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Introduction: We developed a new treatment method that combines tympanoplasty with transplantation of autologous cultured nasal mucosal epithelial cell sheets to regenerate the mucosa of patients with adhesive otitis media, which has been difficult to treat effectively. We verified whether this procedure could be performed safely and measured its therapeutic efficacy. Methods: Autologous nasal mucosal epithelial cell sheets were manufactured at a good manufacturing practice-compliant cell processing facility using autologous nasal mucosal tissue. We performed tympanoplasty and transplanted the cell sheets into the middle ear cavity in six patients with adhesive otitis media. Results: The manufactured autologous cultured epithelial cell sheets met the predetermined quality standards and were successfully transplanted safely in all cases. Computed tomography findings after cell sheet transplantation showed that aeration in the tympanic cavity was maintained or restored in five of the six patients (83.3%). Four of the six (66.7%) patients had postoperative air-bone gap within 20 dB, which is considered a postoperative success in tympanoplasty for chronic middle ear disease. Conclusions: The results of this clinical study suggest that tympanoplasty with cell sheet transplantation can be used to treat adhesive otitis media by reliably preventing re-adhesion of the tympanic membrane.

Introduction We previously reported a new cell transplantation therapy for patients with intractable otitis media using autologous nasal mucosal epithelial cell sheets, manufactured using temperature-responsive cell culture inserts. The current study aimed to verify whether the transplantable cell sheets could be manufactured for application in clinical trials, according to standard operational procedures (SOP), in a cell processing facility (CPF). Methods Human nasal mucosal epithelial cells from four volunteer donors were aseptically cultured and transplantable cell sheets successfully manufactured, with reproducibility, using temperature-responsive cell culture inserts in the CPF. During the manufacture of cell sheets, the CPF environment was confirmed to be aseptic by sterilization tests. Purity of the cell sheets was confirmed by histological analysis and flow cytometry. Both safety and quality of the human nasal mucosal epithelial cell sheets were validated. Results The cultured and manipulated human nasal mucosal epithelial cells showed no evidence of malignant transformation in vitro. The study confirmed the safety and suitability of the manufactured human nasal mucosal epithelial cell sheets for use in clinical trials. Conclusions The results led to the establishment of a coherent system in which transplantation could be achieved smoothly.

The proliferation and differentiation of cultured epithelial cells may be modified by Rho-associated kinase (ROCK) inhibition and extracellular Ca2+ concentration. However, it was not known whether a combination would influence the behavior of cultured epithelial cells through changes in the phosphorylation of non-muscle myosin light chain II (MLC). Here we show that the combination of ROCK inhibition with Ca2+ elevation regulated the phosphorylation of MLC and improved both cell expansion and cell–cell adhesion during the culture of human nasal mucosal epithelial cell sheets. During explant culture, Ca2+ enhanced the adhesion of nasal mucosal tissue, while ROCK inhibition downregulated MLC phosphorylation and promoted cell proliferation. During cell sheet culture, an elevation of extracellular Ca2+ promoted MLC phosphorylation and formation of cell–cell junctions, allowing the harvesting of cell sheets without collapse. Moreover, an in vitro grafting assay revealed that ROCK inhibition increased the expansion of cell sheets three-fold (an effect maintained when Ca2+ was also elevated), implying better wound healing potential. We suggest that combining ROCK inhibition with elevation of Ca2+ could facilitate the fabrication of many types of cell graft.

Temporary storage of nasal tissues and nasal cell sheets, which entails transportation between hospitals and cell culture facilities, is an important issue in regenerative medicine. Herein, we investigated the preservation of chilled and frozen nasal tissues and expiry dates of ready-to-use nasal cell sheets. Although the cell number in preserved tissues was lower than that in fresh tissue, nasal cell sheets could be fabricated from tissues that had been refrigerated for 5 days and frozen–thawed over 5 days. Moreover, the nasal mucosal cell sheets were preserved in a non-hazardous buffer. The cell number, viability, and structure were not maintained in saline containing E-cadherin for 2 days; however, these were maintained in Hank’s balanced salt solution for 2 days, but not for 5 days. To assess the proliferation capacity of cells in the stored cell sheets, we performed cell sheet grafting assays in vitro. Cell sheets stored in Hank’s balanced salt solution for 2 days adhered to collagen gel and expanded normally. Our results show that nasal tissues can be stored temporarily in refrigerators or deep freezers, and Hank’s balanced salt solution can be used for preservation of ready-to-use cell sheets for a few days.

Previously, we succeeded in transplanting autologous nasal mucosal cell sheets in the middle ears of 5 patients, who underwent cholesteatoma resection, which prevents recurrence of cholesteatoma in clinical settings. Current good manufacturing practice (GMP) standards for human cell cultivation requires the establishment of cell processing centers (CPC) which act as germ-free facilities. However, due to practical difficulties involved in establishing and maintaining such facilities at each individual hospital, a functional transport system is felt to be needed for the continuation of effective regenerative therapy. In the current study, nasal mucosal tissue and autologous blood obtained from 3 human volunteers were transported for over 3 h. Disinfected nasal tissues were cultured using keratinocyte culture medium, which included autologous serum prepared from blood. After 24 d, cultured nasal mucosal cells were transported for over 3 h and subsequently assessed for cell number, viability and purity. Moreover, CK4, CK8, and CK18 were analyzed the suitability of these nasal mucosal cell sheets for middle ear regenerative therapy. Overall, we confirmed that nasal mucosal cell sheets can be fabricated using transported nasal mucosal tissue and blood. This study would be contribute to establish a new regenerative therapy for clinical application, accompanied with transportation between companies and hospitals., Highlights • Transportation of nasal mucosal tissue and blood. • Characterizing of nasal mucosal cell sheet by immunohistology. • Transportation of nasal mucosal cell sheet as pre-clinical study.

HLA-G is a natural tolerogenic molecule and has the following unique features: seven isoforms (HLA-G1 to HLA-G7), formation of disulfide-linked homodimers, and β2-microglobulin (β2m)-free forms. Interestingly, individuals null for the major isoform, HLA-G1, are healthy and expressed the α2 domain–deleted isoform, HLA-G2, which presumably compensates for HLA-G1 function. However, the molecular characteristics of HLA-G2 are largely unknown. In this study, we unexpectedly found that HLA-G2 naturally forms a β2m-free and nondisulfide-linked homodimer, which is in contrast to the disulfide-bonded β2m-associated HLA-G1 homodimer. Furthermore, single-particle analysis, using electron microscopy, revealed that the overall structure and domain organization of the HLA-G2 homodimer resemble those of the HLA class II heterodimer. The HLA-G2 homodimer binds to leukocyte Ig-like receptor B2 with slow dissociation and a significant avidity effect. These findings provide novel insights into leukocyte Ig-like receptor B2–mediated immune regulation by the HLA-G2 isoform, as well as the gene evolution of HLA classes.

Introduction Cultured stratified epithelial cell sheets have been clinically utilized as transplantable grafts for the regeneration of epithelial tissues, such as the esophagus, cornea, skin, and intraoral cavity. These cell sheets are expected to gain widespread use as regenerative medicine products and save many patients. For this purpose, establishing and disseminating the stale protocol of fabricating the cell sheet is crucial. The fabrication of cultured stratified epithelial cell sheets consists of many important steps, and since the patients’ epithelial cell conditions vary widely and are sometimes unstable, the qualities of the epithelial cell grafts are likewise potentially unstable. Therefore, in this paper, we report the stable protocol for fabrication of the transplantable cell sheet particularly from patient-derived oral mucosal tissues. Methods Serum extracted from blood and buccal mucosal tissue were collected in Nagasaki University and transported to Tokyo Women's Medical University. Oral mucosal epithelial cells were collected by minimum trypsin method, and this treatment was studied whether to be a critical procedure. After 14 days cultivation, cultured cells were examined whether to be transplantable as cell sheets. Results We successfully transported buccal mucosal tissue and serum without damage and contamination. Oral mucosal epithelial cells were collected with high viability by minimum trypsin method. Finally, we succeeded to stably fabricate oral mucosal epithelial cell sheets in all 10 patients. Conclusions We established a stable protocol for the fabrication of human oral mucosal epithelial cell sheets and their transportation in clinical settings in this study. These methodologies could also be basis for transplantation therapy using cultured cell sheets of various types other than oral mucosal epithelial cell and will contribute largely to the future development of regenerative medicine., Highlights • A stable protocol for fabricating oral mucosal cell sheets.

Extracellular vesicles (EVs), including exosomes, are small membrane-bound particles released by cells. From a therapeutic point of view, EVs can often convey similar biological function as their parent cell. Grafts originating from oral mucosa have frequently been used in regenerative medicine, and we have previously described the use of oral cell sheets to prevent stricture formation of the esophagus. Further, we recently found that exosomes derived from these cell sheets have pro-regenerative effect on skin wound healing. Here, we have isolated exosomes from conditioned media from oral keratinocyte (“OKEx”) and dermal fibroblast (“FEx”) cultures. The exosomes were probed for classical EV-markers by western blot (CD9, annexin V and Flotillin-1), FEx were positive for all markers while OKEx were positive only for CD9. Tunable resistive pulse sensing indicated a mean size of around 110 nm and transmission electron microscopy showed a spherical morphology, for both groups. After fluorescent labelling, we studied the uptake of exosomes co-cultured with fibroblasts or keratinocytes. Signal from OKEx could be detected after 90 min, and signal could be detected in all groups after 16 h. Finally we studied the exosomes’ modulation of cell proliferation. Both groups suppressed proliferation of healthy keratinocyte and fibroblasts, at some doses to similar levels as dexamethasone (a drug commonly used to prevent stricture formation). In contrast, the exosomes also suppressed the proliferation of the carcinoma cell line TR146, while dexamethasone had no effect. In conclusion, we believe that exosome-signaling might be one of the mode-of-actions of cell sheet-therapy for stricture prevention.

The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer.

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and beta 2-microglobulin (beta 2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with beta 2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 mu g/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is classified into two subtypes: eosinophilic (ECRSwNP) and non-eosinophilic (NECRSwNP). Although the inflammatory patterns of ECRSwNP have been elucidated, NECRSwNP is poorly understood.The metalloproteinase ADAM-like decysin 1 (ADAMDEC1) has been reported to play a role in the early stages of the inflammatory response. We investigated the role of ADAMDEC1 in the pathogenesis of CRSwNP.We compared ADAMDEC1 expression in nasal polyp tissue from CRS patients using immunohistochemistry and RT-qPCR. Macrophages were cultured and ADAMDEC1 expression was determined at baseline and after exposure to lipopolysaccharide (LPS).ADAMDEC1 was virtually undetectable in tissues from control patients but was highly expressed in the NECRSwNP group compared with the ECRSwNP group. In nasal polyp tissues, ADAMDEC1 was expressed by CD68-positive cells, with a positive correlation between ADAMDEC1-positive and CD68-positive cells, and also between ADAMDEC1 and CD68 mRNA levels. Furthermore, stimulation of monocyte-derived macrophages with LPS induced ADAMDEC1 expression.This study demonstrates that ADAMDEC1 is involved in the pathogenesis of NECRSwNP, and also bacterial endotoxin signalling in macrophages; however, the underlying mechanism remains to be elucidated.

Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended β-sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.

Autologous stratified squamous epithelial cell sheets have been successfully used to treat epithelial defects in tissues such as the cornea and the esophagus. However, the regenerative cellular events occurring in the grafted epithelial cells are unclear in the early stages of wound healing. In this study, we created an in vitro grafting model using cultured normal human epidermal keratinocyte (NHEK) sheets and a type I collagen gel to investigate the cellular processes that occur within the grafted cell sheet. Cultured NHEK cells successfully became a stratified squamous cell sheet resembling epithelial tissue, retained expression of cellular integrins and adhesion proteins, and adhered successfully to a type I collagen gel. After culture on the collagen gel, expression of E-cadherin, and β-catenin decreased in the cells of the basal layer of the grafted cell sheet, resembling events characteristic of a partial epithelial-mesenchymal transition (EMT). These basal cells also induced migration of the cell sheet. Those phenomena are consistent with the essential events that occur in the wound-healing process observed previously in cell studies. Therefore, the epithelial cell sheet grafted onto a type I collagen gel is a suitable model in vitro to study cellular events and behaviors. Furthermore, we also addressed the therapeutic mechanisms by which the epithelial cell sheet promotes wound healing.

Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended β-sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.

Endoscopic submucosal dissection (ESD) permits en bloc removal of superficial oesophageal squamous cell carcinoma (ESCC). However, post-procedure stricture is common after ESD for widespread tumours, and multiple endoscopic balloon dilation (EBD) procedures are required. We aimed to evaluate the safety and effectiveness of endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets that had been transported by air over a distance of 1200?km in controlling postprocedural oesophageal stricture. Ten patients who underwent complete circular or semicircular ESD for ESCC were transplanted with cell sheets. The safety of the entire process including cell sheet preparation, transport, ESD and cell sheet transplantation was assessed. The incidence of oesophageal stricture, number of EBD sessions, and time until epithelialization were investigated. Each ESD was successfully performed, with subsequent cell sheet engrafting carried out safely. Following cell sheet transplantation, the luminal stenosis rate was 40%, while the median number of EBD sessions was 0. The median post-ESD ulcer healing period was rather short at 36 days. There were no significant complications at any stage of the process. Cell sheet transplantation and preparation at distant sites and transportation by air could be a safe and promising regenerative medicine technology., Scientific Reports, 7, 17460; 2017

This paper proposes a new analytical approach for assessing local damage to reinforced concrete structures subjected to impact load, by applying the discrete element method (DEM). It first outlines the basis concept and analytical formulation of the DEM. Next, it discusses the results of simulation analyses of concrete material tests, uni-axial compression tests and tensile splitting tests conducted to determine appropriate analytical parameters such as material constants, failure criteria and strength increase factors depending on strain rate. Finally, the adaptability of the DEM to local damage to reinforced concrete structures impacted by rigid and deformable missiles is verified through simulation analyses of various types of impact tests. Furthermore, the various impact response characteristics and failure mechanisms, such as impact forces, penetration behavior, reduction in missile velocity and energy transfer process, which are difficult to obtain experimentally, are analytically evaluated by the DEM.

The objective of this study was to clarify experimentally and analytically the loading capacities, deformations and failure modes of various types of reinforced-concrete structure subjected to loads applied at various rates. Flat slabs, slabs with beams and cylindrical walls were tested under static, low speed and high speed loading. Analysis was applied to estimate the test results by the finite element method using a layered shell element. The analysis closely simulated the experimental results until punching shear failure occured.

Three sets of impact tests, small-, intermediate-, and full-scale tests, have been executed to determine local damage to reinforced concrete structures caused by the impact of aircraft engine missiles. The results of the test program showed that (1) the use of the similarity law is appropriate, (2) suitable empirical formulas exist for predicting the local damage caused by rigid missiles, (3) reduction factors may be used for evaluating the reduction in local damage due to the deformability of the engines, (4) the reinforcement ratio has no effect on local damage, and (5) the test results could be adequately predicted using nonlinear response analysis.

Structural damage induced by an aircraft crashing into a reinforced concrete structure includes local damage caused by the deformable engines, and global damage caused by the entire aircraft. Local damage to the target may consist of spalling of concrete from its front face together with missile penetration into it, scabbing of concrete from its rear face, and perforation of missile through it. Until now, local damage to concrete structures has been mainly evaluated by rigid missile impact tests. Past research work regarding local damage caused by impact of deformable missiles has been limited. This paper presents the results of a series of impact tests of small-, intermediate-, and full-scale engine models into reinforced concrete panels. The purpose of the tests was to determine the local damage to a reinforced concrete structure caused by the impact of a deformable aircraft engine.

Previously, estimates of the force caused by aircraft impact into rigid structures have been determined using theoretical methods based on the aircraft's calculated mass and strength distribution. However, these methods required many assumptions to be made and they left many questions unanswered. Because of the uncertainty involved in these analytical predictions of impact force, a full-scale aircraft impact test was performed and an extensive set of response measurements was analyzed to evaluate the impact force against a rigid target. The analysis and evaluation gave an accurate impact force-time curve under the test condition and confirmed the practical use of an existing analytical method. An analysis with a lumped mass spring model also provided good agreement with the test results.

Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have recently been accepted as less invasive methods for treating patients with early esophageal cancers such as squamous cell carcinoma and dysplasia of Barrett's esophagus. However, the large defects in the esophageal mucosa often cause severe esophageal strictures, which dramatically reduce the patient's quality of life. Although preventive endoscopic balloon dilatation can reduce dysphagia and the frequency of dilatation, other approaches are necessary to prevent esophageal strictures after ESD. This review describes several strategies for preventing esophageal strictures after ESD, with a particular focus on anti-inflammatory and tissue engineering approaches. The local injection of triamcinolone acetonide and other systemic steroid therapies are frequently used to prevent esophageal strictures after ESD. Tissue engineering approaches for preventing esophageal strictures have recently been applied in basic research studies. Scaffolds with temporary stents have been applied in five cases, and this technique has been shown to be safe and is anticipated to prevent esophageal strictures. Fabricated autologous oral mucosal epithelial cell sheets to cover the defective mucosa similarly to how commercially available skin products fabricated from epidermal cells are used for skin defects or in cases of intractable ulcers. Fabricated autologous oralmucosal- epithelial cell sheets have already been shown to be safe., World Journal of Gastroenterology, 20(41), 15098; 2014

This paper describes the system modification through the improvement of pulse air penetration into the DPF cell channels in respect to the development of a wall-flow type diesel particulate filter (DPF) system with reverse pulse air regeneration for diesel vehicles. In this system, regeneration becomes more difficult with low exhaust gas temperatures and increased DPF volume. The pressure increase in the DPF cell channels was monitored as a parameter of pulse air penetration when reverse pulse air was injected into the DPF. By maximizing the pressure increase, the pulse air injection system was modified. The modification includes various changes in the air pipe arrangement and the air injecting time. The ratio of the length to the diameter of the DPF was also evaluated in relation to the regeneration efficiency. In this study, the high aspect ratio, i.e. small diameter and long DPF, showed better regeneration efficiency. The results of this study indicate that this system can be enlarged with the above modification despite low exhaust gas temperatures.