Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We previously reported that the cell and colony motion of oral keratinocytes are correlated with proliferative capacity, and speculated that this may be a specific index for monitoring cell quality. However, how cell motility and proliferation are regulated by signaling pathways remains unelucidated. Here, we found that the regulation of cell motility and proliferative capacity of oral keratinocytes can be attributed to the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis. The EGFR downstream cascade involving the Src/PI3K/Akt/mTOR signaling pathway showed a major effect on cell motility and proliferative capacity in oral keratinocytes. Furthermore, both EGFR and Src attenuated E-cadherin expression. Taken together, these findings provide a potential basis for future quality control of cells for therapeutic use.

DOI： 10.1002/2211-5463.13653

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

As function preservation cancer therapy, targeted radiation therapies have been developed for the quality of life of cancer patients. However, preclinical animal studies evaluating the safety and efficacy of targeted radiation therapy is challenging from the viewpoints of animal welfare and animal protection, as well as the management of animal in radiation-controlled areas under the regulations. We fabricated the human 3D oral cancer model that considers the time axis of the follow up in cancer treatment. Therefore, in this study, the 3D model with human oral cancer cells and normal oral fibroblasts was treated based on clinical protocol. After cancer treatment, the histological findings of the 3D oral cancer model indicated the clinical correlation between tumor response and surrounding normal tissue. This 3D model has potential as a tool for preclinical studies alternative to animal studies.

DOI： 10.3390/biotech12020035

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms24076035

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

BACKGROUND: We previously reported a novel technique for fabricating dermo-epidermal junction (DEJ)-like micropatterned collagen scaffolds to manufacture an ex vivo produced oral mucosa equivalent (EVPOME) for clinical translation; however, more biomimetic micropatterns are required to promote oral keratinocyte-based tissue engineering/regenerative medicine. In addition, in-process monitoring for quality control of tissue-engineered products is key to successful clinical outcomes. However, evaluating three-dimensional tissue-engineered constructs such as EVPOME is challenging. This study aimed to update our technique to fabricate a more biomimetic DEJ structure of oral mucosa and to investigate the efficacy of optical coherence tomography (OCT) in combination with deep learning for non-invasive EVPOME monitoring. METHODS: A picosecond laser-textured microstructure mimicking DEJ on stainless steel was used as a negative mould to fabricate the micropatterned collagen scaffold. During EVPOME manufacturing, OCT was applied twice to monitor the EVPOME and evaluate its epithelial thickness. FINDINGS: Our moulding system resulted in successful micropattern replication on the curved collagen scaffold. OCT imaging visualised the epithelial layer and the underlying micropatterned scaffold in EVPOME, enabling to non-invasively detect specific defects not found before the histological examination. Additionally, a gradual increase in epithelial thickness was observed over time. CONCLUSION: These findings demonstrate the feasibility of using a stainless-steel negative mould to create a more biomimetic micropattern on collagen scaffolds and the potential of OCT imaging for quality control in oral keratinocyte-based tissue engineering/regenerative medicine.

DOI： 10.1016/j.heliyon.2022.e11468

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Institute of Electronics Packaging  

In this study, we developed a novel tissue-engineered oral mucosa with micropatterned fish scale collagen as a biomaterial scaffold. Marine collagen derived from the fish scale is considered to have a significant potential of becoming an alternative transplant material for mucosa regeneration due to its vast resources and minimal risk of transmitting infectious diseases. Eight different types of Grid-Rectangular (GR) negative patterns were manufactured using microelectromechanical systems technologies to evaluate size dependency in collagen replication and epithelial cell layer formation. Among the obtained histological images of the tissue-engineered oral mucosa, the height, width, and channel width of 100 μm showed the most successful formation of the epithelial layer and the most similar structure of oral mucosa <i>in vivo</i>. This study not only established a technique for fabricating a novel biomimetic scaffold but also made an extended approach to structural optimization for mold fabrication. We believe this study will be a crucial step for structural optimization in the future and will have a positive impact on the field of tissue regeneration.

DOI： 10.5104/jiepeng.15.e21-008-1

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Authorship：Last author  

DOI： 10.18939/jsdmd.41.2_162

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Development of effective in vitro human lip models, specific to the vermilion epithelium, has not progressed as much as that of skin and oral mucosa/gingiva models in vitro. Our histologic examination demonstrated that a Japanese macaque (male, 7 years and 9 months old) had vermilion in the lip distinct from adjacent skin and oral mucosa, resembling histological characteristics of the human lip. Therefore, in this study, we examined the gene expression profile of the three distinct epithelia (skin/vermilion/oral mucosa) within the lip of a Japanese macaque to explore a single potential marker of human vermilion epithelium. Six pairwise comparisons in the skin/vermilion/oral mucosa epithelium in vitro and in vivo revealed 69 differentially up-regulated genes in vermilion epithelium in vivo, in which a few unique genes were highly expressed when compared with both skin and oral mucosa epithelium in vivo using clustering analysis. However, we could not detect a single marker specific to vermilion epithelium supported by the gene expression profile of a Japanese macaque. Instead, the pair of keratin 10 and small proline-rich protein 3 resulted in a potential marker of vermilion epithelium in the human lip (female, 53-year-old) via a double-immunostaining technique. Nonetheless, our result may provide further clues leading to other potential markers of the vermilion epithelium.

DOI： 10.3390/anatomia1010002

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Language：English   Publishing type：Research paper (scientific journal)  

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/β/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3β3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

DOI： 10.4049/immunohorizons.2100110

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

<title>Abstract</title>Cells/colony motion determined by non-invasive, quantitative measurements using the optical flow (OF) algorithm can indicate the oral keratinocyte proliferative capacity in early-phase primary cultures. This study aimed to determine a threshold for the cells/colony motion index to detect substandard cell populations in a subsequent subculture before manufacturing a tissue-engineered oral mucosa graft and to investigate the correlation with the epithelial regenerative capacity. The distinctive proliferating pattern of first-passage [passage 1 (p1)] cells reveals the motion of p1 cells/colonies, which can be measured in a non-invasive, quantitative manner using OF with fewer full-screen imaging analyses and cell segmentations. Our results demonstrate that the motion index lower than 40 μm/h reflects cellular damages by experimental metabolic challenges although this value shall only apply in case of our culture system. Nonetheless, the motion index can be used as the threshold to determine the quality of cultured cells while it may be affected by any different culture conditions. Because the p1 cells/colony motion index is correlated with epithelial regenerative capacity, it is a reliable index for quality control of oral keratinocytes.

DOI： 10.1038/s41598-021-89073-y

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Other Link： http://www.nature.com/articles/s41598-021-89073-y

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.tranon.2021.101236

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and α6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFRα. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

DOI： 10.3791/62820

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: The vermilion of the human lip presents characteristic features and undergoes aging faster than the skin. Therefore, knowledge of the vermilion surface-specific functional molecules is important to understand lip aging and formulate lip care products. Previously, we analyzed the free fatty acids distributions and showed that docosahexaenoic acid highly accumulated in the vermilion's epithelium than in the skin. OBJECTIVE: We aimed to explore the functional molecules other than the free fatty acids on the vermilion's surface. METHODS: Human lip tissues from children and tape-stripped samples from smooth and rough lips of adults were measured by desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). RESULTS: DESI-MSI of children's lip sections revealed a major distribution of five phospholipid species in the viable layer, but not in the superficial area, of both the vermilion and the skin than that in the underlying tissue. Interestingly, a remarkably higher distribution of cholesterol sulfate was observed in the vermilion's superficial area compared to that in the skin in all subjects under this study. Furthermore, theDESI-MSI of tape-stripped lip sample showed an overall higher accumulation of cholesterol sulfate in the stratum corneum of the rough lip than that in the smooth lips showed an overall higher accumulation of cholesterol sulfate in the stratum corneum of the rough lips than that in the smooth lips. CONCLUSION: Our study concluded that cholesterol sulfate has a characteristic distribution to the vermilion's surface and showed an association with the roughness of the lip.

DOI： 10.1016/j.jdermsci.2021.07.008

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.bbrc.2021.04.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Hh signaling has been shown to be activated in intact and injured peripheral nerve. However, the role of Hh signaling in peripheral nerve is not fully understood. In the present study, we observed that Hh signaling responsive cells [Gli1(+) cells] in both the perineurium and endoneurium. In the endoneurium, Gli1(+) cells were classified as blood vessel associated or non-associated. After injury, Gli1(+) cells around blood vessels mainly proliferated to then accumulate into the injury site along with endothelial cells. Hh signaling activity was retained in Gli1(+) cells during nerve regeneration. To understand the role of Hedgehog signaling in Gli1(+) cells during nerve regeneration, we examined mice with Gli1(+) cells-specific inactivation of Hh signaling (Smo cKO). After injury, Smo cKO mice showed significantly reduced numbers of accumulated Gli1(+) cells along with disorganized vascularization at an early stage of nerve regeneration, which subsequently led to an abnormal extension of the axon. Thus, Hh signaling in Gli1(+) cells appears to be involved in nerve regeneration through controlling new blood vessel formation at an early stage.

DOI： 10.1016/j.neures.2021.06.003

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.bbrc.2021.02.074

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Language：English   Publishing type：Research paper (scientific journal)  

This study investigates the effect of pre-coating with methyl methacrylate (MMA) containing ultraviolet (UV) photoinitiators on the bond strength of poly(ether ether ketone) (PEEK). Cylindrical PEEK blocks were irradiated with 365 nm UV light for 5-20 s after they were coated with MMA containing 0.4-3.0 wt% UV photoinitiators: [1-phenyl-1,2-propanedione (PPD)], [diphenyl(2,4,6-trimethylbenzoyl) (TMDPO)], and [phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide (BTMPO)]. Pre-coated PEEKs were bonded to PEEK blocks with a MMA-based adhesive resin. The shear bond strength was measured using a universal testing machine. Secondary electron images were captured to observe failure surfaces. The data were analyzed with one- and two-way ANOVA and Tukey's post hoc tests (p<0.05). The highest bond strength (20.7±5.1 MPa) was observed for pre-coating with MMA containing 0.4 wt% BTMPO, for 20 s of UV irradiation. Cohesive failure of the adhesive resin was observed. The use of this pre-coating led to improved bonding performance of PEEK.

DOI： 10.4012/dmj.2020-068

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

The oxygen concentration in normal human tissue under physiologic conditions is lower than the atmospheric oxygen concentration. The more hypoxic condition has been observed in the cells with wound healing and cancer. Somatic stem cells reside in a hypoxic microenvironment in vivo and prefer hypoxic culture conditions in vitro. Oral mucosa contains tissue-specific stem cells, which is an excellent tissue source for regenerative medicine. For clinical usage, maintaining the stem cell in cultured cells is important. We previously reported that hypoxic culture conditions maintained primary oral keratinocytes in an undifferentiated and quiescent state and enhanced their clonogenicity. However, the metabolic mechanism of these cells is unclear. Stem cell biological and pathological findings have shown that metabolic reprogramming is important in hypoxic culture conditions, but there has been no report on oral mucosal keratinocytes and fibroblasts. Herein, we conducted metabolomic analyses of oral mucosal keratinocytes and fibroblasts under hypoxic conditions. Hypoxic oral keratinocytes and fibroblasts showed a drastic change of metabolite concentrations in urea cycle metabolites and polyamine pathways. The changes of metabolic profiles in glycolysis and the pentose phosphate pathway under hypoxic conditions in the oral keratinocytes were consistent with those of other somatic stem cells. The metabolic profiles in oral fibroblasts showed only little changes in any pathway under hypoxia except for a significant increase in the antioxidant 2-oxoglutaric acid. This report firstly provides the holistic changes of various metabolic pathways of hypoxic cultured oral keratinocytes and fibroblasts.

DOI： 10.3390/jcm10061156

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

Fish-derived collagen is considered a promising material for oral mucosa tissue engineering because it is free from risk of transmissible infectious diseases. We developed a novel fish scale collagen scaffold with micropatterns on which oral keratinocytes are cultured to create a tissue-engineered oral mucosa. Anisotropic and isotropic etching were used to fabricate negative patterns on the Si substrate and soft lithography was used to translate the patterns to PDMS and fish scale collagen. The histological evaluation of the tissue-engineered oral mucosa confirmed the successful formation of the epithelial layer mimicking oral mucosa in vivo. This study suggests that our novel approach for fabricating biomimetic scaffold is promising and will make a huge contribution to the field of oral mucosa regeneration.

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>The junction between the epithelium and the underlying connective tissue undulates, constituting of rete ridges, which lack currently available soft tissue constructs. In this study, using a micro electro mechanical systems process and soft lithography, fifteen negative molds, with different dimensions and aspect ratios in grid- and pillar-type configurations, were designed and fabricated to create three-dimensional micropatterns and replicated onto fish-scale type I collagen scaffolds treated with chemical crosslinking. Image analyses showed the micropatterns were well-transferred onto the scaffold surfaces, showing the versatility of our manufacturing system. With the help of rheological test, the collagen scaffold manufactured in this study was confirmed to be an ideal gel and have visco-elastic features. As compared with our previous study, its mechanical and handling properties were improved by chemical cross-linking, which is beneficial for grafting and suturing into the complex structures of oral cavity. Histologic evaluation of a tissue-engineered oral mucosa showed the topographical microstructures of grid-type were well-preserved, rather than pillar-type, a well-stratified epithelial layer was regenerated on all scaffolds and the epithelial rete ridge-like structure was developed. As this three-dimensional microstructure is valuable for maintaining epithelial integrity, our micropatterned collagen scaffolds can be used not only intraorally but extraorally as a graft material for human use.

DOI： 10.1038/s41598-020-79114-3

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Other Link： http://www.nature.com/articles/s41598-020-79114-3

Language：English   Publishing type：Research paper (scientific journal)  

The vermilion of the human lip is a unique facial area because of certain distinguishing features from the adjacent tissues such as the white lip (skin) and oral mucosa. However, the distinction in terms of molecular distribution between the vermilion and skin has remained unexplored. Therefore, we aimed to map the human lip by mass spectrometry imaging to gain understanding of the free fatty acid distribution in the vermilion. The lip specimens trimmed off during cheiloplasty were analyzed using desorption electrospray ionization-mass spectrometry imaging. Distributions of two monounsaturated fatty acids and three polyunsaturated fatty acids were observed in the human lip tissue: palmitoleic acid (POA) and oleic acid (OA) and linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA), respectively. Although POA, OA, LA, and AA were differentially distributed across the vermilion and skin, DHA showed a higher accumulation in the epithelium of the vermilion compared to that in the skin. Our results clearly demonstrated the difference in fatty acid distributions between the vermilion and skin. The highly abundant DHA in the epithelium of the vermilion may have an antioxidant role and may thus protect the lip from aging. Our findings can provide a novel strategy for treating lip disorders.

DOI： 10.3390/ijms21082807

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The present study aimed to develop a more biomimetic tissue-engineered oral mucosa equivalent comprising 1% type I tilapia scale collagen scaffold having microstructures mimicking the dermal-epidermal junction of oral mucosa and oral keratinocytes as graft materials for human use. We designed four micropattern prototypes mimicking the dermal-epidermal junction. Using a semiconductor process and soft lithography, negative molds were fabricated to develop microstructures using both polydimethylsiloxane and silicon substrates. Micropattern configurations of dermal-epidermal junctions manufactured from fish collagen consisting of a fibril network using our micropatterning system were well preserved, although the internal fibril network of the pillar pattern was sparse. Mixing 1% chondroitin sulfate with the collagen matrix minimized tissue-engineered oral mucosa equivalent contraction. Histologic examinations showed a flattening of the vertical dimensions of all microstructures and expansion of their pitches, indicating changes in the originally designed configurations. Nonetheless, histologic examinations revealed that a fully differentiated and stratified epithelial layer was developed on all scaffolds, suggesting that the microstructured fish scale collagen scaffolds have potential in the manufacturing of tissue-engineered oral mucosa equivalents for clinical use; however, enhancement of the mechanical properties of micropatterns is required. Our micropatterning technology can also apply to the development of oral mucosa in vitro models.

DOI： 10.1080/09205063.2019.1706147

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：SAGE Publications  

Image-based cell/colony analyses offer promising solutions to compensate for the lack of quality control (QC) tools for noninvasive monitoring of cultured cells, a regulatory challenge in regenerative medicine. Here, the feasibility of two image analysis algorithms, optical flow and normalised cross-correlation, to noninvasively measure cell/colony motion in human primary oral keratinocytes for screening the proliferative capacity of cells in the early phases of cell culture were examined. We applied our software to movies converted from 96 consecutive time-lapse phase-contrast images of an oral keratinocyte culture. After segmenting the growing colonies, two indices were calculated based on each algorithm. The correlation between each index of the colonies and their proliferative capacity was evaluated. The software was able to assess cell/colony motion noninvasively, and each index reflected the observed cell kinetics. A positive linear correlation was found between cell/colony motion and proliferative capacity, indicating that both algorithms are potential tools for QC.

DOI： 10.1177/2041731419881528

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Other Link： http://journals.sagepub.com/doi/full-xml/10.1177/2041731419881528

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Inc.  

Oral mucosa primarily acts as a barrier against the external harmful environments. Loss of its barrier function due to diseases or injury will cause significant dysfunction within the oral cavity. Surgeons are frequently confronted with finding an acceptable source of autologous grafts for reconstruction of oral mucosa defects. Thus, there is a need to overcome these shortcomings of limited supply and donor site morbidity in the current surgical management/reconstruction of oral mucosa defects. Tissue engineering/regenerative medicine is an interdisciplinary field of developmental biology, life sciences, and engineering efforts that attempts to address challenges in the clinical arena. The understanding of the growth and functions of cells, the principles and methods of engineering, and the signals regulating cellular responses drives the fabrication of matrices and the design of tissue assembly to generate tissue-engineered products for in vivo and in vitro applications. Progress has been made over the years in the development of tissue-engineered substitutes that mimic human oral mucosa, either to be used as grafts for the replacement of mucosa defects, or for the in vitro oral mucosa models while tissue engineering of oral mucosa is still in its infancy. An increased understanding of stem cells, scaffolding, and signaling with extracellular matrix interactions will make its future possible. This chapter gives a comprehensive overview of the developments and future prospects of tissue-engineered constructs as oral mucosa substitutes for tissue repair and regeneration.

DOI： 10.1016/B978-0-12-397157-9.00077-1

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O-2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G(0)/G(1) phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated beta-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3 sigma, p75(NTR) and alpha 6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G(0)/G(1) phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer beta-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1 alpha favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state. (C) 2015 S. Karger AG, Basel

DOI： 10.1159/000371342

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：QUINTESSENCE PUBLISHING CO INC  

Purpose: The primary objective of this study was to evaluate the safety of a tissue-engineered human ex vivo-produced oral mucosa equivalent (EVPOME) in intraoral grafting procedures. The secondary objective was to assess the efficacy of the grafted EVPOME in producing a keratinized mucosal surface epithelium. Materials and Methods: Five patients who met the inclusion criteria of having one mucogingival defect or a lack of keratinized gingiva on a nonmolar tooth, along with radiographic evidence of sufficient interdental bone height, were recruited as subjects to increase the width of keratinized gingiva at the defect site. A punch biopsy specimen of the hard palate was taken to acquire oral keratinocytes, which were expanded, seeded, and cultured on an acellular dermal matrix for fabrication of an EVPOME. EVPOME grafts were applied directly over an intact periosteal bed and secured in place. At baseline (biopsy specimen retrieval) and at 7, 14, 30, 90, and 180 days postsurgery, Plaque Index and Gingival Index were recorded for each subject. In addition, probing depths, keratinized gingival width, and keratinized gingival thickness were recorded at baseline, 30, 90, and 180 days. Results: No complications or adverse reactions to EVPOME were observed in any subjects during the study. The mean gain in keratinized gingival width was 3 mm (range, 3 to 4 mm). The mean gain in keratinized gingival thickness was 1 mm (range, 1 to 2 mm). No significant changes in probing depths were observed. Conclusion: Based on these findings, it can be concluded that EVPOME is safe for intraoral use and has the ability to augment keratinized tissue around teeth. Future clinical trials are needed to further explore this potential.

DOI： 10.11607/jomi.te11

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Language：Japanese   Publisher：(一社)日本歯科医学会連合  

アメリカ合衆国(米国)には1980年当時,123社の航空会社があり,アメリカン航空(American Airlines,AA)はその中で中位クラスであったが,クランドール(Robert L. Crandall)が考え出した戦略的情報システム(Strategic Information System,SIS)により業績が上がり,他社を次々に淘汰した。AAのSISに気づいて戦略的に手を打ったユナイテッド航空とデルタ航空は生き残り,目先の戦術に追われた他社はことごとく倒産した。結局,米国の航空会社は3社系列にまで統合された。また,アメリカン・ホスピタル・サプライは独自の情報システムを用い,米国に2,000店あった医薬品医療機器流通業者を40店にまで淘汰した。このようにコンピュータシステムを戦略的に活用したSISの事例が研究された結果,事業を成功に導くには,好印象を持たれるように人の心に訴えることが重要だと考えられるようになった。(著者抄録)

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Language：Japanese   Publisher：(一社)日本人工臓器学会  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The differences between oral mucosa and skin wound healing involving hypoxic responses of fibroblasts are poorly elucidated. In this study, we aimed to study the different hypoxic responses between oral and skin fibroblasts embedded in a three-dimensional (3D) collagen matrix to address the early stage of wound healing. Primary oral mucosa fibroblasts (OMFs) obtained from the retromolar area and skin fibroblasts (SFs) obtained from the abdomen were cultured in the 3D 'floating model' under either 21%, 5% or 1% O2 for 2 days. Cell viability under hypoxia was higher in the OMFs than in the SFs. Collagen gel contraction was suppressed under hypoxic conditions in both fibroblasts, consistent with the reduction of alpha smooth muscle actin expression, except for SFs under 1% O2. Subsequently, their gene expression profiles between 21 and 1% O2 concentrations were compared via microarray technology, and the expression profiles of the extracellular matrix (ECM)-associated proteins, including matrix metalloproteinases and collagens, were evaluated. The OMFs were more susceptible to 1% O2, and more of their genes were downregulated than the SFs'. Although the production and expression levels of ECM-associated proteins in both fibroblasts diminished under hypoxia, those levels in OMFs were significantly higher than those in SFs. In the case of single origin OMFs and SFs, our findings suggest that OMFs possess a higher baseline production capacity of several ECM-associated proteins than SFs, except type III collagen. The intrinsic hypoxic responses of OMFs may be attributed to a more favourable wound healing in oral mucosa.

DOI： 10.1007/s11626-020-00458-1

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<jats:title>Abstract</jats:title><jats:p>
Objective The aim of this study was to examine the potential of periodontal ligament (PDL) cells sheet and arginine-glycyl-aspartic acid (RGD)-modified chitosan scaffold for periodontal tissue regeneration in horizontal periodontal defect model.</jats:p><jats:p>
Materials and Methods PDL cell cytotoxicity was tested with 3–[4,5- dimethylthiazol-2yl]–2,5-diphenyl-2H-tetrazolium bromide assay. Cell migration toward the chitosan-based materials was analyzed with trans-well migration assay. Horizontal periodontal defect model was created in four maxillary and mandibular lateral incisors of Macaque nemestrina. Following periodontal therapy, the sites were transplanted with various regenerative materials: (1) chitosan, (2) RGD-modified chitosan, (3) PDL cell sheet with chitosan, (4) PDL cell sheet with RGD-modified chitosan. The periodontal tissue regeneration was evaluated clinically and radiographically. Gingival crevicular fluids were collected each week to evaluate cementum protein-1 (CEMP-1) expression with enzyme-linked immunosorbent assay, while the biopsies were retrieved after 4 weeks for histological and microcomputed tomography evaluation.</jats:p><jats:p>
Statistical Analysis Data was statistically analyzed using GraphPad Prism 6 for MacOS X. Normality was tested using the Shapiro–Wilk normality test. The Kruskal–Wallis test was used to compare the groups. Significance was accepted when p &lt; 0.05.</jats:p><jats:p>
Results Clinical examination revealed more epithelial attachment was formed in the group with PDL cell sheet with RGD-modified chitosan. Similarly, digital subtraction radiography analysis showed higher gray scale, an indication of higher alveolar bone density surrounded the transplanted area, as well as higher CEMP-1 protein expression in this group. The incorporation of RGD peptide to chitosan scaffold in the group with or without PDL cells sheet reduced the distance of cement–enamel junction to the alveolar bone crest; hence, more periodontal tissue formed.</jats:p><jats:p>
Conclusions Horizontal periodontal defect model could be successfully created in M. nemestrina model. Combination of PDL cell sheet and RGD-modified chitosan resulted in the higher potential for periodontal tissue regeneration. The results of this study highlight the PDL cell sheet and RGD-modified chitosan as a promising approach for future clinical use in periodontal regeneration.</jats:p>

DOI： 10.1055/s-0040-1709955

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Apoptotic cell death frequently occurs in human cancer tissues including oral squamous cell carcinoma (SCC), wherein apoptotic tumor cells are phagocytosed not only by macrophages but also by neighboring tumor cells. We previously reported that the engulfment of apoptotic SCC cells by neighboring SCC cells frequently occurs at the invading front. Therefore, we hypothesized that the phagocytosis of these apoptotic cells by tumor cells contributes to disease progression. Herein, using cultured oral SCC cells, we aimed to confirm whether tumor cells actually phagocytose apoptotic cells and to examine whether cellular activities are regulated by the phagocytosis of apoptotic cells. Co-culture experiments showed that living cells could ingest apoptotic cells into phagolysosomes. NSC23766, an inhibitor of Rac1, which is a key regulator of phagocytic cup formation in professional phagocytes, dramatically suppressed the phagocytosis of apoptotic cells by living cells. Additionally, cell migration and the secretion of DKK1, a tumor-promoting protein, were enhanced by co-culture with apoptotic cells, whereas NSC23766 inhibited these effects. These results show that tumor cells can actively phagocytose apoptotic neighbors in a Rac1-dependent manner and that such activity increases their migration. The regulation of apoptotic cell phagocytosis thus represents new directions for therapeutic intervention for oral cancer.

DOI： 10.1016/j.yexcr.2020.112013

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Osteoclast and osteoblast are essential for proper bone development and remodeling as well as recovery of bone fracture. In this study, we seek chemical compounds that enhance turnover of bone metabolism for promoting bone healing. First, we screen a chemical library which includes 378 compounds by using murine pre-osteoclastic RAW264.7 cells to identify compounds that promote osteoclastic differentiation. We find that two ROCK (Rho-associated coiled-coil kinase) inhibitors, HA-1077 (Fasudil) and Y-27632, enhance osteoclastogenesis. Subsequently, we identify that these two compounds also increase osteoblastic differentiation of MC3T3-E1 cells. Finally, our in vivo experiment shows that the local administration of ROCK inhibitors accelerate the bone healing of the rat calvarial defect.

DOI： 10.1016/j.bbrc.2020.03.033

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The micro- and nanosize surface topography of dental implants has been shown to affect the growth of surrounding cells. In this study, standardized and controlled periodic nanopatterns were fabricated with nanosized surface roughness on titanium substrates, and their influence on bone marrow stromal cells investigated. Cell proliferation assays revealed that the bare substrate with a 1.7 nm surface roughness has lower hydrophilicity but higher proliferation ability than that with a 0.6 nm surface roughness. Further, with the latter substrate, directional cell growth was observed for line and groove patterns with a width of 100 nm and a height of 50 or 100 nm, but not for those with a height of 10 or 25 nm. With the smooth substrate, time-lapse microscopic analyses showed that more than 80% of the bone marrow cells on the line and groove pattern with a height of 100 nm grew and divided along the lines. As the nanosized grain structure controls the cell proliferation rate and the nanosized line and groove structure (50-100 nm) controls cell migration, division, and growth orientation, these standardized nanosized titanium structures can be used to elucidate the mechanisms by which surface topography regulates tissue responses to biomaterials.

DOI： 10.1038/s41598-020-59395-4

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Publishing type：Research paper (scientific journal)   Publisher：SAGE Publications  

Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68⁺CD86⁺cells (M1 phenotype) and CD68⁺CD163⁺cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68⁺cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.

DOI： 10.1177/0022034519894957

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Other Link： http://journals.sagepub.com/doi/full-xml/10.1177/0022034519894957

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Quintessence Publishing  

PURPOSE:This study aimed to evaluate the safety and efficacy of newly designed, laser-perforated pure titanium membranes for guided bone regeneration and to compare them with an existing product, the FRIOS BoneShield (FBS). MATERIALS AND METHODS:Acute-type lateral ridge defects were bilaterally created in the mandibles of 13 dogs (two defects per animal). The defects were randomly divided into three groups and were reconstructed with particulate autologous bone (PAB) in combination with three different titanium membranes: (1) F001M0 (prototype without a frame), (2) F001M1 (prototype with a frame), and (3) FBS as a standard membrane. All animals were observed periodically and sacrificed 26 or 27 weeks postoperatively. At 26 weeks, approximately half of the dogs in each group underwent membrane removal to examine the postoperative condition of the titanium membranes. The samples were dissected and processed for radiographic, histologic, and histomorphometric analyses. RESULTS:Membrane exposure was not found in the F001M0 or F001M1 groups, and their membranes were removed easily without adhesion to the surrounding tissue. Regenerated bone tissue volume was largest in the F001M1 group, followed by the F001M0 and FBS groups. A significant difference was observed only between the F001M1 and FBS groups (P = .047). In contrast, bone mineral density was similar among the three groups. Histologically, a layer of fibrous tissue was present underneath the titanium membrane, overlying the regenerated cortical bone in all the groups. Notably, the tissue was highly vascular in the F001M1 and F001M0 groups compared with the FBS group. In addition, there was little difference in the semiquantitative soft tissue evaluation and histologic findings of bone regeneration among the three groups. The histomorphometric analysis revealed that the regenerated bone area was larger in the F001M1 and F001M0 groups than in the FBS group, and a significant difference was observed only between the F001M1 and FBS groups (P = .045). Calcific osseous area was similar among the three groups. CONCLUSION:The safety and efficacy of both F001M0 and F001M1 were equivalent to or greater than those of FBS, thereby indicating their potential for future clinical applications.

DOI： 10.11607/jomi.6777

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PURPOSE:This study evaluated the efficacy of newly designed, laser-perforated pure titanium membranes for guided bone regeneration using beta-tricalcium phosphate (β-TCP), and compared them with the existing membrane. MATERIALS AND METHODS:Bilateral acute lateral ridge defects were created in the mandibles of 12 dogs (four defects per animal), which were then randomly divided into two groups (six dogs each). The twenty-four bone defects in each group were then further divided into five groups. The groups were as follows: (1) F001M0, a prototype membrane without a frame plus β-TCP (n = 5); (2) F001M1, a prototype membrane with a frame plus β-TCP (n = 5); (3) FBS, an existing control membrane plus β-TCP (n = 5); (4) control 1, β-TCP without membrane and with covering flap only (n = 5); and (5) control 2, no treatment (no β-TCP and no membrane) (n = 4). In all groups where β-TCP was used, it was mixed with peripheral blood. The animals were necropsied at 6 or 12 weeks postoperatively (six dogs each), and samples were collected and processed for radiographic, histologic, and histomorphometric analyses. RESULTS:Among the three membrane groups, regenerated tissue and bone volume was greatest in the F001M1 group at both 6 and 12 weeks postoperatively, although differences among groups were not statistically significant. Bone mineral density was similar among the membrane groups. Histologic analysis revealed that immature fibroblasts were present on the laser-perforated portion at 6 weeks, which induced vascularization. In addition, more calcified bone was replaced beneath the prototypes than beneath the FBS membrane at 12 weeks. Histomorphometric analyses revealed that the calcific osseous areas at 12 weeks after surgery were significantly greater in the F001M1 and F001M0 groups than in the FBS group (P = .021, P = .032). Furthermore, the fibrous tissue areas beneath the membrane at 12 weeks postoperatively were significantly smaller in the prototype groups than in the FBS group (P = .02, P = .02). CONCLUSION:The efficacies of both prototype membranes were not inferior to that of the FBS membrane, indicating that they may facilitate bone regeneration and maturation when β-TCP mixed with autologous blood is employed.

DOI： 10.11607/jomi.6776

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DOI： 10.4236/ojst.2018.82005

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DOI： 10.1678/rheology.46.179

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Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Identifying substandard tissue-engineered oral mucosa grafts with a poor epithelium before clinical use is critical to ensure quality assurance/control in regenerative medicine, leading to success of grafting. This study investigated the effects of one of the C-xylopyranoside derivatives, -D-xylopyranoside-n-propane-2-one (XPP), on oral epithelial regeneration. Using a three-dimensional oral mucosa model, we analyzed changes of the epithelial structure, glycosaminoglycan (GAG) synthesis, the expression levels of basement membrane zone markers, and substrates of Akt/mTOR signaling. Compared with the control, 2mM XPP treatment increased the mean and minimal epithelial thickness, and reduced the variation of epithelial thickness. It also stimulated expressions of decorin and syndecan-1 with change of GAG amount and/or composition, and enhanced the expressions of integrin 6, CD44, and Akt/mTOR signaling substrates. These findings suggest that XPP supplementation contributes to consistent epithelial regeneration. Moreover, upregulation of those markers may play a role in increasing the quality of the oral mucosal epithelium.

DOI： 10.1080/09168451.2016.1153957

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Denture-wearing affects the quality and quantity of epithelial cells in the underlying healthy oral mucosa. The physiologic mechanisms, however, are poorly understood. This study aimed to compare histologic changes and cellular responses of an epithelial cell layer to cyclic mechanical pressure-loading mimicking denture-wearing using an organotypic culture system to develop a three-dimensional in vitro oral mucosa model (3DOMM). Primary human oral keratinocytes and fibroblasts were serially grown in a monolayer culture, and cell viability was measured under continuous cyclic mechanical pressure (50kPa) for 7days (cycles of 60min on, 20s off to degas and inject air). Upon initiation of an air-liquid interface culture for epithelial stratification, the cyclic pressure, set to the mode above mentioned, was applied to the 3DOMMs for 7days. Paraffin-embedded 3DOMMs were examined histologically and immunohistochemically. In the monolayer culture, the pressure did not affect the viability of oral keratinocytes or fibroblasts. Few histologic changes were observed in the epithelial layer of the control and pressure-loaded 3DOMMs. Immunohistochemical examination, however, revealed a significant decrease in Ki-67 labelling and an increase in filaggrin and involucrin expression in the suprabasal layer of the pressure-loaded 3DOMMs. Pressure-loading attenuated integrin 1 expression and increased matrix metalloproteinase-9 activity. Incomplete deposition of laminin and type IV collagen beneath the basal cells was observed only in the pressure-loaded 3DOMM. Cyclic pressure-loading appeared to disrupt multiple functions of the basal cells in the 3DOMM, resulting in a predisposition towards terminal differentiation. Thus, denture-wearing could compromise oral epithelial homeostasis.

DOI： 10.1111/joor.12254

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

This study examined the negative effects of zoledronic acid on the re-epithelialization of oral mucosa in a three-dimensional in vitro oral mucosa wound healing model. A living oral mucosa equivalent was constructed by seeding a mixture of primary human oral keratinocytes and fibroblasts, at a cell density of 1.5 x 10(5) cm(2) each, onto human cadaver dermis. This was cultured in a submerged condition in 1.2 mM Ca2+ EpiLife for 5 days, and then in an air liquid interface for 14 days. The equivalent was wounded by excising a linear 2-mm-wide epithelial layer on day 8 and subsequently incubated with 10 mu M zoledronic acid for an additional 11 days. Histological and immunohistochemical observations revealed zoledronic acid to significantly suppress the epithelial thickness and Ki-67-labelling index. Zoledronic acid also abolished integrin alpha v beta 6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-beta 1) production into the supernatant, but down-regulated TGF-beta receptor types I and II expression and Smad3 phosphorylation, as was also confirmed by immunofluorescence microscopy. This study therefore showed zoledronic acid to abrogate integrin alpha v beta 6 expression, cause the down-regulation of TGF-beta/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.

DOI： 10.1016/j.ijom.2013.06.016

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.

DOI： 10.1007/s00418-012-1064-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Mary Ann Liebert Inc.  

A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by H&amp
E histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2 nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator. © 2013, Mary Ann Liebert, Inc.

DOI： 10.1089/ten.tec.2012.0287

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The isolation of human oral mucosa/skin keratinocytes progenitor/stem cells is clinically important to regenerate epithelial tissues for the treatment of oral mucosa/skin defects. Researchers have attempted to isolate a keratinocyte progenitor/stem cell population using cell markers, rapid adherence to collagen type IV, and other methods. In this regard, one of the specific characteristics of keratinocyte progenitor/stem cells is that these cells have a smaller diameter than differentiated cells. This chapter describes methods used in our laboratory to set up primary human oral mucosa and skin keratinocytes in a chemically defined culture system devoid of animal derived products. We utilized the cells in a FDA-approved human clinical trial that involved the intraoral grafting of an ex vivo produced oral mucosa equivalent to increase keratinized tissue around teeth. We also provide two protocols on how to sort keratinocytes using physical criterion, cell size, using a cell sorter and a serial filtration system. © 2013 Springer Science+Business Media New York.

DOI： 10.1007/978-1-62703-330-5-23

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

This study was designed to (1) assess the in vitro biocompatibility of a chitosancollagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm (R) (EVPOME-A), BioMend (R) Extend (TM) (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the beta 1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine. (C) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.

DOI： 10.1002/jbm.b.32746

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Objective: This study aimed to clarify the effects of zoledronic acid (ZOL) on human primary oral mucosal keratinocytes growing in a monolayer culture and on a tissue-engineered oral mucosal construct.
Design: Changes in the viability and proliferation of oral keratinocytes incubated with ZOL were measured. Following treatment with 10 mu M ZOL, histological examinations and immunohistochemical analyses for Ki-67, Geminin, and phospho-histone (gamma)-H2A.X were performed on tissue-engineered oral mucosa. Cell cycle distribution and the degree of apoptosis were also measured by flow cytometry. Additionally, we measured the expression of cell cycle regulatory proteins as well as phospho-Chk1 and -Chk2.
Results: ZOL treatment suppressed cell viability and proliferation in a dose-dependent manner. Compared with untreated tissue-engineered oral mucosa, ZOL treatment resulted in a thinner epithelium in which the basal cells appeared less-organised. This is consistent with the observed significant reduction in the Ki-67 labelling index. In contrast, the Geminin labelling index was significantly higher than that in the untreated sample. In spite of the presence of a few apoptotic cells, ZOL induced an arrest in S-phase, which was confirmed by our observed alterations in the expression levels of cyclin A, B1, p27(KIP1), Rb and phospho-Rb. When the proteasome inhibitor MG132 was added to the ZOL-treated cells, we observed a partial restoration of the expression of cyclin A, cyclin B1, and p27(KIP1). Expression of phospho-Chk1 was detected, and a significant increase in the labelling index of gamma-H2A.X was also seen.
Conclusions: These results indicate that a 10-mu M ZOL treatment induces a DNA damage response in oral keratinocytes that activates the ubiguitin-mediated proteolysis of cell cycle regulators, resulting in cell cycle arrest and repressive effects on cell viability, proliferation, and epithelial turnover. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2011.11.015

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Language：English   Publishing type：Research paper (international conference proceedings)  

We show the application of near-infrared Raman Spectroscopy to in-vitro monitoring of the viability of tissue constructs (EVPOMEs). During their two week production period EVPOME may encounter thermal, chemical or biochemical stresses that could cause development to cease, rendering the affected constructs useless. We discuss the development of a Raman spectroscopic technique to study EVPOMEs noninvasively, with the ultimate goal of applying it in-vivo. We identify Raman spectroscopic failure indicators for EVPOMEs, which are stressed by temperature, and discuss the implications of varying calcium concentration and pre-treatment of the human keratinocytes with Rapamycin. In particular, Raman spectra show correlation of the peak height ratios of CH 2 deformation to phenylalanine ring breathing, providing a Raman metric to distinguish between viable and nonviable constructs. We also show the results of singular value decomposition analysis, demonstrating the applicability of Raman spectroscopic technique to both distinguish between stressed and non-stressed EVPOME constructs, as well as between EVPOMEs and bare AlloDerm substrates, on which the oral keratinocytes have been cultured. We also discuss complications arising from non-uniform thickness of the AlloDerm® substrate and the cultured constructs, as well as sampling protocols used to detect local stress and other problems that may be encountered in the constructs. © 2012 SPIE.

DOI： 10.1117/12.908281

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPIE-INT SOC OPTICAL ENGINEERING  

We show the application of near-infrared Raman Spectroscopy to in-vitro monitoring of the viability of tissue constructs (EVPOMEs). During their two week production period EVPOME may encounter thermal, chemical or biochemical stresses that could cause development to cease, rendering the affected constructs useless. We discuss the development of a Raman spectroscopic technique to study EVPOMEs noninvasively, with the ultimate goal of applying it in-vivo. We identify Raman spectroscopic failure indicators for EVPOMEs, which are stressed by temperature, and discuss the implications of varying calcium concentration and pre-treatment of the human keratinocytes with Rapamycin. In particular, Raman spectra show correlation of the peak height ratios of CH2 deformation to phenylalanine ring breathing, providing a Raman metric to distinguish between viable and nonviable constructs. We also show the results of singular value decomposition analysis, demonstrating the applicability of Raman spectroscopic technique to both distinguish between stressed and non-stressed EVPOME constructs, as well as between EVPOMEs and bare AlloDerm (R) substrates, on which the oral keratinocytes have been cultured. We also discuss complications arising from non-uniform thickness of the AlloDerm (R) substrate and the cultured constructs, as well as sampling protocols used to detect local stress and other problems that may be encountered in the constructs.

DOI： 10.1117/12.908281

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Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier Inc.  

DOI： 10.1016/B978-1-4160-2527-6.00009-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Acoustic microscopy was used to monitor an ex vivo produced oral mucosal equivalent (EVPOME) developed on acellular cadaveric dermis (AlloDerm (R)). As seeded cells adhered and grew, they filled in and smoothed out the surface irregularities, followed by the production of a keratinized protective outermost layer. If noninvasive in vitro ultrasonic monitoring of these cellular changes could be developed, then tissue cultivation could be adjusted in-process to account for biologic variations in the development of these stratified cell layers. Cultured keratinocytes (from freshly obtained oral mucosa) were harvested and seeded onto AlloDerm (R) coated with human type IV collagen and cultured 11 days. EVPOMEs were imaged on the 11th day post-seeding using a scanning acoustic microscope (SAM) that consists of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f-number. The specimen surface was determined by thresholding the magnitude of the signal at the first axial incidence of a value safely above noise: 20-40 dB above the signal for the water and 2-dimensional (2-D) ultrasonic images were created using confocal image reconstruction. A known area from each micrograph was divided into 12-40 even segments and examined for surface irregularities. These irregularities were quantified and one-way analysis of variance (ANOVA) and linear regression analysis were performed to correlate the surface profiles for both the AlloDerm (R) and EVPOME specimens imaged by SAM. Histology micrographs of the AlloDerm (R) and EVPOME specimens were also prepared and examined for surface irregularities. Unseeded AlloDerm (R) averaged seven to nine surface changes per 400 mu m. The number of changes in surface irregularities decreased to two to three per 400 mu m on the mature EVPOMEs. The numbers of surface irregularities between the unseeded AlloDerm (R) vs. developing EVPOME are similar for both histology and SAM 2-D B-scan images. For the EVPOME 2-D B-scan micrographs produced by SAM, the decrease in surface irregularities is indicative of the stratified epithelium formed by seeded oral keratinocytes; verified in the histology images between the AlloDerm (R) and EVPOME. A near 1: 1 linear correlation shows the similarities between the two imaging modalities. SAM demonstrates its ability to discern the cell development and differentiation occurring on the EVPOME devices. Unlike histology, SAM measurements are noninvasive and can be used to monitor tissue graft development without damaging any cells/tissues. (E-mail: fwinterr@umich.edu) (C) 2011 World Federation for Ultrasound in Medicine & Biology.

DOI： 10.1016/j.ultrasmedbio.2011.06.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

To ensure quality control and assurance in tissue engineering, noninvasive, real-time and aseptic evaluation of cell-based devices is required before product release. In this study, Raman spectroscopy was applied to monitor the cellular activities of an oral mucosa equivalent (EVPOME) produced ex vivo from cultured autogenous oral keratinocytes and acellular dermis - AlloDerm. Raman spectra showed a positive correlation of the peak area ratio of amide I (1655 cm(-1))/phenylalanine (1004 cm(-1)) with a negative linear regression (R(2) &gt; 0.95) according to the number of cultured days, especially on the 14th and 21st day. This work demonstrates the successful application of Raman spectroscopy for quantitatively monitoring and evaluating the maturity of EVPOME. Copyright (C) 2010 John Wiley & Sons, Ltd.

DOI： 10.1002/jrs.2688

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The acid-sensing ion channel 3 (ASIC3), a member of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily, has been reported to participate in acid sensing, mechanosensation, and nociception. However, no information is available regarding the precise localization and function of this molecule in the periodontal ligament, which contains abundant sensory nerves originating from the trigeminal ganglion. The present study examined the expression of ASIC3 in the lingual periodontal ligament of mouse incisors by immunohistochemistry. Furthermore, the expression of ASIC3 in the trigeminal ganglion which innervates the periodontal ligament - was investigated at protein (immunohistochemistry and quantitative analysis) and mRNA levels (RT-PCR technique and in situ hybridization histochemistry). Immunohistochemistry for ASIC3 was able to demonstrate dendritic profiles of the periodontal Ruffini endings in the mouse incisors. No thin fibers terminating as nociceptive free nerve endings exhibited ASIC3 immunoreactivity. Double immunofluorescent staining revealed ASIC3 immunoreaction in the axoplasm but not in the ordinary Schwann cells - including the associated terminal Schwann cells. Observation of the trigeminal ganglia showed variously sized neurons expressing ASIC3 immunoreaction; the most intense immunopositivity was found in the small and medium-sized neurons, as confirmed by in situ hybridization histochemistry using a specific cRNA probe. Quantitative analysis on trigeminal ganglion neurons showed that 38.0% of ASIC3 neurons could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that ASIC3 functions as a molecule for mechanosensation in the periodontal Ruffini endings. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2010.11.023

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Optical Society of America (OSA)  

Endogenous fluorescence redox imaging was developed to noninvasively assess cell viability in 3-dimensional tissue-engineered constructs prior to implantation. A lower redox ratio was observed from samples with higher proliferation. © 2011 OSA: BODA/NTM/OMP/OTA.

DOI： 10.1364/omp.2011.omc1

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Language：Japanese   Publisher：一般社団法人 日本口蓋裂学会  

This report presents the use of &lt;I&gt;ex vivo&lt;/I&gt; produced oral mucosa equivalent (EVPOME) applied to an oral mucosa defect in two patients with cleft lip and palate. Autologous keratinocytes were placed and propagated in a chemically-defined, animal product and a xenogeneic feeder layer free culture system. Cells were then seeded and cultured on an acellular dermal substrate, AlloDerm&lt;sup&gt;&amp;reg;&lt;/sup&gt;, which provides strength, durability, and elasticity to the graft.&lt;br&gt;In case 1, a 20-year-old female underwent closure of the oronasal fistula in the midline of the hard palate using a rotational palatal flap. A circular EVPOME graft (approximately 20 mm in diameter) was grafted onto the bone surface of the donor site of the flap. In case 2, a 16-year-old male underwent vestibuloplasty in the premaxilla region. After trimming, two circular EVPOME grafts were placed over the intact periosteum.&lt;br&gt;The grafts were easily fixed in place to the surrounding scarred mucosa. Although the underlying tissue of the EVPOME graft sutures was scarred, graft adherence was achieved within a week. The successful clinical outcome reported here suggests the efficacy of EVPOME grafts onto a less-vascularized, scarred tissue, and that the use of EVPOME grafts is highly applicable for patients with cleft lip and palate. EVPOME grafting could be extended especially toward pediatric patients with cleft lip and palate for reconstruction of oral mucosa defects by using cryopreserved autologous keratinocytes and by developing a novel material to ensure a steady supply of scaffold instead of the man-made AlloDerm&lt;sup&gt;&amp;reg;&lt;/sup&gt;.

DOI： 10.11224/cleftpalate.35.235

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DOI： 10.1016/j.ijom.2009.12.020

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Oral mucosa progenitor/stem cells reside as a small-sized cell population that eventually differentiates concurrently with an increase in cell size. Activation of the mammalian target of rapamycin (mTOR) leads to an increase in cell size. We hypothesized that rapamycin, a specific inhibitor of mTOR, will maintain primary human oral keratinocytes as a small-sized, undifferentiated cell population capable of retaining their proliferative capacity. Primary, rapamycin-treated (2 nM, 20 nM) oral keratinocytes showed a diminished cell size that correlated with a higher clonogenicity, a longer-term proliferative potential, and a slower cycling cell population concurrent with decreased expression of a differentiation marker when compared with untreated cells. Only the 2-nM rapamycin-treated oral keratinocytes maintained their ability to regenerate oral mucosa in vitro after 15 weeks of culture. Rapamycin, a Food and Drug Administration-approved drug, may have applicability for use in creating a highly proliferative cell population for use in regenerative medicine.

DOI： 10.1177/0022034509350559

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Background: Small diameter characterizes epidermal progenitor/stem cells. We have developed Gravity Assisted Cell Sorting (GACS) to simply enrich small-sized epidermal progenitor/stem cells.
Objective: The cells sorted by GACS were characterized by fluorescence-activated cell sorting analysis, and cultured for up to 7 weeks. The cultured cells were then used for reconstruction of skin equivalent.
Methods: GACS was performed on primary cultures (primary cell) and passage 6-7 cultures (cultured cell) of keratinocytes. A keratinocyte suspension was sized into two groups: cells trapped by a 20 mu m filter (trapped cells), and cells flowing through both a 20 and 11 mu m filter (non-trapped cells).
Results: In the primary cell groups, viability of the trapped cells was 62.5 +/- 7.2% compared to 77.0 +/- 3.7% for the non-trapped cells. In the cultured cell groups, viability of the trapped cells was 64.3 +/- 14.9%, compared to the non-trapped cells (93.1 +/- 2.0%). Flow cytometric analysis showed better discrimination by cell size between trapped and non-trapped cells in culture than in the primary cell suspension. Non-trapped cells contained a larger number of cells with high levels of alpha 6 integrin and low levels of CD71 (alpha 6 integrin(bri)CD71(dim),), indicating an enriched progenitor/stem cell population. The difference in these markers between the non-trapped and trapped cells was seen in both the primary and cultured cell groups although this difference was more distinct in cultured cells. Culture of both groups showed that cultures originating from the trapped cells senesced after approximately 15 days while the non-trapped keratinocytes grew for up to 40 days. Manufacture of an epidermis/dermal device (artificial skin) showed that non-trapped cells formed a significantly thicker epithelial layer than the trapped cells, demonstrating the enhanced regenerative capability of the smaller diameter, alpha 6 integrin(bri)CD71(dim) cells separated by GACS.
Conclusion: These results indicate that GACS is simple and useful technique to enrich for epidermal progenitor/stem cell populations, and is more efficient when used on cells in culture. (C) 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.jdermsci.2009.09.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：W B SAUNDERS CO-ELSEVIER INC  

Purpose: The Food and Drug Administration requires an accurate determination of the dose and potency of tissue-engineered or combination products as is required for drugs. This needs to be done as a rapid, quantitative, and noninvasive measurement of biologic function/activity in a way so as not to perturb the tissue-engineered product being developed. The aim of this study was to correlate constitutive release of cytokine(s) from unstimulated cells, at different stages of development, within a 3-dimensional (3D) organotypic ex vivo produced oral mucosa equivalent (EVPOME) to be used for intraoral grafting, with oral keratinocyte cell viability of the EVPOME.
Materials and Methods: Tissue culture medium was assayed with an enzyme-linked immunosorbent assay from monolayer culture of oral keratinocytes and a 3D EVPOME to determine the constitutive release of interleukin (IL) 1 alpha, IL-6, IL-8, and vascular endothelial growth factor (VEGF). VEGF messenger ribonucleic acid expression by oral keratinocytes within the 3D EVPOME was detected by in situ hybridization at days 4, 7, and 11. The number of viable oral keratinocytes within the EVPOME was extrapolated from VEGF release by use of a modified MTT assay.
Results: Both VEGF release level and the number of viable cells in the monolayer cultures and 3D EVPOME as measured by MTT assay significantly increased in a time-dependent manner (P &lt; .001, r = 0.743).
Conclusion: These results suggest that the increasing detectable levels of VEGF associated with the increasing number of Viable cells in the EVPOME may provide a useful noninvasive/nondestructive means of assessing both cellular viability (dose) and biologic function/activity (potency) of a combination cell-based device such as the EVPOME. (C) 2009 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 67:1256-1264, 2009

DOI： 10.1016/j.joms.2009.02.003

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

Angiogenesis is anticipated during wound healing. Vascular endothelial growth factor (VEGF) is a potent direct angiogenic factor that stimulates endothelial cell migration and proliferation in vitro and angiogenesis in vivo. Ex vivo produced oral mucosa equivalent (EVPOME) grafts have been reported to promote revascularization in the underlying tissue after grafting. The aim of this study was to evaluating the following: VEGF mRNA and its protein expression in EVPOME grafts, the protein levels in conditioned media produced by EVPOMEs, and the ability of VEGF to stimulate the growth of microvascular endothelial cells. VEGF mRNA expression and immunoreaction were found in basal and suprabasal layers. VEGF secreted by EVPOME was detected throughout the period of manufacture of the grafts, and became elevated for the first week at an air-liquid interface. Both EVPOME-conditioned media and a medium containing 10 ng/mL recombinant human VEGF induced endothelial cell proliferation, while neutralization of VEGF by an antibody blocked cell growth. These results suggest that VEGF secreted by EVPOME grafts might assist initial vascularization after grafting, which is critical to subsequent graft survival.

DOI： 10.1016/j.ijom.2007.06.013

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Language：Japanese   Publisher：(株)メディカルレビュー社  

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Other Link： http://search.jamas.or.jp/link/ui/2007269760

Publishing type：Research paper (scientific journal)  

DOI： 10.1177/154405910708600408

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPIE-INT SOC OPTICAL ENGINEERING  

Tissue engineered constructs can be employed to graft wounds or replace diseased tissue. Non-invasive methods are required to assess cellular viability in these constructs both pre- and post- implantation into patients. In this study, Monte Carlo simulations and fluorescence experiments were executed on ex vivo produced oral mucosa equivalent (EVPOME) constructs to investigate the fluorescence emitted at 355 nm excitation from these constructs. Both simulations and experiments indicated the need to investigate alternative excitation wavelengths in order to increase the cellular fluorescence from these constructs, while decreasing contributions from extra-cellular fluorophores.

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Language：Japanese   Publisher：(一社)日本頭頸部癌学会  

われわれの開発した培養複合上皮(皮膚、口腔粘膜)は、動物由来蛋白や3T3マウスfeeder layerを使用せずに完全無血清培地でkeratinocyteを培養した移植材料である。培養細胞を無細胞真皮上に播種して作製される培養複合上皮は、文部科学省高度先進医療開発経費Bの支援のもと、口腔粘膜上皮に関して新潟大学、富山大学、神戸大学の三施設合同で臨床応用され、平成14年〜16年の三年間で106症例を数えた。その内52症例が口腔癌もしくはin situであり、結果として90症例で成功し、何らかの理由により16症例で不成功となった。この方法の利点は、異種移植と定義されないこと、理論上将来のプリオン病への危惧がほぼ皆無であること、把持縫合可能な操作性を有すことである。欠点として、無細胞であるが他人の真皮をscaffoldとして使用すること、作製に約一ヵ月を要することである。(著者抄録)

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In order to more clarify the delayed wound healing in diabetes mellitus, we cultured the human epidermal keratinocytes in both 6 mM (control group) and 12 mM glucose (high-glucose group) of 'complete' MCDB 153 medium. Hyperglycaemia slowed the rate of their proliferation and inhibited their DNA synthesis and the production of total proteins. By 1 month after primary seeding in high-glucose group, the cells ceased their proliferation, whereas the cells in control group grew for more than 40 days. Mean population doublings in high-glucose group was 5.27 (vs. 7.25 in control, P = 0.001), and mean population doubling time during 1 month in high glucose group was 5.43 days (vs. 3.65 days in control, P = 0.02). They indicate that prolonged exposure to high glucose decreases the replicative life span of human epidermal keratinocytes in vitro. Furthermore, analysis of fatty acid contents in membrane phospholipids with thin-layer and gas chromatography showed no difference between the cultured keratinocytes in both conditions. Immunocytochemical staining of glucose transporter 1 shows that 28.1% of cells in high-glucose group were almost twice positive of those in control group (13.2%, P = 0.008). The mechanism of the ill effects of high glucose on epidermal keratinocytes is not so far clear, but it indicates the possibility of any direct effect of hyperglycaemia on glucose metabolism without changing lipid metabolism on cell membrane. The high-glucose group presented in this report can be available as an in vitro valuable study model of skin epidermal condition on diabetes mellitus. © Blackwell Publishing Ltd and Medicalhelplines.com Inc 2005.

DOI： 10.1111/j.1742-4801.2005.00148.x

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DOI： 10.1002/jbm.a.30144

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The main objective of this publication is to make the reader aware of the complexity and steps that are necessary to make a Food and Drug Administration (FDA)-approved laboratory produced cell-based device, for use in clinical trials for reconstructive surgery. Most tissue-engineered cell-based devices are considered as 'human somatic cell therapy' and fall under the auspices of the Center of Biologic Evaluation and Research (CBER) and are considered a combination product by the FDA. We have illustrated the algorithm that is necessary to follow an Independent New Drug (IND) application by using our ex vivo produced oral mucosa equivalents (EVPOME), a tissue-engineered oral mucosa, as an example of a cell-based device that needs FDA approval prior to clinical application. By illustrating the experimental approach and presenting resulting data we attempt to explain each step that we address along the way. Copyright (C) 2004 S. Karger AG, Basel.

DOI： 10.1159/000075034

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We present a unique case of malignant fibrous histiocytoma (MFH) developing around a masseter muscle close to the surgical margin from which a benign fibrous histiocytoma (BFH) of the mandible was excised 3 years and 10 months earlier. In the interval between the surgical removal of the original BFH and the MFH, several surgical procedures were performed in the related region. Clinical and histopathological evidence showed that this case represented two separate lesions rather than a single disease process. The clinical history suggests that the chronic repair process might have contributed to the tumorigenic factor of a MFH. © 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ooe.2003.12.001

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

The objective of this study was to assess the efficacy of the use of an ex vivo produced oral mucosa equivalent (EVPOME) for intraoral grafting procedures. Autogenous keratinocytes were harvested from a punch biopsy 4 weeks phor to surgery. placed in a serum-free culture system and seeded onto a human cadaveric dermal equivalent, AlloDerm (R). Thirty patients with either a premalignant or cancerous lesion were triaged into two groups, depending on the stage of disease: Group 1: EVPOME or Group 2: AlloDerm (R), control without an epithelial layer. Clinically, EVPOME grafts were easy to handle and showed excellent compliance on grafting. Both, EVPOME and AlloDerm (R) grafts, showed a 100% take rate, At 6 days post-grafting, the EVPOME clinically showed changes indicating vascular ingrowth and had cytologic evidence of the persistence of grafted cultured keratinocytes on the surface. The EVPOME grafts had enhanced maturation of the underlying submucosal layer associated with rapid epithelial coverage when compared to the AlloDerm (R) grafts at biopsies taken at 28 days post-grafting. In summary, EVPOME appears to be an acceptable oral mucosal substitute for human intraoral grafting procedures and results in a more favorable wound healing response than AlloDerm (R) alone.

DOI： 10.1054/ijom.2002.0365

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

The aim of this study was to determine the optimal stage of development at which transplant human ex vivo-produced oral mucosa equivalents (EVPOMEs) in vivo. EVPOMEs were generated in a serum-free culture system, without the use of an irradiated xenogeneic feeder layer, by seeding human oral keratinocytes onto a human cadaveric dermal equivalent, AlloDerm. EVPOMEs were cultured for 4 days submerged and then for 7 or 14 days at an air-liquid interface to initiate stratification before transplantation into SCID mice. AlloDerm, without epithelium, was used as a control. Mice were killed on days 3, 10, and 21 posttransplantation. Epithelium of the transplanted EVPOMEs was evaluated with the differentiation marker keratin 10/13. Dermal microvessel ingrowth was determined by immunohistochemistry with a mouse vascular marker, lectin binding from Triticum vulgaris. The presence and stratification of the epithelium were correlated with revascularization of the underlying dermis. The microvessel density of AlloDerm without epithelium was less than that of EVPOMEs with an epithelial layer. Microvessel density of the dermis varied directly with the degree of epithelial stratification of the EVPOMEs. The EVPOMEs cultured at an air-liquid interface for 7 days had the optimal balance of neoangiogenesis and epithelial differentiation necessary for in vivo grafting.

DOI： 10.1089/107632703762687645

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Language：Japanese   Publisher：新潟大学  

Distraction osteogenesis is a method of increasing bone length, and is now being used in the craniofacial region. The authors report two cases in which segmental mandibular defects were reconstructed by transport distraction osteogenesis and bone transplantation. The patients were a 67-year-old man and a 54-year-old man with squamous cell carcinoma of the lower gingiva and their mandible had been resected from the first molar on the right side to the mandibular angle region on the left side. In the first patient, transport distraction osteogenesis in the mandible was performed at the both sides and low-intensity pulsed ultrasound was used to accelerate bone healing. In the second patient, transport distraction osteogenesis was performed at the right side. Although bone transplants were needed for complete continuity in both cases, segmental mandibular defects were decreased by the regenerated bone. Successful new bone formations in the lengthened areas were recognized by radiographic observation, especially in the old patient used low-intensity pulsed ultrasound. Low-intensity pulsed ultrasound may be effective measures to accelerate bone healing after distraction osteogenesis.骨延長法は、骨そのものを牽引延長することにより骨の増生をはかる方法で、近年顎顔面領域においても広く応用されるようになってきた。今回われわれは、下顎骨欠損に対してトランスポート骨延長と骨移植を組み合わせた下顎骨再建を2症例に施行したので報告する。患者は、67歳と54歳の男性で、ともに下顎歯肉癌の診断にて右側第一大臼歯から左側下顎角部の下顎骨区域切除を施行した｡症例1では、両側において骨トランスポート法による下顎骨延長を行い、低出力超音波パルス照射を応用して骨形成の促進を囲った｡症例2では、右側において骨トランスポート法による下顎骨延長を行った｡両症例とも骨の延長に伴い欠損範囲を縮小し、骨移植を容易にすることができた｡特に、低出力超音波パルス照射を応用した症例1では、高齢にも関わらず良好な骨形成が認められ、骨延長における骨形成促進に低出力超音波パルス照射は有用ではないかと思われた｡

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Cleft Palate Association  

Orthodontic space closure of a cleft in the maxillary arch without prosthetic rehabilitation has been enabled by secondary bone grafting. It is easily achieved when the canine has not fully erupted at the time of bone grafting. However, not a few bone grafting procedures must be performed after the canine has erupted. The aim of this research was to investigate the outcome of dental occlusion in patients with bone-grafted alveolar clefts. Subjects were 88 patients who underwent secondary bone grafting, in our department, from 1987 to 1998, and they were divided into two groups, of the canine not fully erupted, and fully erupted, at the time of surgery. The numbers of the various types of clefts are as follows: 28cases of cleft of the lip and alveolus (unilateral,26; bilateral,2); 60 cases of cleft of the lip and palate (unilateral,52; bilateral,8). Of the total of 99 clefts,51 were in the group of the canine not fully erupted, and 48 were in that of fully erupted. Dental occlusion was classified into nine categories as follows: orthodontic space closure with the canine or the lateral incisor (C), space closure with restoration of the dwarf lateral incisor (Cr), bridgework (B), partial denture (D), autotransplantation of a tooth (T), application of an implant (I), no prosthetic treatment (N), unknown (U), and under orthodontic treatment or observation (O). In the group of the canine not fully erupted, the outcome of dental occlusion was as follows: C,28; Cr,2; B,1; T,1; N,1; U,2, and 0,16 cases. In the group of the canine fully erupted, the outcome of dental occlusion was as follows C,22; Cr,1; B,14; D,1; T,3; I,1; N,2; U,2, and 0,2 cases. According to the types of clefts, the major dental occlusion in patients with unilateral and bilateral cleft lip and alveolus and unilateral cleft lip and palate, was C, while it was B for bilateral cleft lip and palate. Non-prosthetic space closure (categories C, Cr, T, I) was accomplished in 61% of cleft sites in which the canine was not fully erupted at surgery, and in 52% of cleft sites in which the canine was fully erupted at surgery. Orthodontic space closure without prosthetic rehabilitation was the major procedure in both groups. In particular, in the not fully eruption group, the prospect that cases of category Oare likely to move into category C will result in the achievement of space closure in 92% of cleft sites, sustaining the clinical significance of bone grafting before the canine eruption. Bridgework was the second major procedure in the canine fully eruption group, as well as in bilateral cleft lip and palate patients. Desirably, a primary goal in the canine fully eruption group is also achieving orthodontic closure of the cleft space, or the application of tooth autotransplantation and dental implant to bone-grafted alveolar clefts, without prosthetic rehabilitation.

DOI： 10.11224/cleftpalate1976.27.1_58

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

To date, successfully developed EVPOMEs in a serum-free culture system without a feeder layer are the most acceptable and promising oral mucosal substitutes for human intraoral grafting because of minimal risk of foreign contaminants, easy handling and stitching, subsequent rapid revascularization into dermal component after transplantation, and contribution to favorable open wound closure without functional compromise, although several types of oral mucosal substitutes described in this article have been used in patients.

DOI： 10.1016/S1042-3699(02)00010-9

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Language：Japanese   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

A rare case of myoepithelioma originating from the submandibular gland is reported. A 28-yearold woman was referred for treatment of an asymptomatic mass in the left submandibular region. On clinical examination, a 30×15mm hard, mobile, non-tender mass was noted. Ultrasonography and computed tomography demonstrated a large, well-demarcated mass that was not distinct from the submandibular gland. The tumor was removed surgically in March 1998. Histopathologically, the tumor consisted of both spindle-shaped cells and plasmacytoid cells with proliferation of many vessels in the stroma. The neoplastic cells expressed S-100 protein, DF-3, and EMA immunohistochemically. The histopathological diagnosis was myoepithelioma. There was no evidence of recurrence 2 years postoperatively.

DOI： 10.5794/jjoms.46.677

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Other Link： http://search.jamas.or.jp/link/ui/2001121697

Language：Japanese   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

A case of ranula in a neonate is reported. A 2-month-old infant was found to have a small swelling in the floor of the mouth at birth. Initial examination revealed a fluctuant, translucent swelling with protrusion from the mouth. The lesion arose in the left side of the mouth floor. A computed tomographic examination confirmed the presence of a cystic lesion, un associated with the sublingual gland. The clinical diagnosis was a sublingual ranula. Although marsupialization was performed three times, the swelling recurred and returned to its original size soon after treatment. Three months later, extirpation of the cyst and the sublingual gland was performed. The postoperative course was uncomplicated, with no signs of recurrence for 6 months. The histological diagnosis was mucous retension cyst. To prevent recurrence, extirpation of the sublingual gland was effective, even in an infant.

DOI： 10.5794/jjoms.46.536

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Other Link： http://search.jamas.or.jp/link/ui/2001052352

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The phospholipid component of the cellular membrane is crucial to the structure and function of cells. Basal cells from three epithelial tissues, adult human skin epidermis, oral mucosa, and hair follicles, grow rapidly in serum- and lipid-free medium. Analysis of phospholipid extracts from the above three types of stratified squamous epithelium in both in vivo and in vitro was done to relate fatty acid cell composition to cell function. The fatty acid composition of hair follicles in vivo was analyzed in plucked scalp hairs, and those of skin epidermis and oral mucosa in vivo were analyzed after separating the tissue into suprabasal and basal layers. The fatty acid composition of the in vivo cells from hair follicles shows a partial essential fatty acid (EFA)-deficient state. There was no significant difference between the skill epidermis and the oral mucosa in the fatty acid composition of the in vivo cells from each basal layer. However, in the suprabasal layers, the percent of linoleic acid (18:2) from the skin epidermis was higher than that from the oral mucosa. This study shows that total fatty acid composition in cell membranes of stratified squamous epithelium varies with their keratinization pattern. When cultured, the three types of rapidly growing keratinocytes showed the same essential fatty acid deficient pattern in the membrane phospholipids. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

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Language：Japanese   Publisher：社団法人 日本口腔外科学会  

The relationship between recipient condition and the period required for periodontal healing of autotransplanted teeth was studied. The period for periodontal healing was evaluated on the basis of regeneration of the periodontal space and lamina dura on dental radiographs. Age, sex, site of transplantation, period after tooth extraction at the recipient site, and the timing of occlusal function were the recipient factors examined. The mean periods required for regeneration of the periodontal space and lamina dura were 3.7 months and 7.6 months, respectively. Regeneration of the periodontal space was not influenced by any recipient factor, whereas regeneration of lamina dura was associated with the period after tooth extraction, site of transplantation, and timing of occlusal function. It was suggested that regeneration of the lamina dura may not totally depend on that of the periodontal space.

DOI： 10.5794/jjoms.43.733

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：THE JAPANESE SOCIETY FOR JAW DEFORMITIES  

Unfavorable fractures and blood vessel injuries encountered during 196 operations on 185 patients who underwent surgical correction of jaw deformities were studied to analyze the causes. There were 12 unfavorable fractures (6.1%) and 9 blood vessel injuries (4.6%). All complications occurred in two-jaw surgery and sagittal splitting ramus osteotomy (SSRO). Blood loss and operation time were significantly higher in cases with complications than in those without complication in SSRO.<BR>SSRO. With the exception of one case, all fractures were encountered during SSRO. Most of them were minor fractures occurring along the osteotomy line, but relatively large segments of the proximal segment were fractured in 3 cases, 2 of which were not repositioned. Blood vessel injury of the descending palatine artery was encountered in 2 cases of Le Fort I osteotomy. In SSRO, injuries of the inferior alveolar artery, facial artery, and unidentified artery occurred in 3, 1, and 3 cases, respectively. Ligation of the arteries was required to stop bleeding in 2 cases, whereas in the other cases, bleeding was controlled by packing of gauze or oxidized regenerated cellulose.<BR>Insufficient bony cut, forcible splitting, and careless handling of surgical instruments were the main causes of the complications. It is thought that the complications can be avoided by precise analysis of structures at the operation site by computed tomography, sufficient bony cut, and careful handling of surgical instruments.

DOI： 10.5927/jjjd1991.7.141

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Language：Japanese   Publisher：特定非営利活動法人 日本顎変形症学会  

A case of unilateral ankylosis of the temporomandibular joint, which caused micrognathia with facial asymmetry, was reported. A 3-year-old female visited our department because of facial asymmetry She was placed on conservative therapy using a functional appliance to improve facial asymmetry and occlusal relationship. Because of mandibular trismus, condylectomy was performed at the age of 13 years 10 months. Interincisal distance increased to 38mm, but micrognathia with facial asymmetry remained. Therefore, Le Fort I osteotomy combined with extraoral vertical ramus osteotomy on the affected side and sagittal split ramus osteotomy on the opposite side was performed at the age of 16 years 10 months. In addition, augmentation genioplasty with a porous hydroxyapatite block was performed at the age of 17 years 6 months. Four years later, the patient was satisfied with recovery of jaw opening and improvement in facial appearance.

DOI： 10.5927/jjjd1991.7.57

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：社団法人 日本口腔外科学会  

The relationship between root canal filling and the periodontal healing of autotransplantated teeth was studied clinically and radiographically in 21 autotransplanted teeth with complete root formation that were followed up for more than 6 months. The teeth were classified into three types according to the status of root canal filling immediately after transplantation: type A) teeth with root canal (s) wide enough to be detected on radiographs and root canal filling (s) to the apex (es); type B) teeth having incomplete root canal filling (s) because of narrow root canal (s); and type C) teeth having root canal (s) detectable on radiographs but with incomplete filling (s). The periodontal healing was examined more than 6 months postoperatively. Ten teeth classified as type A had no clinical symptoms and showed regeneration of the periodontium on radiographs. Eight teeth classified as type B had no clinical symptoms but the periodontal spaces were indistinct in 5 teeth. No clinical symptoms were associated with 2 of 3 teeth classified as type C, but the periodontal spaces were not clearly seen in the apical regions. The presence of apical lesions and inflammatory resorption of the root was observed on radiographs of the other tooth. These findings suggest that periodontal healing is associated with the width of the root canal and the status of root canal filling.

DOI： 10.5794/jjoms.42.684

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：MONDUZZI EDITORE  

The effect of damage to the periodontium on the outcome of tooth transplantation was studied by examining the relationship of macroscopic condition of the root surface to the clinical course in 27 human autotransplanted teeth. The macroscopic conditions were classified into the areas with A) remnants of periodontal tissue, B) cementum exposure, and C) cementum loss. The macroscopic evaluation was confirmed to be well correlated with the histological findings or 10 healthy teeth. Most of the root surfaces of autotransplanted teeth consisted of the areas with cementum exposure and attachment of periodontal tissue in varying proportions. These teeth showed normal periodontal healing both clinically and radiographically. Replacement resorption in the area of cementum loss was observed in one tooth. The results were consistent with those of previous reports which showed that cementum is important for normal periodontal healing.

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DOI： 10.2330/joralbiosci1965.34.239

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Authorship：Lead author   Language：Japanese   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

Innervation of carcinomas induced by application of 9, 10-dimethy1-1, 2-benzanthracene (DMBA) was investigated in the hamster tongue by using immunohistochemistry concerning neurofilament protein (NFP) and glia specific S-100 protein.<BR>The tumor induced by application of DMBA was a well-differentiated squamous cell carcinoma having cord-like invasion of the underlying fibrcus tissue and muscle layer.<BR>The characteristic feature of the innervation was the extreme scarcity of NFP-and S-100 pro tein-immunopositive neural elements recognized in the stroma and around blood vessels of the carcinomas.<BR>A few NFP-positive neural elements which were observed in the carcinoma invading zone were very fine and also found not to bear any relation to blood vesseles nor tumor nests.<BR>Unlike calcitonin gene-related peptide-positive nerves, a dense distribution of S-100 protein-positive neural elements characteristic of arterioles in the surrounding muscle layer was also not seen around arterioles abundant in the carcinoma invading zone.<BR>The results indicate a high pain threshold and the absence of autoregulatory mechanism in the blood vessels of the carcinoma tissue.

DOI： 10.5794/jjoms.38.896

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Other Link： http://search.jamas.or.jp/link/ui/1993109170

Language：English   Publisher：Biomedical Research Press  

In Fig. 8 in the above article (page 387), some specified lines should have been printed in red, but printed in black due to an editorial error. On this page, the figure is correctly printed as was intended by the authors.

DOI： 10.2220/biomedres.13.85

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Biomedical Research Press  

DOI： 10.2220/biomedres.12.377

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：The Japanese Society for Dental Materials and Devices (JSDMD)  

The aim of this study was to characterize the surface properties of PEEK and CAD/CAM resins, CAM-A and CAM-B, by SiC polishing. Two way polishing unit was used with a waterproof P#1200-SiC paper. Mean wear rate was calculated based on the polishing pressure and sliding distance. The surface properties were analyzed with AFM. The mean wear rate of PEEK, CAM-A and CAM-B was about 0.5, 1.9 and 0.5 mm^3/Nm, respectively. The surface roughness (Ra) of these specimens was around 0.51, 0.29 and 0.23 μm, respectively. These results indicated that PEEK can be used as a material for CAD/CAM resin.

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Language：Japanese  

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Language：Japanese   Publisher：The Japanese Society for Dental Materials and Devices (JSDMD)  

The present study examined the feasibility of hexagonal boron nitride (hBN) as a wax pattern coating material for gold alloy casting in air. After wax patterns were coated with hBN, they were outer-invested with a gypsum-bonded investment. The thin specimens made of the gold alloy, 0.55 mm in thickness, were cast in air. The color of the cast surface by using hBN was rusty red. The X-ray diffractometer analysis revealed that copper oxide produced on the hBN cast surface was the only Cu_2O, and CuO produced on the control was not detected. This means that hBN remarkably inhibited the oxidation during casting. Thus, the use of hBN could be effective in hardness of the cast surface.

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Language：Japanese  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：MARY ANN LIEBERT, INC  

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Language：Japanese   Publisher：The Japanese Society for Dental Materials and Devices (JSDMD)  

The aim of this study was to characterize the adhesion of mineral particles to a metal (titanium) plate by electrolysis. MMA including a conductive monomer and zircon powder treated with stearic acid was used as an electrolyte. Titanium and carbon plates were used as electrodes. Twenty volts DC were applied between the electrodes for 3,600 s. EPMA revealed the adhesion of zircon particles to the metal surface. In addition, S, O elements were concurrently detected on and around the particles. Since both elements were derived from the conductive monomer, the present study indicated the formation of an organic material including conductive monomer on the metal surface preceded the adhesion of zircon particles.

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Language：Japanese   Publisher：The Japanese Society for Dental Materials and Devices (JSDMD)  

The water-powder ratio (W/P ratio) affects compressive strength of dental stone. In this study, we investigated the effects of water droplets adhering on a mechanical spatulator with vacuum and hand spatula on W/P ratio. The amount of water droplets was measured using an electronic balance. The compressive strength of dental stone was calculated based on the loaded weight. The total amount of water droplets adhering onto spatulator (volume; 250 and 500 mL) and a hand spatula were 0.90 mL (SD 0.10) and 1.33 mL (SD 0.12), respectively. The compressive strength of the dental stone were 10.23 kg/mm^2 (SD 0.27) and 6.89 kg/mm^2 (SD 1.31) at W/P ratio 0.20 and 0.23, respectively. Thus, the total amount of water droplets adhering on those instruments should be taken into consideration when mixing dental stone.

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Language：Japanese  

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Language：Japanese   Publisher：The Japanese Society for Dental Materials and Devices (JSDMD)  

Japanese society for dental materials and devices has title system of Dental Materials Adviser (DMA) and Dental Materials Senior Adviser (DMSA), which are titles about dental materials and devices. Dentists use dental materials and devices in their clinics. Therefore, it appears to be beneficial for patients that dentists take DMA and DMSA. In this study, we explored the strategy beneficial for patients by utilizing this title system. Since Japan dental association (JDA) is the largest association of dentists including general practitioners, we will be able to inform dentists of our title system by using the advertising media in the JDA. If the JDA agreed with our plan, the number of DMA and DMSA would increase dramatically. Consequently, our strategy would result in the benefit for thousands of patients.

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Language：Japanese   Publisher：The Japanese Society for Dental Materials and Devices (JSDMD)  

The aim of this study was to prepare organo-mineral complex by electrolysis and to characterize it. MMA including conductive monomer and oxide with a stearic acid treatment was used as an electrolyte. Titanium and stainless substrate were used as electrodes. Twenty volts DC were applied between the electrode and Pt counter electrode in the electrolyte for 3,600 s. FTIR results confirmed the preparation material to be a PMMA. Mineral particles were well dispersed on the PMMA matrix according to the EPMA results. The mineral particle size was the order of submicron by AFM image. Consequently, the organo-mineral complex was easily prepared by the electrolysis.

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Language：Japanese   Publisher：The Japanese Society for Dental Materials and Devices (JSDMD)  

The aim of this study was to investigate the effect on corrosion products due to difference in Cu content. We prepared three kinds of Ag-Pd-Cu-Au alloys with difference Cu content. After casting in 10 × 20 × 1 mm^3, the contacted casting plates were immersed in corrosion solution for 8 weeks at 37℃. After immersed, we analyzed alloy surface by XPS. In spite of Cu content, a peak derived from PdO was confirmed in Pd3d XPS spectra of all specimens.

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Language：Japanese  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：NATURE PUBLISHING GROUP  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：Japanese   Publisher：新潟県医師会  

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Language：Japanese   Publisher：(公社)日本口腔外科学会  

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Language：Japanese   Publisher：(一社)日本口腔内科学会  

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Language：Japanese   Publisher：(NPO)日本口腔科学会  

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Language：Japanese   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

Recent studies have revealed that glucose transporter 1 (GLUT 1) expression can be detected in basal layer cells of skin. Expression levels of GLUT 1 are thought to be regulated by glucose transport activity within cells. Glucose metabolism in the epithelial layers may affect keratinocyte differentiation. We studied the localization of GLUT 1 expression in the epithelial layers of oral mucosa equivalents, grown in a defined serum-free culture medium without a feeder layer. The oral mucosa produced in this system is minimally exposed to potential foreign contaminants and is thus more acceptable for use in humans. Samples of native human gingiva and oral mucosa equivalents on days 4, 11, and 18 were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-micrometer-thick sections. For immunohistochemical studies, sections were stained by the ABC method using polyclonal antibodies to rabbit GLUT-1. Well-stratified, differentiated epithelial layers in day 18 oral mucosa equivalents had a similar keratinization pattern to that of native tissue. GLUT 1 was expressed in both the basal cell layer and suprabasal cell layer of oral mucosa epithelium, but not in the superficial layer. Immunoreactivity for GLUT-1 was detected in all epithelial layers of oral mucosa equivalents, except for the superficial layer of day 18 equivalents. In conclusion, keratinocytes of all layers of oral mucosa equivalents were in a highly active metabolic state and showed increased glycolysis, suggesting active proliferative and a less differentiated state as comnared with the basal and na.ra.ba.sa.1 cell lavers of native tissue.

DOI： 10.5794/jjoms.47.289

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Other Link： http://search.jamas.or.jp/link/ui/2001265829

Language：Japanese   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

Recent studies have revealed that glucose transporter 1 (GLUT 1) expression can be detected in basal layer cells of skin. Expression levels of GLUT 1 are thought to be regulated by glucose transport activity within cells. Glucose metabolism in the epithelial layers may affect keratinocyte differentiation. We studied the localization of GLUT 1 expression in the epithelial layers of oral mucosa equivalents, grown in a defined serum-free culture medium without a feeder layer. The oral mucosa produced in this system is minimally exposed to potential foreign contaminants and is thus more acceptable for use in humans. Samples of native human gingiva and oral mucosa equivalents on days 4, 11, and 18 were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-micrometer-thick sections. For immunohistochemical studies, sections were stained by the ABC method using polyclonal antibodies to rabbit GLUT-1. Well-stratified, differentiated epithelial layers in day 18 oral mucosa equivalents had a similar keratinization pattern to that of native tissue. GLUT 1 was expressed in both the basal cell layer and suprabasal cell layer of oral mucosa epithelium, but not in the superficial layer. Immunoreactivity for GLUT-1 was detected in all epithelial layers of oral mucosa equivalents, except for the superficial layer of day 18 equivalents. In conclusion, keratinocytes of all layers of oral mucosa equivalents were in a highly active metabolic state and showed increased glycolysis, suggesting active proliferative and a less differentiated state as comnared with the basal and na.ra.ba.sa.1 cell lavers of native tissue.

DOI： 10.5794/jjoms.47.289

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Other Link： http://search.jamas.or.jp/link/ui/2001265829

Language：English   Publisher：AMER ASSOC DENTAL RESEARCH  

A problem maxillofacial surgeons face is a lack of sufficient autogenous oral mucosa for reconstruction of the oral cavity. Split-thickness or oral mucosa grafts require more than one surgical procedure and can result in donor site morbidity. Skin has disadvantages of adnexal structures and a different keratinization pattern than oral mucosa. In this study, we successfully assembled, ex vivo, a human oral mucosa equivalent, consisting of epidermal and dermal components, in a defined, essential-fatty-acid-deficient, serum-free culture medium without a feeder layer, that could be used for intra-oral grafting in humans. Autogenous oral keratinocytes were seeded onto a cadaveric dermis, AlloDerm(TM). The oral mucosa equivalent was cultured at an air-liquid interface for 2 wks. The resulting equivalent had a well-stratified parakeratinized epithelial layer similar to native oral keratinized mucosa. Expression of differentiation markers, filaggrin and cytokeratin 10/13, suggested a premature keratinized state. The presence of proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67, suggested a state of hyperproliferation. Fatty acid composition of the equivalent was similar to that of in vitro cultured oral keratinocytes but differed from the that of in vivo native tissue, showing a lower content of 18:2 and 20:4, and a higher content of 16:1 and 18:1 fatty acids, respectively. The keratinocytes of the equivalent appeared to be in a more active and proliferative state than native keratinized mucosa. The dynamic nature of the cell population on the oral mucosa equivalent may be beneficial for intra-oral grafting procedures and for transfection of the keratinocytes.

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Language：Japanese   Publisher：Niigata University  

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Other Link： http://search.jamas.or.jp/link/ui/2000148581

Language：English   Publisher：MOSBY-YEAR BOOK INC  

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Language：English   Publisher：W B SAUNDERS CO  

Purpose: The aim of this study was the ex vivo development of a composite oral mucosal equivalent composed of a continuous stratified layer of human oral keratinocytes grown on a cadaveric human dermal matrix in a defined medium without a feeder layer.
Materials and Methods: Enzymatically dissociated human oral keratinocytes from keratinized oral mucosa were cultured, submerged in a serum-free, low-calcium (0.15 mmol/L) supplemented medium, and expanded through several passages. Once a sufficient population of keratinocytes was reached, they were seeded on 1-cm(2) pieces of AlloDerm (LifeCell Co, Woodlands, TX), an acellular nonimmunogenic cadaveric human dermis, at cell densities of 2.5 x 10(4), 5.0 x 10(4), 1.25 x 10(5), or 2.5 x 10(5). The oral keratinocyte-AlloDerm composites were cultured while submerged in a high-calcium (1.8 mmol/L) medium for 4 days. After 4 days, the composites were raised to an air-liquid interface. Samples of the composites were taken for histologic examination at 4, 11, and 18 days postseeding of the keratinocytes on the AlloDerm.
Results: At day 4, only the seeded cell density of 2.5 x 10(5) cells/cm(2) formed a continuous monolayer on the AlloDerm. At day 11, a continuous stratified epithelium was seen, and at day 18 a well-differentiated, confluent parakeratotic epithelial layer was developed at cell densities of 5.0 x 10(4), 1.25 x 10(5), and 2.5 x 10(5) cells/cm(2).
Conclusion: With the method used, it was possible to successfully develop an ex vivo composite oral mucosal equivalent that consisted of a stratified epidermis on a dermal matrix.

DOI： 10.1016/S0278-2391(99)90077-0

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Language：English   Publisher：Churchill Livingstone  

Our objective was to find out the incidence of signs and symptoms of temporomandibular joint (TMJ) and disc displacement in patients with mandibular prognathism. Fifty-one patients were examined clinically and by axial computed tomography (CT). The incidence of TMJ signs and symptoms was 6/25 (24) in patients with simple mandibular prognathism and 12/26 (46) in patients with mandibular prognathism and asymmetry. No discs were displaced in patients with simple mandibular prognathism, but 15 (58) of the patients with mandibular prognathism and asymmetry had displaced discs. There was no association between signs and symptoms of TMJ and disc displacement. Patients with mild protrusion and severe asymmetry of the mandible had a high incidence of disc displacement, which interestingly was on the deviated side in 14 of the 15 patients affected. We conclude that skeletal morphology may have a role in the development of TMJ disorders but the mechanism is obscure.

DOI： 10.1054/bjom.1999.0016

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Language：Japanese   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

Seventeen patients with 23 surgically treated ankylotic temporomandibular joints were studied clinically. Including 2 patients with recurrence, high operations, low operations, and gap surgery were performed on 12, 8, and 6 joints, respectively, in patients 6 to 60 years of age. Joints were reconstructed by costochondral grafts and artificial joints in 3 patients (4 joints). Interpositional materials were used in 7 joints in 5 patients. With the exception of 3 patients who underwent joint reconstruction, all patients started postoperative mouth opening exercises within a week after surgery. The maximum mouth opening increased by 6 to 30 mm 4 months to 7 years 9 months postoperatively. The average postoperative maximum mouth opening was greater in patients younger than 16 years of age than in those older than 20 years of age, and there was no re-ankylosis in the former group. Intermaxillary fixation was maintained for 3 weeks postoperatively in the patients who un-derwent reconstruction. Postoperative open bite was encountered in 5 patients. Open bite was controlled by mouth closing exercises, the use of a chin cap and class II elastics, and occlusal adjustment by prosthetic devices. In conclusion, surgical treatment of ankylotic joints should be performed as early as possible in young patients.

DOI： 10.5794/jjoms.45.296

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Language：Japanese   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

Seventeen patients with 23 surgically treated ankylotic temporomandibular joints were studied clinically. Including 2 patients with recurrence, high operations, low operations, and gap surgery were performed on 12, 8, and 6 joints, respectively, in patients 6 to 60 years of age. Joints were reconstructed by costochondral grafts and artificial joints in 3 patients (4 joints). Interpositional materials were used in 7 joints in 5 patients. With the exception of 3 patients who underwent joint reconstruction, all patients started postoperative mouth opening exercises within a week after surgery. The maximum mouth opening increased by 6 to 30 mm 4 months to 7 years 9 months postoperatively. The average postoperative maximum mouth opening was greater in patients younger than 16 years of age than in those older than 20 years of age, and there was no re-ankylosis in the former group. Intermaxillary fixation was maintained for 3 weeks postoperatively in the patients who un-derwent reconstruction. Postoperative open bite was encountered in 5 patients. Open bite was controlled by mouth closing exercises, the use of a chin cap and class II elastics, and occlusal adjustment by prosthetic devices. In conclusion, surgical treatment of ankylotic joints should be performed as early as possible in young patients.

DOI： 10.5794/jjoms.45.296

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：MOSBY-YEAR BOOK INC  

A rare case of oral adenosquamous carcinoma in a 78-year-old woman is reported. The tumor occurred in her tongue and metastasized to the submandibular and cervical lymph node;. Histologically, the tumor showed invasive growth involving the submucosal and muscle lavers. Its solid carcinomatous nests exhibited ductal differentiation in the deeper aspects and squamous differentiation toward the surface. Histochemical examination revealed an accumulation of acid mucopolysaccharide in the ductal luminal and the ductal cells were immunohistochemically positive for carcinoembryonic antigen, epithelial membrane antigen, cancer antigen 15-3 and Ulex europaeus agglutinin I. Ultrastructurally, tonotibrils, desmosomes and numerous cytoplasmic processes were common features of the tumor cells. In addition, true glandular structures and pseudocysts were seen in areas. Clinical features of 13 adenosquamous carcinomas in the literature were analyzed.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC DENTAL RESEARCH  

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Language：Japanese   Publisher：新潟医学会  

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Language：English   Publisher：MOSBY-YEAR BOOK INC  

A rare case of oral leiomyosarcoma diagnosed with the aid of immunohistochemical and electron microscopic examinations together with a review of the literature are reported. The patient was a 70-year-old Japanese man. The primary tumor involved the maxillary gingiva and bone and metastasized to the cervical lymph nodes. On histologic examination the tumor showed invasive growth into the maxillary bone. It was composed of interlacing fascicles of spindle-shaped cells with eosinophilic cytoplasm and elongated, blunt-ended nuclei. The tumor formed extensive metastatic foci in the cervical lymph nodes. On immunohistochemical examination most of the tumor cells were positive for desmin, smooth muscle-specific actin, and myosin. The ultrastructural characteristics of the tumor cells were abundant microfilaments, pinocytotic vesicles, and basement membrane formation. The findings were indicative of a tumor demonstrating myogenic differentiation. A review of the literature during the past 50 years disclosed a total of 60 oral leiomyosarcomas, including our case.

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Language：Japanese   Publisher：新潟医学会  

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Other Link： http://hdl.handle.net/10191/42442

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Application no：特願2018-149488  Date applied：2018.8

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Application no：特願2018-145182  Date applied：2018.8

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Applicant：国立大学法人 新潟大学, 多木化学株式会社

Application no：特願2016-031777  Date applied：2016.2

Announcement no：特開2017-147951  Date announced：2017.8

Patent/Registration no：特許6758616  Date registered：2020.9 

Rights holder：国立大学法人 新潟大学, 多木化学株式会社

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