Objective This study aimed to investigate whether various SARS-CoV-2 infection-related molecules, including ACE2, are expressed in oral tissues and clarify the effects of smoking on these. Methods The expressions of the SARS-CoV-2 infection-related molecules ACE2, NRP-1, TMPRSS, and Furin in normal oral mucosa and salivary gland tissues were immunohistochemically examined. This study also investigated the expression of SARS-CoV-2 infection-related molecules when cigarette smoke extract (CSE) was applied to oral mucosal epithelial cells in vitro and examined differences in the expression of these molecules at the mRNA and protein levels in the oral tissues of smokers and nonsmokers. Results The expression of ACE2, NRP-1, TMPRSS2, and Furin was observed in the oral mucosal tissues and salivary gland tissues. When oral mucosal epithelial cells were exposed to CSE, the mRNA expression of ACE2 and Furin increased in a dose- and time-dependent manner, whereas the expression of NRP-1 increased up to 1% CSE but decreased to 5% CSE. In terms of protein expression, only NRP-1 increased up to 1% CSE, as did its mRNA. The expression of these molecules in the oral mucosal tissues of smokers and nonsmokers tended to be higher in smokers; however, the difference was not significant. Conclusions This study revealed that the SARS-CoV-2 infection-related molecules ACE2, NRP-1, TMPRSS, and Furin are expressed in the oral mucosa and salivary gland tissue. These molecules may be affected by smoking and indicate the importance of oral health and are thought to be one of the scientific grounds for future antismoking campaigns. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request.

This study aimed to determine whether the positional relationship between the underside of the screw head and the surface of the alveolar bone could alter the stress on the two surfaces and affect the stability of implanted anchor screws. First, in order to confirm the extent of the gap between the mini-screw and the bone surface, a mini-screw was placed in the palate of rabbits and examined histologically. As a result, in the conventional screw implantation procedure, oral mucosa between the base of the screw head and the bone creates a spatial gap. Removal of the oral mucosa eliminates this gap. Then, we compared the positional difference of the screw in a contact and gap group by analyzing stress distribution on the bone and screw. Analysis using the finite element method showed that more stress was loaded on both the bone and screw in the gap group than in the contact group. Cortical bone thickness did not affect stress in either group. The effects of different load strengths were similar between groups. A surgical procedure in which mucosal coverings are removed so that implanted anchor mini-screws are in contact with the bone surface was found to reduce the stress load on both the bone and screw. This procedure can be used to prevent undesirable dislodgement of implanted mini-screws.

Melatonin exerts various physiological effects through melatonin receptors and their ability to scavenge free radicals. Radiotherapy is a common treatment for head and neck tumors, but stomatitis, a side effect affecting irradiated oral mucosa, can impact treatment outcomes. This study investigated the preventive effect of melatonin, a potent free radical scavenger, on radiation-induced oral mucositis. Mice were irradiated with 15 Gy of X-ray radiation to the head and neck, and the oral mucosa was histologically compared between a melatonin-administered group and a control group. The results showed that radiation-induced oral mucositis was suppressed in mice administered melatonin before and after irradiation. It was suggested that the mechanism involved the inhibition of apoptosis and the inhibition of DNA damage. From these findings, we confirmed that melatonin has a protective effect against radiation-induced oral mucositis.

Dental pulp stem cells (DPSCs) are considered a valuable cell source for regenerative medicine because of their high proliferative potential, multipotency, and availability. We established a new cryopreservation method (NCM) for collecting DPSCs, in which the tissue itself is cryopreserved and DPSCs are collected after thawing. We improved the NCM and developed a new method for collecting and preserving DPSCs more efficiently. Dental pulp tissue was collected from an extracted tooth, divided into two pieces, sandwiched from above and below using cell culture inserts, and cultured. As a result, the cells in the pulp tissue migrated vertically over time and localized near the upper and lower membranes over 2–3 days. With regard to the underlying molecular mechanism, SDF1 was predominantly involved in cell migration. This improved method is valuable and enables the more efficient collection and reliable preservation of DPSCs. It has the potential to procure a large number of DPSCs stably.

Masks are effective for preventing the spread of COVID-19 and other respiratory infections. If antimicrobial properties can be applied to the non-woven fabric filters in masks, they can become a more effective countermeasure against human-to-human and environmental infections. We investigated the possibilities of carrying antimicrobial agents on the fiber surfaces of non-woven fabric filters by applying silica-resin coating technology, which can form silica-resin layers on such fabrics at normal temperature and pressure. Scanning electron microscopy and electron probe microanalysis showed that a silica-resin layer was formed on the fiber surface of non-woven fabric filters. Bioassays for coronavirus and quantitative reverse transcription-polymerase chain reactions (RT-PCR) revealed that all antimicrobial agents tested loaded successfully onto non-woven fabric filters without losing their inactivation effects against the human coronavirus (inhibition efficacy: >99.999%). These results indicate that this technology could be used to load a functional substance onto a non-woven fabric filter by vitrifying its surface. Silica-resin coating technology also has the potential of becoming an important breakthrough not only in the prevention of infection but also in various fields, such as prevention of building aging, protection of various cultural properties, the realization of a plastic-free society, and prevention of environmental pollution.

The early diagnosis and isolation of infected individuals with coronavirus disease 2019 (COVID-19) remain important. Although quantitative polymerase chain reaction (qPCR) testing is considered the most accurate test available for COVID-19 diagnosis, it has some limitations, such as the need for specialized laboratory technicians and a long turnaround time. Therefore, we have established and reported a rapid diagnostic method using a small amount of saliva as a sample using a lightweight mobile qPCR device. This study aimed to improve the existing method and increase the detection sensitivity and specificity. The detection specificity of CDC N1 and N2 was examined by improving qPCR reagents and polymerase chain reaction conditions for the previously reported method. Furthermore, the feasibility of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA was examined using both the previous method and the improved method in patients with COVID-19. The results showed that the improved method increased the specificity and sensitivity. This improved method is useful for the rapid diagnosis of SARS-CoV-2.

Containment of SARS-CoV-2 has become an urgent global issue. To overcome the problems of conventional quantitative polymerase chain reaction (qPCR) tests, we verified the usefulness of a mobile qPCR device that utilizes mouthwash to obtain a saliva sample with the aim of developing a rapid diagnostic method for SARS-CoV-2. First, we examined whether anyone could easily operate this device. Then, we examined whether RNA in the mouthwash could be detected in a short time. In addition, we investigated whether it was possible to diagnose SARS-CoV-2 infection using mouthwash obtained from COVID-19 patients undergoing hospitalization. The results revealed that all subjects were able to complete the operation properly without error. In addition, RNase P was detected in the mouthwash without pretreatment. The average detection time was 18 min, which is significantly shorter than conventional qPCR devices. Furthermore, this device detected SARS-CoV-2 in the mouthwash of a COVID-19 patient undergoing hospitalization. The above findings verified the efficacy of this diagnostic method, which had a low risk of infection, was technically simple, and provided stable results. Therefore, this method is useful for the rapid detection of SARS-CoV-2.

Considering that every tissue/organ has the most suitable microenvironment for its functional cells, controlling induced pluripotent stem cell (iPSC) differentiation by culture on frozen sections having a suitable microenvironment is possible. Induced PSCs were cultured on frozen sections of the liver, the brain, the spinal cord, and cover glasses (control) for 9 days. The iPSCs cultured on the sections of the liver resembled hepatocytes, whereas those on sections of the brain and the spinal cord resembled neuronal cells. The percentage of hepatocytic marker-positive cells in the iPSCs cultured on the sections of the liver was statistically higher than that of those in the iPSCs cultured on the sections of the brain and the spinal cord or on cover glasses. In contrast, the iPSCs cultured on the sections of the brain and the spinal cord revealed a high percentage of neural marker-positive cells. Thus, iPSCs can be differentiated into a specific cell lineage in response to specific factors within frozen sections of tissues/organs. Differentiation efficacy of the frozen sections markedly differed between the iPSC clones. Therefore, our induction method could be simple and effective for evaluating the iPSC quality.

This study investigated (i) the degradation effect of 405-nm blue light-emitting diode (LED) light irradiation on Candida albicans and C. glabrata biofilms formed on denture base resin and (ii) the effects of 405-nm blue LED light irradiation on the mechanical and surface characteristics of the resin. Polymethyl methacrylate denture base resin discs were prepared, and C. albicans or C. glabrata biofilms formed on the denture base resin discs. Each biofilm was irradiated with 405-nm blue LED light under a constant output power (280 mW/cm(2)) for different times in a moisture chamber with 100% relative humidity. Postirradiation, each biofilm was analyzed using a colony-forming unit assay, fluorescence microscopy, and scanning electron microscopy (SEM). Parallelepiped specimens of acrylic resin were prepared, and changes in their flexural strength (FS), flexural modulus (FM), and surface roughness (R-a) preirradiation and postirradiation with 405-nm blue LED light were evaluated. Irradiation for 30 min completely inhibited colony formation in both Candida species. Fluorescence microscopy showed that almost all Candida cells were killed because of irradiation. SEM images showed various cell damage patterns, such as wrinkles, shrinkage, and cell surface damage. An increase in FS was noted postirradiation, but no significant changes were observed in FM and R-a preirradiation and postirradiation. In conclusion, irradiation with 405-nm blue LED light induces degradation of C. albicans and C. glabrata biofilms on denture base resin, even in the absence of photosensitizers, without resin surface deterioration.

A pleomorphic lipoma is a rare benign lipomatous tumor that has been defined by Enzinger as a new lipomatous entity. Pleomorphic lipoma mainly arises in the subcutaneous tissue of the neck and shoulders in elderly men, with relatively few reported cases of the tumor in the oral maxillofacial region. We report a case of pleomorphic lipoma arising in the left hard palate mucosa of a 67-year-old man. The lesion was a tender, sharp and pedunculated mass measuring 7 × 7 mm. The tumor was surgically removed including the surrounding normal tissue, and the histopathological diagnosis was pleomorphic lipoma. Microscopic examination histologically revealed a subepithelial admixture of mature adipose tissue and overgrowth of fibrous tissue, scattered with CD34-positive large and multinucleated cells. Four years after surgery, the patient remains recurrence-free and has a good clinical course.

This study aimed to investigate the cleansing effects of grapefruit seed extract (GSE) on biofilms of Candida albicans (C. albicans) formed on denture-base resin and the influence of GSE on the mechanical and surface characteristics of the resin. GSE solution diluted with distilled water to 0.1% (0.1% GSE) and 1% (1% GSE) and solutions with Polident® denture cleansing tablet dissolved in distilled water (Polident) or in 0.1% GSE solution (0.1% G+P) were prepared as cleansing solutions. Discs of acrylic resin were prepared, and the biofilm of C. albicans was formed on the discs. The discs with the biofilm were treated with each solution for 5 min at 25°C. After the treatment, the biofilm on the discs was analyzed using a colony forming unit (CFU) assay, fluorescence microscopy, and scanning electron microscopy (SEM). In order to assess the persistent cleansing effect, the discs treated with each solution for 5 min were aerobically incubated in Yeast Nitrogen Base medium for another 24 h. After incubation, the persistent effect was assessed by CFU assay. Some specimens of acrylic resin were immersed in each solution for 7 days, and changes in surface roughness (Ra), Vickers hardness (VH), flexural strength (FS), and flexural modulus (FM) were evaluated. As a result, the treatment with 1% GSE for 5 min almost completely eliminated the biofilm formed on the resin; whereas, the treatment with 0.1% GSE, Polident, and 0.1% G+P for 5 min showed a statistically significant inhibitory effect on biofilms. In addition, 0.1% GSE and 0.1% G+P exerted a persistent inhibitory effect on biofilms. Fluorescence microscopy indicated that Polident mainly induced the death of yeast, while the cleansing solutions containing at least 0.1% GSE induced the death of hyphae as well as yeast. SEM also revealed that Polident caused wrinkles, shrinkage, and some deep craters predominantly on the cell surfaces of yeast, while the solutions containing at least 0.1% GSE induced wrinkles, shrinkage, and some damage on cell surfaces of not only yeasts but also hyphae. No significant changes in Ra, VH, FS, or FM were observed after immersion in any of the solutions. Taken together, GSE solution is capable of cleansing C. albicans biofilms on denture-base resin and has a persistent inhibitory effect on biofilm development, without any deteriorations of resin surface.

In the present study, a scaffold-free tissue construct was developed as an approach for the regeneration of tissue defects, which produced good outcomes. We fabricated a scaffold-free tissue construct from human dental pulp stem cells (hDPSCs construct), and examined the characteristics of the construct. For its fabrication, basal sheets prepared by 4-week hDPSCs culturing were subjected to 1-week three-dimensional culture, with or without osteogenic induction, whereas hDPSC sheets (control) were fabricated by 1-week culturing of basal sheets on monolayer culture. The hDPSC constructs formed a spherical structure and calcified matrix that are absent in the control. The expression levels for bone-related genes in the hDPSC constructs were significantly upregulated compared with those in the control. Moreover, the hDPSC constructs with osteogenic induction had a higher degree of calcified matrix formation, and higher expression levels for bone-related genes, than those for the hDPSC constructs without osteogenic induction. These results suggest that the hDPSC constructs with osteogenic induction are composed of cells and extracellular and calcified matrices, and that they can be a possible scaffold-free material for bone regeneration.

Delayed postoperative infection is known as a major complication after bone surgeries using osteosynthetic biomaterial such as titanium (Ti) and bioresorbable organic materials. However, the precise cause of this type of infection is still unclear and no effective prevention has been established. The purpose of this study is to investigate the effect of irradiation with a 405 nm blue-violet laser on the bacteria adhered on the Ti and hydroxyapatite-poly-L-lactic acid- (HA-PLLA) based material surfaces and to verify the possibility of its clinical application to prevent the delayed postoperative infection after bone surgeries using osteosynthetic biomaterial. The suspension of Staphylococcus aureus FDA 209P was delivered onto the surface of disks composed of Ti or HA-PLLA. Bacterial adhesion on each disk was observed using a scanning electron microscope (SEM). After thorough washing with distilled water, the growth of bacteria attached to the material surfaces was examined with an alamar blue-based redox indicator. Moreover, a bactericidal effect of 405 nm blue-violet laser irradiation on residual bacteria on both materials was investigated using colony-forming assay. As a result, there was no significant difference in the bacterial adhesion between Ti and HA-PLLA materials. In contrast, 45 J/cm 2 of irradiation with 405 nm blue-violet laser inhibited the bacterial growth at approximately 93% on Ti disks and at approximately 99% on HA-PLLA disks. This study clearly demonstrated the possibility that the irradiation with a 405 nm blue-violet laser is useful as an alternative management strategy for the prevention of delayed postoperative infection after bone surgeries using osteosynthetic biomaterials.

Dental pulp stem cells (DPSCs) are an attractive cell source for use in cell-based therapy, regenerative medicine, and tissue engineering because DPSCs have a high cell proliferation ability and multidifferentiation capacity. However, several problems are associated with the collection and preservation of DPSCs for use in future cell-based therapy. In particular, the isolation of DPSCs for cryopreservation is time consuming and expensive. In this study, we developed a novel cryopreservation method (NCM) for dental pulp tissues to isolate suitable DPSCs after thawing cryopreserved tissue. Using the NCM, dental pulp tissues were cultured on adhesion culture dishes for 5 days and then cryopreserved. After thawing, the cryopreserved dental pulp tissue fragments exhibited cell migration. We evaluated each property of DPSCs isolated using the NCM (DPSCs-NCM) and the explant method alone without cryopreservation (DPSCs-C). DPSCs-NCM had the same proliferation capacity as DPSCs-C. Flow cytometry (FACS) analysis indicated that both DPSCs-NCM and DPSCs-C were positive for mesenchymal stem cell markers at the same level but negative for hematopoietic cell markers. Moreover, both DPSCs-NCM and DPSCs-C could differentiate into osteogenic, chondrogenic, and adipogenic cells during culture in each induction medium. These results suggest that DPSCs-NCM may be mesenchymal stem cells. Therefore, our novel method might facilitate the less expensive cryopreservation of DPSCs, thereby providing suitable DPSCs for use in patients in future cell-based therapies.

Leptin, a 16 kDa circulating anti-obesity hormone, has many physiological properties such as body weight homeostasis, lipid metabolism, hematopoiesis, thermogenesis, ovarian function, bone formation, and angiogenesis. Interestingly, a certain study showed that skin wound healing delayed in leptin deficient ob/ob mice. However, little has been known about the physiological role of leptin in skin wound healing. In this chapter, we introduce whether local and single-dose administration of leptin exerted a promotive influence on the skin wound healing. Immunohistochemical analysis revealed that leptin receptor was expressed in mouse epidermal cells. In addition, topical administration of leptin promoted the healing of chemical burn wounds created on the back skin of mice without any side effects. Then, the mechanisms of the promotive effect of leptin on the wound healing of the skin were demonstrated immunohistochemical and biological analysis; namely, leptin stimulated angiogenesis in the connective tissue beneath the wounded area and the cell proliferation, differentiation/function, and migration of human epidermal keratinocytes. These findings revealed the possible and promising usefulness of leptin as a new wound-healing promoting agent.

IntroductionLeptin, a 16 kDa anti-obesity hormone, exhibits various physiological properties. Interestingly, skin wound healing was proven to delay in leptin-deficient ob/ob mice. However, little is known on the mechanisms of this phenomenon. In this study, we attempted to elucidate a role of leptin in wound healing of skin.MethodsImmunohistochemical analysis was performed to confirm the expression of the leptin receptor (Ob-R) in human and mouse skin. Leptin was topically administered to chemical wounds created in mouse back skin along with sustained-release absorbable hydrogel. The process of wound repair was histologically observed and the area of ulceration was measured over time. The effect of leptin on the proliferation, differentiation and migration of human epidermal keratinocytes was investigated.ResultsOb-R was expressed in epidermal cells of human and mouse skin. Topical administration of leptin significantly promoted wound healing. Histological analysis showed more blood vessels in the dermal connective tissues in the leptin-treated group. The proliferation, differentiation/function and migration of human epidermal keratinocytes were enhanced by exogenous leptin.ConclusionTopically administered leptin was proven to promote wound healing in the skin by accelerating proliferation, differentiation/function and migration of epidermal keratinocytes and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the skin.

The purpose of this study is to evaluate the usefulness of 405 nm monochromatic laser irradiation as an alternative management for prevention and/or treatment of endodontic infections. A monochromatic laser-emitting device equipped with a 405-nm laser diode was developed. Using this device, the effect of 405 nm laser irradiation on the growth of Porphyromonas gingivalis, Prevotella intermedia, Enterococcus faecalis, and Candida albicans, which are microorganisms associated with persistent endodontic infections, was evaluated by viable colony counting. As a result, the irradiation with a 405 nm laser had a significant bactericidal/fungicidal effect on P. gingivalis, P. intermedia, and C. albicans, whereas the growth of E. faecalis was not affected by the irradiation. The inhibition rate in P. gingivalis and P. intermedia was ~60% and ~80%, respectively, following irradiation at 0.2 W for 300 sec. The inhibition rate in C. albicans was ~90% following irradiation at 0.2 W for 1200 sec. These results indicate that 405 nm monochromatic laser irradiation exerts a bactericidal/fungicidal effect on these microorganisms. The present study clearly demonstrates that 405 nm laser irradiation is a promising alternative management strategy for prevention and/or treatment of endodontic infections.

Melatonin regulates a variety of biological processes, which are the control of circadian rhythms, regulation of seasonal reproductive function and body temperature, free radical scavenging and so on. Our previous studies have shown that various cells exist in human and mouse tooth germs that express the melatonin 1a receptor (Mel1aR). However, little is known about the effects of melatonin on tooth development and growth. The present study was performed to examine the possibility that melatonin might exert its influence on tooth development. DP-805 cells, a human dental papilla cell line, were shown to express Mel1aR. Expression levels of mRNA for Mel1aR in DP-805 cells increased until 3 days after reaching confluence and decreased thereafter. Real-time reverse transcription-polymerase chain reaction showed that melatonin increased the expression of mRNAs for osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotin (DSPP). Melatonin also enhanced the mineralized matrix formation in DP-805 cell cultures in a dose-dependent manner. These results strongly suggest that melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.

Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is a lymphoid proliferation or lymphoma in an immunosuppressed patient administered with MTX. Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a clinical condition characterized by bone exposure in the oromaxillofacial region of the patients administered with BPs. Here, we report a case of MTX-LPD complicated by BRONJ occurred in a 67-year-old Japanese woman with RA, who received MTX and alendronate sodium hydrate for several years. She had a mass with ulcer formation in the area of hard palate. Histopathologically, large atypical lymphoid cells were found to infiltrate into granulation tissue in the ulcerative oral mucosa. Immunohistochemistry showed that the large atypical lymphoid cells were positive for Ki-67, CD3, CD30, and CD79a and negative for CD15, ALK1, and AE1/AE3. Withdrawal of MTX therapy led to complete remission within 4 weeks. However, 9 weeks later, necrotic bone was formed on hard palate and sequestrectomy was performed twice. Thereafter, the operation for closing naso-oral fistula was performed. The patient has been free of recurrence for 1 year since fistula closure. This is a very rare case in which the oral cavity was the initial site of manifestation of MTX-LPD followed by BRONJ in the patient with RA.