resultant growth factor yield was determined in vitro and in vivo., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

During the current coronavirus disease 2019 (COVID-19) pandemic, dental education and training requiring face-to-face interaction must prioritize infection prevention and the safety of students, staff, and patients. In July 2020, the Association for Dental Education, Asia Pacific (ADEAP) published safety guidelines for safe dental education during the COVID-19 pandemic. These guidelines summarize ADEAP recommendations for classroom-based courses, reopening of simulated training courses and dental clinics, and provision of clinical skills training courses in dentistry during the COIVID-19 pandemic. They have been formulated to ensure the safety of students and teaching staff, dental auxiliary staff, and patients. However, the present guidelines are considered appropriate only when the number of COVID-19 epidemic cases has been significantly reduced, i.e. when the epidemiological curve has flattened in the area concerned. The criteria for lifting restrictions on activities should be consistent with relevant jurisdictional guidelines.

The recapitulation of cartilage/bone formation via guiding induced pluripotent stem cells (iPSCs) differentiation toward chondrogenic mesoderm lineage is an ideal approach to investigate cartilage/bone development and also for cartilage/bone regeneration. However, current induction protocols are time-consuming and complicated to follow. Here, we established a rapid and efficient approach that directly induce iPSCs differentiation toward chondrogenic mesoderm lineage by regulating the crucial Bmp-4 and FGF-2 signaling pathways using a 3D rotary suspension culture system. The mechanical stimulation from 3D rotary suspension accelerates iPSCs differentiation toward mesodermal and subsequent chondrogenic lineage via the Bmp-4-Smad1 and Tgf-β-Smad2/3 signaling pathways, respectively. The scaffold-free homogenous cartilaginous pellets or hypertrophic cartilaginous pellets derived from iPSCs within 28 days were capable of articular cartilage regeneration or vascularized bone regeneration via endochondral ossification in vivo, respectively. This biomimetic culture approach will contribute to research related to cartilage/bone development, regeneration, and hence to therapeutic applications in cartilage-/bone-related diseases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal/stem cells (MSCs), which generally expand into adherent monolayers, readily lose their proliferative and multilineage potential following repeated passages. Floating culture systems can be used to generate MSC spheroids, which are expected to overcome limitations associated with conventional adherent cultures while facilitating scaffold-free cell transplantation. However, the phenotypic characteristics of spheroids after long-term culture are unknown. In addition, regenerative therapies require new culture systems to maintain their undifferentiated state. In this study, we established a novel culture method employing three-dimensional (3D) "shaking" to generate MSC spheroids using bone marrow derived MSCs. Floating 3D cultures of mouse or human MSCs formed spheroids after shaking (85-95 rpm), within 1 month. These spheroids maintained their osteogenic-, adipogenic-, and chondrogenic-differentiation capacity. The adipogenic-differentiation capacity of adherent cultured mouse and human MSCs, which is lost following several passages, was remarkedly restored by shaking-culture. Notably, human MSC spheroids exhibited a renewable "undifferentiated MSC-pool" property, wherein undifferentiated MSCs grew from spheroids seeded repeatedly on a plastic culture dish. These data suggest that the shaking-culture method maintains and restores multipotency that is lost following monolayer expansion and thereby shows potential as a promising strategy for regenerative therapies with mesenchymal tissues., (Copyright © 2020 Niibe, Ohori-Morita, Zhang, Mabuchi, Matsuzaki and Egusa.)

Mechanical loading on articular cartilage induces various mechanical stresses and strains. In vitro hydrodynamic forces such as compression, shear and tension impact various cellular properties including chondrogenic differentiation, leading us to hypothesize that shaking culture might affect the chondrogenic induction of induced pluripotent stem cell (iPSC) constructs. Three-dimensional mouse iPSC constructs were fabricated in a day using U-bottom 96-well plates, and were subjected to preliminary chondrogenic induction for 3 days in static condition, followed by chondrogenic induction culture using a see-saw shaker for 17 days. After 21 days, chondrogenically induced iPSC (CI-iPSC) constructs contained chondrocyte-like cells with abundant ECM components. Shaking culture significantly promoted cell aggregation, and induced significantly higher expression of chondrogenic-related marker genes than static culture at day 21. Immunohistochemical analysis also revealed higher chondrogenic protein expression. Furthemore, in the shaking groups, CI-iPSCs showed upregulation of TGF-β and Wnt signaling-related genes, which are known to play an important role in regulating cartilage development. These results suggest that shaking culture activates TGF-β expression and Wnt signaling to promote chondrogenic differentiation in mouse iPSCs in vitro. Shaking culture, a simple and convenient approach, could provide a promising strategy for iPSC-based cartilage bioengineering for study of disease mechanisms and new therapies.

Jagged1 activates Notch signaling and subsequently promotes osteogenic differentiation in human periodontal ligament cells (hPDLs). The present study investigated the participation of the Notch receptor, NOTCH2, in the Jagged1-induced osteogenic differentiation in hPDLs. NOTCH2 and NOTCH4 mRNA expression levels increased during hPDL osteogenic differentiation. However, the endogenous NOTCH2 expression levels were markedly higher compared with NOTCH4. NOTCH2 expression knockdown using shRNA in hPDLs did not dramatically alter their proliferation or osteogenic differentiation compared with the shRNA control. After seeding on Jagged1-immobilized surfaces and maintaining the hPDLs in osteogenic medium, HES1 and HEY1 mRNA levels were markedly reduced in the shNOTCH2-transduced cells compared with the shControl group. Further, shNOTCH2-transduced cells exhibited less alkaline phosphatase enzymatic activity and in vitro mineralization than the shControl cells when exposed to Jagged1. MSX2 and COL1A1 mRNA expression after Jagged1 activation were reduced in shNOTCH2-transduced cells. Endogenous Notch signaling inhibition using a γ-secretase inhibitor (DAPT) attenuated mineralization in hPDLs. DAPT treatment significantly promoted TWIST1, but decreased ALP, mRNA expression, compared with the control. In conclusion, Notch signaling is involved in hPDL osteogenic differentiation. Moreover, NOTCH2 participates in the mechanism by which Jagged1 induced osteogenic differentiation in hPDLs.

Stem cell-based developmental engineering has been considered as a promising strategy for tissue/organ regeneration. Tooth is formed by sequential reciprocal interactions between epithelium derived from surface ectoderm and mesenchymal cells derived from cranial neural crest. The neural crest cell is an appealing cell source for tooth development and regeneration research. In this study, we investigated the odontogenic differentiation and dentin-pulp complex regeneration potential of neural crest cells. Our results showed that neural crest cells (O9-1 mouse cranial neural crest cell line) can sequentially differentiate into dentin matrix acidic phosphoprotein 1 (DMP-1)-positive odontoblasts within a developing tooth germ in vitro . Moreover, O9-1 cells and induced pluripotent stem cell (iPSC)-derived neural crest-like cells (iNCLCs) can form well-organized vascularized dentin-pulp complex when transplanted in vivo with tooth scaffold. Furthermore, both O9-1 cells and iNCLCs can be differentiated into odontoblast-like cells, positive staining with odontogenic-related markers DMP-1 and dentin sialophosphoprotein (DSPP), under odontogenic induction with the administration of bone morphogenetic protein 4 (BMP-4). These results demonstrated that neural crest cells, especially the unlimited iNCLCs, are a promising cell source for tooth development and dental tissue/tooth organ regeneration studies., (Copyright © 2020 Zhang, Zhang, Luo, Yan, Niibe, Egusa, Zhang, Xie and Jiang.)

Background: The use of silver-formulation as microbicide to arrest dentinal caries is gaining popularity. The primary objective of the present appraisal was to systematically review the clinical (in vivo) applications and antimicrobial potential of silver-containing formulations in arresting dentinal caries. Our secondary aim was to sum up the available in vitro applications of silver-containing formulations against cariogenic microbes isolated from dentine lesions., Methods: Ovid MEDLINE, EBSCO host, Web of Science, and Cochrane Library databases was searched between January 2009-May 2019., Results: In vivo: We observed conflicting evidence of antimicrobial efficacy of SDF on a diverse array of microbial taxa present in carious dentine of primary and permanent teeth. Moreover, there is insufficient evidence on the application of AgNP-fluoride as an effective microbicidal against cariogens of dentine lesions. In vitro: We found a good evidence of microbicidal efficacy of silver diamine fluoride (SDF) on selective cariogenic microbes in human dentine model. Additionally, a good evidence was noted of in vitro application of silver nanoparticles (AgNPs) as a useful microbicidal against S. mutans adhesion, growth and subsequent biofilm formation in human dentine models., Conclusions: Taken together, in vitro evidence indicates the promising antimicrobial potential of silver-based formulations (SDF and nanosilver) against the predominant cariogenic flora, particularly from dentine lesions. Post-treatment clinical data of either the bactericidal and bacteriostatic effects of SDF or nanosilver are sparse. Furthermore, the current understanding of the specific size, concentration, antimicrobial mechanisms, and toxicological aspects of nano-silver compounds is inadequate to draw firm conclusions on their clinical utility.

Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro . The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560  μ m (Elp400), 500/700  μ m (Elp500), and 900/700  μ m (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2 , Osterix , Collagen 1a1 , Osteocalcin , Bone sialoprotein , and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat's pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Phoonsuk Limraksasin et al.)

Background: The microbiome of Severe-Early Childhood Caries (S-ECC), is characterized by an ecosystem comprising bacterial and fungal species, with a predominance of Candida species. Hence, an anti-cariogen effective against both bacteria and fungi would be valuable in the management of S-ECC. Here we evaluate the antifungal effect of silver diamine fluoride (SDF) against 35-clinical yeast isolates (Ten-each of C. albicans, C. krusei, C. tropicalis and five C. glabrata strains) from dentinal caries-lesions from S-ECC., Results: Disc-diffusion and time-kill assays as well as MIC 50 and MIC 90 evaluations against therapeutic concentrations confirmed the broad-spectrum anti-candidal potency of SDF. Ultrastructural images revealed morphologic aberrations of yeast-cell walls on exposure to SDF. All C. krusei and C. glabrata isolates were significantly more sensitive to SDF, relative to the standard antifungal fluconazole. Further, SDF appears to effectively abrogate filamentation of C. albicans even at very low concentrations., Conclusions: Our data, for the first time, elucidate the antifungal potency of SDF, in addition to its known antibacterial activity, in the management of S-ECC.

The protected niche of deep-caries lesions is a distinctive ecosystem. We assessed the Candida biome and its cariogenic traits from dentin samples of 50 children with severe-early childhood caries (S-ECC). Asymptomatic, primary molars belonging to International Caries Detection and Assessment-ICDAS caries-code 5 and 6 were analyzed, and C. albicans (10-isolates), C. tropicalis (10), C. krusei (10), and C. glabrata (5) isolated from the lesions were then evaluated for their biofilm formation, acidogenicity, and the production of secreted hydrolases: hemolysins, phospholipase, proteinase and DNase. Candida were isolated from 14/43 ICDAS-5 lesions (32.5%) and 44/57 ICDAS-6 lesions (77.2%). Compared to, ICDAS-5, a significantly higher frequency of multi-species infestation was observed in ICDAS-6 lesions (p=0.001). All four candidal species (above) showed prolific biofilm growth, and an equal potency for tooth demineralization. A significant interspecies difference in the mean phospholipase, as well as proteinase activity was noted (p < 0.05), with C. albicans being the predominant hydrolase producer. Further, a positive correlation between phospholipase and proteinase activity of Candida -isolates was noted (r = 0.818, p < 0.001). Our data suggest that candidal mycobiota with their potent cariogenic traits may significantly contribute to the development and progression of S-ECC., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1 . Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

Bone tissue engineering has been widely studied and proposed as a promising platform for correcting the bone defects. The applications of mesenchymal stem cell (MSC)-based bone tissue engineering have been investigated in various in vitro and in vivo models. In this regard, the promising animal bone defect models have been employed for illustrating the bone regenerative capacity of MSC-based bone tissue engineering. However, most studies aimed for clinical applications in human. These evidences suggest a knowledge gap to fulfill the accomplishment for veterinary implementation. In this review, the fundamental concept, knowledge, and technology of MSC-based bone tissue engineering focusing on veterinary applications are summarized. In addition, the potential canine MSCs resources for veterinary bone tissue engineering are reviewed, including canine bone marrow-derived MSCs, canine adipose-derived MSCs, and canine dental tissue-derived MSCs. This review will provide a basic and current information for studies aiming for the utilization of MSC-based bone tissue engineering in veterinary practice., (© 2019 The Author(s).)

Oxidative stress hinders tissue regeneration in cell therapy by inducing apoptosis and dysfunction in transplanted cells. N -acetyl- L -cysteine (NAC) reinforces cellular antioxidant capabilities by increasing a major cellular endogenous antioxidant molecule, glutathione, and promotes osteogenic differentiation. This study investigates the effects of pretreatment of osteoblast-like cells with NAC on oxidative stress-induced apoptosis and dysfunction and bone regeneration in local transplants. Rat femur bone marrow-derived osteoblast-like cells preincubated for 3 h with and without 5 mM NAC were cultured in a NAC-free osteogenic differentiation medium with continuous exposure to 50 μM hydrogen peroxide to induce oxidative stress. NAC preincubation prevented disruption of intracellular redox balance and alleviated apoptosis and negative impact on osteogenic differentiation, even under oxidative stress. Autologous osteoblast-like cells with and without NAC pretreatment in a collagen sponge vehicle were implanted in critical-size defects in rat femurs. In the third week, NAC-pretreated cells yielded complete defect closure with significantly matured lamellar bone tissue in contrast with poor bone healing by cells without pretreatment. Cell-tracking analysis demonstrated direct bone deposition by transplanted cells pretreated with NAC. Pretreatment of osteoblast-like cells with NAC enhances bone regeneration in local transplantation by preventing oxidative stress-induced apoptosis and dysfunction at the transplanted site.

Mechanical force regulates periodontal ligament cell (PDL) behavior. However, different force types lead to distinct PDL responses. Here, we report that pretreatment with an intermittent compressive force (ICF), but not a continuous compressive force (CCF), promoted human PDL (hPDL) osteogenic differentiation as determined by osteogenic marker gene expression and mineral deposition in vitro. ICF-induced osterix (OSX) expression was inhibited by cycloheximide and monensin. Although CCF and ICF significantly increased extracellular adenosine triphosphate (ATP) levels, pretreatment with exogenous ATP did not affect hPDL osteogenic differentiation. Gene-expression profiling of hPDLs subjected to CCF or ICF revealed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and transforming growth factor beta (TGF-β) signaling pathway genes were commonly upregulated, while calcium signaling pathway genes were downregulated in both CCF- and ICF-treated hPDLs. The TGFB1 mRNA level was significantly increased, while those of TGFB2 and TGFB3 were decreased by ICF treatment. In contrast, CCF did not modify TGFB1 expression. Inhibiting TGF-β receptor type I or adding a TGF-β1 neutralizing antibody attenuated the ICF-induced OSX expression. Exogenous TGF-β1 pretreatment promoted hPDL osteogenic marker gene expression and mineral deposition. Additionally, pretreatment with ICF in the presence of TGF-β receptor type I inhibitor attenuated the ICF-induced mineralization. In conclusion, this study reveals the effects of ICF on osteogenic differentiation in hPDLs and implicates TGF-β signaling as one of its regulatory mechanisms.

Mechanical force regulates numerous biological functions. Application of different force types leads to different cell responses. This data article describes RNA sequencing data identifying gene expression of human periodontal ligament cells (hPDLs) treated with the continuous or intermittent compressive force. These data could be further utilized to investigate the controlling pathways that regulate hPDLs' behaviors by the different force types. Raw RNA sequencing data were deposited in the NCBI Sequence Read Archive (SRP136155) and NCBI Gene Expression Omnibus (GSE112122)., (© 2019 The Authors.)

Oxidative stress on transplanted bone marrow-derived mesenchymal stem cells (BMSCs) during acute inflammation is a critical issue in cell therapies. N-acetyl-L cysteine (NAC) promotes the production of a cellular antioxidant molecule, glutathione (GSH). The aim of this study was to investigate the effects of pre-treatment with NAC on the apoptosis resistance and bone regeneration capability of BMSCs. Rat femur-derived BMSCs were treated in growth medium with or without 5 mM NAC for 6 h, followed by exposure to 100 μM H 2 O 2 for 24 h to induce oxidative stress. Pre-treatment with NAC significantly increased intracellular GSH levels by up to two fold and prevented H 2 O 2 -induced intracellular redox imbalance, apoptosis and senescence. When critical-sized rat femur defects were filled with a collagen sponge containing fluorescent-labeled autologous BMSCs with or without NAC treatment, the number of apoptotic and surviving cells in the transplanted site after 3 days was significantly lower and higher in the NAC pre-treated group, respectively. By the 5th week, significantly enhanced new bone formation was observed in the NAC pre-treated group. These data suggest that pre-treatment of BMSCs with NAC before local transplantation enhances bone regeneration via reinforced resistance to oxidative stress-induced apoptosis at the transplanted site., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Regenerative medicine for bone tissue mainly depends on efficient recruitment of endogenous or transplanted stem cells to guide bone regeneration. Platelet-derived growth factor (PDGF) is a functional factor that has been widely used in tissue regeneration and repair. However, the short half-life of PDGF limits its efficacy, and the mechanism by which PDGF regulates stem cell-based bone regeneration still needs to be elucidated. In this study, we established genetically modified PDGF-B-overexpressing bone marrow stromal cells (BMSCs) using a lentiviral vector and then explored the mechanism by which PDGF-BB regulates BMSC-based vascularized bone regeneration. Our results demonstrated that PDGF-BB increased osteogenic differentiation but inhibited adipogenic differentiation of BMSCs via the extracellular signal-related kinase 1/2 (ERK1/2) signaling pathway. In addition, secreted PDGF-BB significantly enhanced human umbilical vein endothelial cell (HUVEC) migration and angiogenesis via the phosphatidylinositol 3 kinase (PI3K)/AKT and ERK1/2 signaling pathways. We evaluated the effect of PDGF-B-modified BMSCs on bone regeneration using a critical-sized rat calvarial defect model. Radiography, micro-CT, and histological analyses revealed that PDGF-BB overexpression improved BMSC-mediated angiogenesis and osteogenesis during bone regeneration. These results suggest that PDGF-BB facilitates BMSC-based bone regeneration by enhancing the osteogenic and angiogenic abilities of BMSCs.