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Developing remote control and supervision technology can help in addressing current and future safety issues at sea owing to human errors resulting from an individual’s watch-keeping. This paper presents a remote navigational watch-keeping system that uses virtual reality (VR) to reduce the risk of human errors and alleviate officers’ workload. The system creates a VR environment that replicates the state and navigational instruments of a ship, with limited connectivity via satellite, enabling an operator at an office onshore to have a comprehensive understanding of the surroundings as through the operator is on the navigational bridge. Two navigation experiences were used to evaluate the system. The results demonstrate that the proposed system is effective for double-checking purposes.

Abstract For pluripotent stem cell (PSC)-based regenerative therapy against diabetes, the differentiation efficiency to pancreatic lineage cells needs to be improved based on the mechanistic understanding of pancreatic differentiation. Here, we aimed to elucidate the molecular mechanisms underlying pancreatic endoderm differentiation by searching for factors that regulate a crucial pancreatic endoderm marker gene, NKX6.1. Unbiasedly screening an siRNA knockdown library, we identified a candidate transcription factor, HHEX. HHEX knockdown suppressed the expression of another pancreatic endoderm marker gene, PTF1A, as well as NKX6.1, independently of PDX1, a known regulator of NKX6.1 expression. In contrast, the overexpression of HHEX upregulated the expressions of NKX6.1 and PTF1A. RNA-seq analysis showed decreased expressions of several genes related to pancreatic development, such as NKX6.1, PTF1A, ONECUT1 and ONECUT3, in HHEX knockdown pancreatic endoderm. These results suggest that HHEX plays a key role in pancreatic endoderm differentiation.

Abstract To determine the impact of the coverage of risk-reducing salpingo-oophorectomy (RRSO) and mastectomy (RRM) as well as genetic testing for BRCA pathogenic variants by the national insurance system in Japan. We compared the clinical background of women who underwent RRSO at our institution before and after its coverage by the national insurance system. Those who underwent RRSO between January 2017 and December 2019 and between April 2020 and March 2022 were classified as Period. A and B, respectively. Overall, 134 women underwent RRSO during the study period. In Period A and B, 45 and 89 women underwent RRSO for the study period was 36 and 24 months, respectively. Compared with Period A, the number of women who underwent RRSO per month increased by threefold in Period B (p

Abstract Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), have been implicated in the pathogenesis of skin disorders. We previously reported that Il36rn −/− mice exhibit an enhanced contact hypersensitivity (CHS) response through increased neutrophil recruitment. In addition, Il36rn −/− mice show severe imiquimod-induced psoriatic skin lesions and enhanced neutrophil extracellular trap (NET) formation. We hypothesized that NETs may play an important role in the CHS response. To confirm this, we examined the CHS response and NET formation in Il36rn −/− mice. Il36rn −/− mice showed enhanced CHS responses, increased infiltration of inflammatory cells, including neutrophils, CD4+ T cells, and CD8+ T cells, NET formation, and enhanced mRNA expression of cytokines and chemokines, including IL-1β, C-X-C motif chemokine ligand (CXCL)1, CXCL2, and IL-36γ. Furthermore, NET formation blockade improved the CHS response, which consequently decreased inflammatory cell infiltration and NET formation. Consistently, we observed decreased expression of these cytokines and chemokines. These findings indicate that IL-36Ra deficiency aggravates the CHS response caused by excessive inflammatory cell recruitment, NET formation, and cytokine and chemokine production, and that NET formation blockade alleviates the CHS response. Thus, NET formation may play a prominent role in the CHS response.

2-Aza-9-fluorenone derivatives were synthesized and their optical properties were investigated. The UV–visible absrption spectra revealed λmax values in the range of 350–380 nm, with a shift to the longer wavelength when an electron-donating group was substituted on the aryl group at the 3-position adjacent to the nitrogen in the azafluorenone. N-Methyl-2-azafluorenone was synthesized by methylating the nitrogen of azafluorenone. The N-methyl derivative was found to have a maximum absorption at 426 nm from UV-Vis spectral measurements, and electrochemical measurements revealed that it has a lower first reduction potential and higher electron-accepting ability than azafluorenone. In addition, the application of azafluorenone as a photocatalyst was investigated, focusing on its structural and electronic features. Oxidation of toluene with azafluorenone as a photocatalyst yielded benzoic acid in high yield. Electron-transfer dynamics analysis using nanosecond time-resolved laser flash photolysis suggests that azafluorenone and N-methylazafluorenone act as hydrogen transfer and electron transfer catalysts, respectively.

Classes of lipoproteins solubilize lipids in the blood, and their profiles are important for preventing atherosclerotic diseases. These can be identified by gel filtration HPLC, which has been analyzed in a manner that yields the same values as the de facto standard method, i.e., ultracentrifugation; however, previous studies have found that ultracentrifugation and its simplified alternatives, enzymatic methods, yield incorrect values. Here HPLC data of stroke patients and the controls were compared using data-driven analyses, without consideration for ultracentrifugation. The data well-separated patients from controls. In many patients, the level of HDL1 (a cholesterol scavenger) was low. The TG/cholesterol ratio of chylomicrons was found to be low in patients and high in the healthy elderly; the lower level may indicate a larger intake of animal fats. High levels of free glycerol in the elderly were hazardous, suggesting more dependence on lipids as an energy source. Statins had minimal effect on these factors. LDL cholesterol, the commonly-used risk indicator, was not a risk factor actually. Enzymatic methods failed to separate the patients from the control; hence, the existing guidelines for screening methods and medical treatment need to be revised. As an immediate step, glycerol would be an adaptable indicator.

Abstract The serotonin 5-HT2A receptor (5-HT2AR) has been receiving increasing attention because its genetic variants have been associated with a variety of neurological diseases. To elucidate the pathogenesis of the neurological diseases associated with 5-HT2AR gene (HTR2A) variants, we have previously established a protocol to induce HTR2A-expressing neurons from human-induced pluripotent stem cells (hiPSCs). Here, we investigated the maturation stages and electrophysiological properties of HTR2A-positive neurons induced from hiPSCs and constructed an HTR2A promoter-specific reporter lentivirus to label the neurons. We found that neuronal maturity increased over time and that HTR2A expression was induced at the late stage of neuronal maturation. Furthermore, we demonstrated successful labelling of the HTR2A-positive neurons, which had fluorescence and generated repetitive action potentials in response to depolarizing currents and an inward current during the application of TCB-2, a selective agonist of 5-HT2ARs, respectively. These results indicated that our in vitro model mimicked the in vivo dynamics of 5-HT2AR. Therefore, in vitro monitoring of the function of HTR2A-positive neurons induced from hiPSCs could help elucidate the pathophysiological mechanisms of neurological diseases associated with genetic variations of the HTR2A gene.

Huntingtin-associated protein 1 (HAP1) is a determinant marker for the stigmoid body (STB), a neurocytoplasmic physiological inclusion. STB/HAP1 enriched areas in the brain/spinal cord are usually protected from neurodegenerative diseases, whereas the regions with tiny amounts or no STB/HAP1 are affected. In addition to the brain/spinal cord, HAP1 is highly expressed in the myenteric/submucosal plexuses of the enteric nervous system in the gastrointestinal tract. The tongue is attached to the pharynx by the hyoid bone as an extension of the gastrointestinal system. To date, the immunohistochemical distribution and neurochemical characterization of HAP1 have not been elucidated in the lingual ganglia. Using immunohistochemistry and light microscopy, our current study demonstrates the expression and immunohistochemical phenotype of HAP1 in the lingual ganglia of adult mice. We showed that HAP1 was profoundly distributed in the intralingual ganglion (ILG) and the ganglia near the root of the tongue (which we coined as “lingual root ganglion”; LRG). Neurons in ILG and LRG exhibited high coexpression of HAP1 with NOS or ChAT. Furthermore, most HAP1-immunoreactive neurons contained SP, CGRP, and VIP immunoreactivity in both ILG and LRG. The current results might serve as an essential base for future studies to elucidate the pathological/physiological functions of HAP1 in the lingual ganglia.

Abstract Aims/Introduction Non‐alcoholic fatty liver disease (NAFLD) is often observed in individuals with type 2 diabetes mellitus, and it is known that the presence of type 2 diabetes mellitus leads to the aggravation of NAFLD. The aim of this study was to compare the possible effects of three kinds of oral hypoglycemic agents on NAFLD in individuals with type 2 diabetes mellitus. Materials and Methods We carried out a prospective clinical trial (a randomized and open‐label study) in patients with type 2 diabetes mellitus and NAFLD. A total of 98 patients were randomly allocated either to the dapagliflozin (n = 32), pioglitazone (n = 33) or glimepiride (n = 33) group, and the patients took these drugs for 28 weeks. The primary end‐point was the change of the liver‐to‐spleen ratio on abdominal computed tomography. Results There was no difference in baseline clinical characteristics among the three groups. Dapagliflozin, pioglitazone and glimepiride ameliorated hyperglycemia similarly. Bodyweight and visceral fat area were significantly decreased only in the dapagliflozin group. Serum adiponectin levels were markedly increased in the pioglitazone group compared with the other two groups. Dapagliflozin and pioglitazone, but not glimepiride, significantly increased the liver‐to‐spleen ratio, and the effects of dapagliflozin and pioglitazone on the liver‐to‐spleen ratio were comparable. Conclusions The present study showed that the decrease of visceral fat area and the increase of adiponectin level contributed to the improvement of NAFLD in patients with type 2 diabetes mellitus. Furthermore, dapagliflozin and pioglitazone exerted equivalent beneficial effects on NAFLD in patients with type 2 diabetes mellitus, although it seemed that these two drugs had different mechanisms of action.

Sleep bruxism (SB) is a sleep-related movement disorder characterized by grinding and clenching of the teeth during sleep. We previously found a significant association between SB and a single nucleotide polymorphism (SNP), rs6313, in the neuronal serotonin 2A receptor gene (HTR2A), and established human induced pluripotent stem cell (iPSC)-derived neurons from SB patients with a genetic variant. To elucidate the electrophysiological characteristics of SB iPSC-derived neural cells bearing an SB-related genetic variant, we generated ventral hindbrain neurons from SB patients and unaffected controls, and explored the intrinsic membrane properties of these neurons using the patch-clamp technique. We found that the electrophysiological properties of iPSC-derived neurons mature in a time-dependent manner in long-term control cultures. SB neurons exhibited higher action potential firing frequency, higher gain, and shorter action potential half duration. This is the first in vitro modeling of SB using patient-specific iPSCs. The revealed electrophysiological characteristics may serve as a benchmark for further investigation of pathogenic mechanisms underlying SB. Moreover, our results on long-term cultures provide a strategy to define the functional maturity of human neurons in vitro, which can be implemented for stem cell research of neurogenesis, and neurodevelopmental disorders.

Recent studies have reported that plasma levels of tricarboxylic acid (TCA) cycle metabolites and TCA cycle-related metabolite change in patients with chronic fatigue syndrome (CFS) and in healthy humans after exercise. Exogenous dietary citric acid has been reported to alleviate fatigue during daily activities and after exercise. However, it is unknown whether dietary citric acid affects the plasma levels of these metabolites. Therefore, the present study aimed to investigate the effects of exogenously administered citric acid on TCA cycle metabolites and TCA cycle-related metabolites in plasma. Sprague-Dawley rats were divided into control and citric acid groups. We evaluated the effect of exogenous dietary citric acid on the plasma TCA cycle and TCA cycle-related metabolites by metabolome analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). TCA cycle metabolites, including plasma citrate, cis-aconitate, and isocitrate, were significantly elevated after exogenous administration of citric acid. Anaplerotic amino acids, which are converted to TCA cycle metabolites, such as serine, glycine, tryptophan, lysine, leucine, histidine, glutamine, arginine, isoleucine, methionine, valine, and phenylalanine, also showed significantly elevated levels. Citric acid administration significantly increased the levels of initial TCA cycle metabolites in the plasma. This increase after administration of citric acid was shown to be opposite to the metabolic changes observed in patients with CFS. These results contribute novel insight into the fatigue alleviation mechanism of citric acid.

The aim of this study was to determine changes in gene expression associated with glucose metabolism in the liver and soleus muscles of rats exposed to hypoxia to improve work capacity under high altitude conditions. Rats were divided into normobaric normoxia (control) and normobaric hypoxia (hypoxia) groups (n = 7 each), and the hypoxia group was exposed to 10.5% oxygen for 90 min. Glucose metabolism-related gene expression was examined by real-time polymerase chain reaction. In the liver, the expression levels of the glucose utilization-related genes solute carrier family 2 member 1, glucokinase, and liver-type phosphofructokinase and the gluconeogenesis-related gene phosphoenolpyruvate carboxykinase 1 (Pck1) were significantly increased upon hypoxic exposure. In contrast, gene expression in the soleus was unchanged, with the exception of Pck1. The results suggest that under hypoxia, both glucose utilization and gluconeogenesis are accelerated in the liver, and liver glycogen is degraded to maintain blood glucose level.

The balance between neural excitation and inhibition has been shown to be crucial for normal brain function. However, it is unclear whether this balance is maintained through healthy aging. This study investigated the effect of aging on the temporal dynamics of the somatosensory evoked local field potential (LFP) in rats and tested the hypothesis that excitatory and inhibitory post-synaptic activities remain balanced during the aging process. The LFP signal was obtained from the barrel cortex of three different age groups of anesthetized rats (pre-adolescence: 4–6 weeks, young adult: 2–3 months, middle-aged adult: 10–20 months) under whisker pad stimulation. To confirm our previous finding that the initial segment of the evoked LFP was solely associated with excitatory post-synaptic activity, we micro-injected gabazine into the barrel cortex to block inhibition while LFP was collected continuously under the same stimulus condition. As expected, the initial slope of the evoked LFP in the granular layer was unaffected by gabazine injection. We subsequently estimated the excitatory and inhibitory post-synaptic activities through a balanced model of the LFP with delayed inhibition as an explicit constraint, and calculated the amplitude ratio of inhibition to excitation. We found an age-dependent slowing of the temporal dynamics in the somatosensory-evoked post-synaptic activity, as well as a significant age-related decrease in the amplitude of the excitatory component and a decreasing trend in the amplitude of the inhibitory component. Furthermore, the delay of inhibition with respect to excitation was significantly increased with age, but the amplitude ratio was maintained. Our findings suggest that aging reduces the amplitude of neural responses, but the balance between sensory evoked excitatory and inhibitory post-synaptic activities is maintained to support normal brain function during healthy aging. Further whole cell patch clamp experiments will be needed to confirm or refute these findings by measuring sensory evoked synaptic excitatory and inhibitory activities in vivo during the normal aging process.

Summary: There has been increasing success with the generation of pancreatic cells from human induced pluripotent stem cells (hiPSCs); however, the molecular mechanisms of the differentiation remain elusive. The purpose of this study was to reveal novel molecular mechanisms for differentiation to PDX1+NKX6.1+ pancreatic endoderm cells, which are pancreatic committed progenitor cells. PDX1+ posterior foregut cells differentiated from hiPSCs failed to differentiate into pancreatic endoderm cells at low cell density, but Rho-associated kinase (ROCK) or non-muscle myosin II (NM II) inhibitors rescued the differentiation potential. Consistently, the expression of phosphorylated myosin light chain 2 and NM IIA was downregulated in aggregation culture. Notably, the soluble factors we tested were substantially effective only with ROCK-NM II inhibition. The PDX1+NKX6.1+ cells induced with NM II inhibitors were successfully engrafted and maturated in vivo. Taken together, these results suggest that NM IIs play inhibitory roles for the differentiation of hiPSCs to pancreatic endoderm cells. : Toyoda and colleagues show that ROCK or NM II inhibitors facilitate differentiation of PDX1+ posterior foregut cells into PDX1+NKX6.1+ pancreatic endoderm cells in hiPSC cultures. Consistently, NM II expression was elevated at low cell density, but upon aggregation, it was downregulated and associated with NKX6.1+ cell induction. The results provide mechanistic insight into the benefit of three-dimensional cultures for directed differentiation. Keywords: ESC, iPSC, differentiation, pancreas, pancreatic bud, pancreatic endoderm, NKX6.1, ROCK, non-muscle myosin II, cytoskeleton

In the original published PDF version of the article, the labels on the graphs in Figure 2c were inadvertently swapped. The error has been corrected in the PDF.

Duplication of the alimentary tract is a rare congenital malformation that occurs most often in the abdominal region, whereas esophageal duplication cyst develops typically in the thoracic region but occasionally in the neck and abdominal regions. Esophageal duplication cyst is usually diagnosed in early childhood because of symptoms related to bleeding, infection, and displacement of tissue surrounding the lesion. We recently encountered a rare adult case of esophageal duplication cyst in the abdominal esophagus. A 50-year-old man underwent gastroscopy, endoscopic ultrasonography, computed tomography, and magnetic resonance imaging to investigate epigastric pain and dysphagia that started 3 months earlier. Imaging findings suggested esophageal duplication cyst, and the patient underwent laparoscopic resection followed by intraoperative esophagoscopy to reconstruct the esophagus safely and effectively. Histopathological examination of the resected specimen revealed two layers of smooth muscle in the cystic wall, confirming the diagnosis of esophageal duplication cyst.

Previously, we showed silicone nerve conduits containing a vascular bundle and decellularized allogenic basal laminae (DABLs) seeded with bone marrow-derived mesenchymal stem cells (BMSCs) demonstrated successful nerve regeneration. Nerve conduits should be flexible and biodegradable for clinical use. In the current study, we used nerve conduits made of polyglycoric acid (PGA) fiber mesh, which is flexible, biodegradable and capillary-permeable. DABLs were created using chemical surfactants to remove almost all cell debris. In part 1, capillary infiltration capability of the PGA tube was examined. Capillary infiltration into regenerated neural tissue was compared between the PGA tube with blood vessels attached extratubularly (extratubularly vascularized tube) and that containing blood vessels intratubularly (intratubularly vascularized tube). No significant difference was found in capillary formation or nerve regeneration between these two tubes. In part 2, a 20 mm gap created in a rat sciatic nerve model was bridged using the extratubularly vascularized PGA tube containing the DABLs with implantation of isogenic cultured BMSCs (TubeC+ group), that containing the DABLs without implantation of the BMSCs (TubeC- group), and 20 mm-long fresh autologous nerve graft (Auto group). Nerve regeneration in these three groups was assessed electrophysiologically and histomorphometrically. At 24 weeks, there was no significant difference in any electrophysiological parameters between TubeC+ and Auto groups, although all histological parameters in Auto group were significantly greater than those in TubeC+ and TubeC- groups, and TubeC+ group demonstrated significant better nerve regeneration than TubeC- group. The transplanted DABLs showed no signs of immunological rejection and some transplanted BMSCs were differentiated into cells with Schwann cell-like phenotype, which might have promoted nerve regeneration within the conduit. This study indicated that the TubeC+ nerve conduit may become an alternative to nerve autograft., Competing Interests: The authors have declared that no competing interests exist.

Background:  We previously reported the development of a scaffold-free Bio three-dimensional (3D) nerve conduit from normal human dermal fibroblasts (NHDFs). The aim of this study was to investigate the regenerative mechanism of peripheral nerve cells using a Bio 3D conduit in a rat sciatic nerve defect model., Methods:  Bio 3D conduits composed of NHDFs were developed, and cell viability was evaluated using a LIVE/DEAD cell viability assay immediately before transplantation and 1-week post-surgery. Tracking analysis using PKH26-labeled NHDFs was performed to assess the distribution of NHDFs within the regenerated nerve and the differentiation of NHDFs into functional Schwann cells (SCs)., Results:  The assessment of the viability of cells within the Bio 3D conduit showed high cell viability both immediately before transplantation and 1-week post-surgery (88.56 ± 1.70 and 87.58 ± 9.11, respectively). A modified Masson's trichrome staining of the Bio 3D conduit revealed the formation of a prominent extracellular matrix (ECM) in between the cells. We observed, via tracking analysis, that the tube-like distribution of the NHDFs remained stable, the majority of the regenerated axons had penetrated this structure and PKH26-labeled cells were also positive for S-100., Conclusion:  Abundant ECM formation resulted in a stable tube-like structure of the Bio 3D conduit with high cell viability. NHDFs in the Bio 3D conduit have the potential to differentiate into SCs-like cells., Competing Interests: K.N. is a co-founder and shareholder of Cyfuse Biomedical K.K. The remaining authors declared no competing interests., (Thieme. All rights reserved.)

Objectives/hypothesis: To regenerate defected recurrent laryngeal nerves (RLNs), various methods have been developed. However, no consistently effective treatments are currently available because of their insufficient functional recovery. RADA16-I, a self-assembling peptide used clinically as a hemostat, reportedly supports neurite outgrowth and functional synapse formation in vitro. The purpose of this study was to investigate the effect of RADA16-I hydrogels on transected RLNs in rats., Study Design: Animal experiments with controls., Methods: Fifteen adult rats were divided into the following three groups: RADA16-I (+), RADA16-I (-), and neurectomy. A 6-mm gap of the left RLN was bridged using an 8-mm silicone tube in the RADA16-I (-) and RADA16-I (+) groups. Subsequently, RADA16-I hydrogel was injected into the tube in the RADA16-I (+) group. The surgical incisions were closed without any further treatment in the neurectomy group. After 8 weeks, laryngoscopy and electrophysiological and histological examinations were performed to evaluate the effect of RADA16-I on nerve regeneration and thyroarytenoid muscle atrophy., Results: Although most rats in the three groups exhibited no improvements of their vocal fold movement, partial recovery was observed in one rat in the RADA16-I (+) group. The neurofilament-positive areas and the number of myelinated nerves in the RADA16-I (+) group were significantly higher than in the RADA16-I (-) group. The area of the left thyroarytenoid muscle in the RADA16-I (+) group was significantly larger than that of the neurectomy group., Conclusions: Our results suggested that RADA16-I hydrogel was effective for RLN regeneration., Level of Evidence: NA Laryngoscope, 130:2420-2427, 2020., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

Introduction: A Bio 3D printed nerve conduit was reported to promote nerve regeneration in a 5 mm nerve gap model. The purpose of this study was to fabricate Bio 3D nerve conduits suitable for a 10 mm nerve gap and to evaluate their capacity for nerve regeneration in a rat sciatic nerve defect model., Materials and Methods: Eighteen F344 rats with immune deficiency (9-10 weeks old; weight, 200-250 g) were divided into three groups: a Bio 3D nerve conduit group (Bio 3D, n = 6), a nerve graft group (NG, n = 6), and a silicon tube group (ST, n = 6). A 12-mm Bio 3D nerve conduit or silicon tube was transplanted into the 10-mm defect of the right sciatic nerve. In the nerve graft group, reverse autografting was performed with an excised 10-mm nerve segment. Assessments were performed at 8 weeks after the surgery., Results: In the region distal to the suture site, the number of myelinated axons in the Bio 3D group were significantly larger compared with the silicon group (2,548 vs. 950, p < .05). The myelinated axon diameter (MAD) and the myelin thickness (MT) of the regenerated axons in the Bio 3D group were significantly larger compared with those of the ST group (MAD: 3.09 vs. 2.36 μm; p < .01; MT: 0.59 vs. 0.40 μm, p < .01)., Conclusions: This study indicates that a Bio 3D nerve conduit can enhance peripheral nerve regeneration even in a 10 mm nerve defect model., (© 2019 Wiley Periodicals, Inc.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background:  The treatment of peripheral nerve defects requires bridging materials. Skeletal muscle grafts have been studied as an alternative to nerve autografts because they contain longitudinally aligned basal laminar tubes that are similar to axons. Several pretreatment methods for muscle grafts have promoted axonal regeneration. Here, a new method of doxorubicin pretreatment was used, and the efficacy of the pretreated muscle graft was evaluated in a rat model of a sciatic nerve defect., Methods:  A rat model of a 10-mm sciatic nerve defect was analyzed in three settings: muscle grafts with and without doxorubicin pretreatment (M-graft-w-Dox and M-graft-w/o-Dox groups, respectively) and a nerve autograft group (N-graft) ( n  = 6/group). The M-graft-w-Dox group was immersed in a doxorubicin solution for 10 minutes and rinsed with saline. Analyses of target muscle atrophy, electrophysiology, and histology were performed 8 weeks after grafting., Results:  Electrophysiological parameters and target muscle atrophy were significantly superior in the M-graft-w-Dox group compared with the M-graft-w/o-Dox group. Histological assessment revealed the presence of a significantly greater number of regenerated axons in the M-graft-w-Dox group versus the M-graft-w/o-Dox group, while there were no significant differences between the M-graft-w-Dox and N-graft groups. The diameter of myelinated axons of the regenerated nerve in the M-graft-w-Dox group was significantly larger than that in the M-graft-w/o-Dox group, while it was not significantly different compared with the N-graft group., Conclusion:  Pretreatment of muscle grafts with doxorubicin promoted significant peripheral nerve regeneration. This method may represent a new option for the treatment of peripheral nerve defects., Competing Interests: None declared., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

We previously reported that a nerve conduit created from fibroblasts promotes nerve regeneration in a rat sciatic nerve model. This study aims to determine whether a nerve conduit created from bone marrow stromal cells (BMSCs) can promote nerve regeneration. Primary BMSCs were isolated from femur bone marrow of two Lewis rats, and cells at passages 4-7 were used. We created seven Bio 3D nerve conduits from BMSCs using a Bio-3D Printer. The conduits were transplanted to other Lewis rats to bridge 5-mm right sciatic nerve gaps (Bio 3D group, n = 7). We created two control groups: a silicone group (S group, n = 5) in which the same nerve gap was bridged with a silicone tube, and a silicone cell group (SC group, n = 5) in which the gap was bridged with a BMSC injection. Twelve weeks after transplantation, nerve regeneration was evaluated functionally and morphologically. In addition, PKH26-labeled BMSCs were used to fabricate a Bio 3D conduit that was transplanted for cell trafficking analysis. Electrophysiological study, kinematic analysis, wet muscle weight, and morphological parameters showed significantly better nerve regeneration in the Bio 3D group than in the S group or SC group. In immunohistochemical studies, sections from the Bio 3D group contained abundant S-100-positive cells. In cell trafficking analysis, PKH26-positive cells stained positive for the Schwann cell markers S-100, p75NTR, and GFAP. Bio 3D nerve conduits created from BMSCs can promote peripheral nerve regeneration in a rat sciatic nerve model through BMSC differentiation into Schwann-like cells.

Background: The reduction of systemic immunosuppressive agents is essential for the expansion of vascularized composite allotransplantation (VCA) in a clinical setting. The purpose of this study is to compare human-induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) with four other types of mesenchymal stem cells (human bone marrow-derived MSCs [BMMSCs], human adipose-derived MSCs [ADMSCs], rat BMMSCs, and rat ADMSCs) in vitro, and to investigate the in vivo immunomodulatory effect of iMSCs in a rat VCA model., Materials and Methods: One Brown Norway (BN) rat, 2 Lewis (LEW) rats, and 1 Wistar rat were used in the mixed lymphocyte reaction (MLR), and 9 BN rats and 3 LEW rats (for donors), and 24 LEW rats (for recipients) were used in the VCA model. The abovementioned five types of MSCs were imaged to examine their morphology and were also tested for suppressor function using a MLR. The 24 recipient LEW rats were divided randomly into four groups, and subjected to orthotopic hind limb transplantation. The three control groups were the Iso group, in which transplantation was performed on from three to six LEW rats without immunosuppressive treatment (n = 6); the FK group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus alone (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) (n = 6); and the UT group, in which transplantation was performed from BN rats to LEW rats without any immunosuppressive treatment (n = 6). The experimental group was the iMSC group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) and injected with iMSCs (2 × 10 6 cells, day 7, intravenously) (n = 6). Hind limb survival was assessed by daily inspection of gross appearance until 50 days postoperatively. Histology of the skin and muscle biopsy were investigated on day 14 postoperatively. A time series of the plasma cytokine level (before transplantation, and at 10, 14, and 17 days after transplantation) was also analyzed., Results: The size of adherent and trypsinized iMSCs was 67.5 ± 8.7 and 9.5 ± 1.1 μm, respectively, which was the smallest among the five types of MSCs (p < .01). The absorbance in MLR was significantly smaller with rat ADMSCs (p = .0001), human iMSCs (p = .0006), rat BMMSCs (p = .0014), human ADMSCs (p = .0039), and human BMMSCs (p = .1191) compared to without MSCs. In vivo, iMSC treatment prolonged hind limb survival up to 12.7 days in macroscopic appearance, which is significantly longer than that of the FK group (p < .01). Histology of the skin and muscle biopsy revealed that mononuclear cell infiltration was significantly reduced by iMSC injection (p < .01). iMSC treatment also affected proinflammatory cytokines (interferon-gamma (IFNγ) and tumor necrosis factor α (TNFα)) and the anti-inflammatory cytokine (interleukin-10 (IL-10)) of the recipient plasma. The IFNγ levels at Δ14 and the TNFα levels at Δ14 and Δ17 of the iMSC group were significantly lower than those of the FK group (p = .0226, .0004, and .004, respectively). The IL-10 levels at Δ10 and Δ14 of the iMSC group were significantly higher than those of the FK group (p = .0013 and .0374, respectively)., Conclusions: iMSCs induce T cell hyporesponsiveness to prolong hind limb survival in a rat VCA model. This immunomodulatory property against acute rejection could provide one of the promising strategies capable of enabling the toxicities of immunosuppressants to be avoided in clinical settings., (© 2019 Wiley Periodicals, Inc.)

Autologous nerve grafting is widely accepted as the gold standard treatment for segmental nerve defects. To overcome the inevitable disadvantages of the original method, alternative methods such as the tubulization technique have been developed. Several studies have investigated the characteristics of an ideal nerve conduit in terms of supportive cells, scaffolds, growth factors, and vascularity. Previously, we confirmed that biological scaffold-free conduits fabricated from human dermal fibroblasts promote nerve regeneration in a rat sciatic nerve injury model. The purpose of this study is to evaluate the feasibility of biological scaffold-free conduits composed of autologous dermal fibroblasts using a large-animal model. Six male beagle dogs were used in this study. Eight weeks before surgery, dermal fibroblasts were harvested from their groin skin and grown in culture. Bio 3D conduits were assembled from proliferating dermal fibroblasts using a Bio 3D printer. The ulnar nerve in each dog's forelimb was exposed under general anesthesia and sharply cut to create a 5 mm interstump gap, which was bridged by the prepared 8 mm Bio 3D conduit. Ten weeks after surgery, nerve regeneration was investigated. Electrophysiological studies detected compound muscle action potentials (CMAPs) of the hypothenar muscles and motor nerve conduction velocity (MNCV) in all animals. Macroscopic observation showed regenerated ulnar nerves. Low-level hypothenar muscle atrophy was confirmed. Immunohistochemical, histological, and morphometric studies confirmed the existence of many myelinated axons through the Bio 3D conduit. No severe adverse event was reported. Hypothenar muscles were re-innervated by regenerated nerve fibers through the Bio 3D conduit. The scaffold-free Bio 3D conduit fabricated from autologous dermal fibroblasts is effective for nerve regeneration in a canine ulnar nerve injury model. This technology was feasible as a treatment for peripheral nerve injury and segmental nerve defects in a preclinical setting.

Background: Skin is considered to be the most antigenic component of all vascularized composite allotransplantation tissues. However, no studies have used methods other than histological assessment to analyze the relative antigenicity of various components. In this study, we analyzed gene expression to investigate the relative antigenicity of each component in the transplanted limb., Methods: Seven Brown Norway rats and 31 Lewis rats were assigned to two groups: an allograft group and a syngeneic (control) group. Brown Norway rats were used as the allogeneic donors, and Lewis rats were used as the syngeneic donors and recipients. About 13 recipients in the allograft group and 12 recipients in the control group were analyzed. Histological assessment was performed in 5 of the recipients in each group, and microRNA expression was analyzed in the remaining recipients, except for 1 recipient in the syngeneic group., Results: In the allograft group, the relative microRNA-146a expression was significantly higher in skin (2.34 ± 0.44) than in muscle (1.25 ± 0.22; p = .034) and bone (1; p = .0081). In the allograft group, microRNA-155 expression was significantly higher in skin (1.91 ± 0.18) than in bone (1; p = .010). Histological assessment showed that some skin tissue in the allograft group showed evidence of severe acute rejection., Conclusions: The microRNA-146a and microRNA-155 seemed to reflect the relative antigenicity during acute rejection of transplanted limbs. Skin seemed to be more antigenic than muscle and bone in both the histological assessment and gene expression analysis., (© 2018 Wiley Periodicals, Inc.)

Background: Replantation of the avulsed nerve root has been proposed for the treatment of severe brachial plexus injury for several decades. However, due to the complexity of the technique and limited functional improvement, practical applications are yet to be implemented., Objective: In the present study, we investigated the effect of pretreatment with resveratrol on nerve autografts used for replantation surgery in a rat model of nerve root avulsion., Methods: Resveratrol pretreatment was performed using an explant culture technique. Two surgical procedures were performed. During the first surgery, Sprague-Dawley rats were subjected to left C6 nerve root avulsion, and nerves were harvested for autografting. The harvested grafts were explant-cultured for 1 week. A second procedure was performed to replant the C6 nerve root using the explant-cultured nerve graft 1 week after the first procedure. Histological and immunohistochemical analyses were performed 8 weeks after the second procedure. We first compared findings between explant-cultured nerve grafts and fresh nerve grafts, following which we compared findings between explant-cultured grafts pretreated with and without resveratrol. Changes induced within nerve grafts by 1 week of explant culture with or without resveratrol were investigated in vitro., Results: There was no significant difference in outcomes between 1 week-explant-cultured and fresh nerve grafts. Addition of resveratrol to the explant culture medium resulted in a significant increase in the number and myelin thickness of regenerated axons, and in the number of regenerating motor neurons in the C6 spinal cord segment. In vitro analyses revealed that nerve grafts pretreated with resveratrol exhibited significant increases in glial cell line-derived neurotrophic factor (GDNF) expression and the number of dedifferentiated Schwann cells., Conclusions: Resveratrol may promote axonal regeneration following replantation surgery for the treatment of nerve root avulsion injury; however, further studies are required to verify these findings in humans.

Background: Limb transplantation is considered to be a treatment option for amputees. Visual skin inspection and histological assessment are used to assess rejection, but these techniques are largely subjective. Using a rat model, we examined the potential of several microRNAs (miRNAs) to be used as objective and minimally invasive biomarkers of acute rejection of transplanted limbs., Methods: Three Brown Norway rats and 15 Lewis rats were assigned to 2 groups. In the allograft group, 3 Brown Norway rats were used as the donors and 6 Lewis rats as the recipients. In the syngeneic (control) group, 9 Lewis rats were used as the donors and recipients. The hind limbs of the donors were orthotopically transplanted to the recipients. Plasma samples were obtained from all recipients before surgery and on posttransplantation days 3, 7, 10, and 14. All recipients were euthanized on day 14, and skin tissues were harvested for histological assessment., Results: On posttransplantation days 10 and 14, the plasma expression of miRNA-146a, miRNA-155, and miRNA-182 was significantly upregulated in the allograft group compared with the syngeneic control group. Of these 3 miRNAs, miRNA-182 had the highest sensitivity and specificity; however the cutoff point for miRNA-182 was close to the baseline and miRNA-155 was considered to be most suitable for identifying rejection. Most skin samples in the allograft group were classified as exhibiting grade III rejection on day 14., Conclusions: Some miRNAs are upregulated during acute rejection of transplanted limbs. These miRNAs are potential biomarkers for the objective, early, minimally invasive diagnosis of rejection., (© 2017 Wiley Periodicals, Inc.)

Background: Recent studies have indicated that bone marrow-derived stromal cells (BMSCs) have immunomodulatory properties that suppress the T cell responses that cause graft rejection. The purpose of this study is to evaluate the effect of recipient BMSCs intravenous infusion for immunomodulation in a rat vascularized composite allotransplantation model., Methods: A total of nine Wistar (WIS) rats and thirty Lewis (LEW) rats were used. BMSCs were harvested from three LEW rats. Twenty-four LEW rats were used as recipients and divided randomly into four groups: BMSC group, FK group, UT group, and Iso group. In the BMSC group, orthotopic rat hind limb transplantation was performed between WIS donor and LEW recipient rats. Recipient rats were injected intravenously with 2 × 10 6 recipient BMSCs on day 6, and with 0.2 mg/kg/day tacrolimus administered over 7 days (n = 6). In the FK group, recipient rats were treated with tacrolimus alone (n = 6). Rats in the UT group received no immunosuppressive treatment (n = 6). In the Iso group, transplantation was performed from three LEW donor rats to six LEW recipient rats without any immunosuppressive treatment (n = 6). Graft survival was assessed by daily inspection and histology. The immunological reactions of recipients were also evaluated., Results: The graft survival of recipient rats in the BMSC group (24.5 days) was significantly prolonged in comparison with that of the FK group (18 days) (P < .01). Cytokine expression analysis of the skin of grafted limbs showed that BMSCs treatment significantly decreased IFN-γ mRNA expression of the BMSC group (0.138 ± 0.045) in comparison with that of the FK group (1.049 ± 0.167) (P = .0001). Recipient rats in the BMSC group had significantly reduced serum IFN-γ cytokine levels (1.571 ± 0.779 pg/ml) in comparison with that of the FK group (7.059 ± 1.522 pg/ml) (P = .001). In in vitro study, BMSCs induce T cell hyporesponsiveness in a mixed lymphocyte reaction., Conclusion: BMSCs induce T cell hyporesponsiveness and prolong graft survival in the rat vascularized composite allotransplantation model. BMSCs exhibit immunomodulatory properties against acute rejection that can be realized without the need for significant recipient immunosuppression., (© 2016 Wiley Periodicals, Inc.)

Cells, scaffolds, growth factors, and vascularity are essential for nerve regeneration. Previously, we reported that the insertion of a vascular bundle and the implantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a nerve conduit promoted peripheral nerve regeneration. In this study, the efficacy of nerve conduits containing a vascular bundle, BM-MSCs, and thermally decellularized allogenic nerve matrix (DANM) was investigated using a rat sciatic nerve model with a 20-mm defect. Lewis rats were used as the sciatic nerve model and for the preparation of BM-MSCs, and Dark Agouti rats were used for the preparation of the DANM. The revascularization and the immunogenicity of the DANM were investigated histologically. The regeneration of nerves through nerve conduits containing vessels, BM-MSCs, and DANM (VBD group) was evaluated based on electrophysiological, morphometric, and reinnervated muscle weight measurements and compared with that of vessel-containing conduits that were implanted with BM-MSCs (VB group). The DANM that was implanted into vessel-containing tubes (VCTs) was revascularized by neovascular vessels that originated from the inserted vascular bundle 5-7 days after surgery. The number of CD8+ cells found in the DANM in the VCT was significantly smaller than that detected in the untreated allogenic nerve segment. The regenerated nerve in the VBD group was significantly superior to that in the VB group with regard to the amplitude of the compound muscle action potential detected in the pedal adductor muscle; the number, diameter, and myelin thickness of the myelinated axons; and the tibialis anterior muscle weight at 12 and 24 weeks. The additional implantation of the DANM into the BM-MSC-implanted VCT optimized the axonal regeneration through the conduit. Nerve conduits constructed with vascularity, cells, and scaffolds could be an effective strategy for the treatment of peripheral nerve injuries with significant segmental defects.

Background: Although autologous nerve grafting is the gold standard treatment of peripheral nerve injuries, several alternative methods have been developed, including nerve conduits that use supportive cells. However, the seeding efficacy and viability of supportive cells injected in nerve grafts remain unclear. Here, we focused on a novel completely biological, tissue-engineered, scaffold-free conduit., Methods: We developed six scaffold-free conduits from human normal dermal fibroblasts using a Bio 3D Printer. Twelve adult male rats with immune deficiency underwent mid-thigh-level transection of the right sciatic nerve. The resulting 5-mm nerve gap was bridged using 8-mm Bio 3D conduits (Bio 3D group, n = 6) and silicone tube (silicone group, n = 6). Several assessments were conducted to examine nerve regeneration eight weeks post-surgery., Results: Kinematic analysis revealed that the toe angle to the metatarsal bone at the final segment of the swing phase was significantly higher in the Bio 3D group than the silicone group (-35.78 ± 10.68 versus -62.48 ± 6.15, respectively; p < 0.01). Electrophysiological studies revealed significantly higher compound muscle action potential in the Bio 3D group than the silicone group (53.60 ± 26.36% versus 2.93 ± 1.84%; p < 0.01). Histological and morphological studies revealed neural cell expression in all regions of the regenerated nerves and the presence of many well-myelinated axons in the Bio 3D group. The wet muscle weight of the tibialis anterior muscle was significantly higher in the Bio 3D group than the silicone group (0.544 ± 0.063 versus 0.396 ± 0.031, respectively; p < 0.01)., Conclusions: We confirmed that scaffold-free Bio 3D conduits composed entirely of fibroblast cells promote nerve regeneration in a rat sciatic nerve model., Competing Interests: There are no patents or marketed products to declare. KN is the co-founder and shareholder of Cyfuse Biomedical K.K., Tokyo, Japan (Cyfuse). SA and MT, who are employees of Cyfuse, contributed to the manufacturing of 3D conduits and Cyfuse provided the bioprinter to manufacture the conduit. The company has the industrial rights related to the bioprinting method used to construct the 3D conduit in this work. Cyfuse provided support in the form of salaries for authors SA, KN, and MT and provided research grants to RI, TA, KN and SM. These competing interests do not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Background: The development of effective immunosuppressive regimens has resulted in many cases of successful hand transplantation. Visual skin inspection and histological evaluation are used to assess the rejection of hand transplants, but these methods are largely subjective. In this study, we aimed to determine the potential of microRNAs (miRNAs) as biomarkers for acute rejection in vascularized composite allotransplants., Methods: In allograft group, 7 male Brown-Norway rats (RT1 n ) were used as donors and 13 male Lewis rats (RT1 l ) were used as recipients. In control group, 8 Lewis rats were used as donors and recipients. The hindlimbs of donor rats were transplanted orthotopically to recipient rats. Skin changes were noted daily. Skin biopsies were obtained from 5 recipients and evaluated histologically. Plasma samples were obtained from the other 8 recipients before transplant and 7, 10, and 14 days posttransplant and used to measure miRNA expression., Results: Skin changes occurred at a mean of 11.0 days posttransplant. Rejection in most skin biopsies taken 7 and 10 days posttransplant was histologically classified as grade 0, whereas that in most biopsies taken 14 days posttransplant was classified as grade 3. We found that expression of miRNA-146a and miRNA-155 was significantly upregulated at 10 and 14 days posttransplant compared with that at 7 days posttransplant. In control group, there were no significant changes in plasma miRNAs expressions., Conclusions: The upregulation of plasma miRNA-146a and miRNA-155 was detected before the histological evaluation methods could diagnose complete rejection in the rat hindlimb transplantation model. Plasma miRNA-146a and miRNA-155 may be potential biomarkers of acute rejection after vascularized composite allotransplantation., Competing Interests: The authors declare no conflicts of interest.

Background: Percutaneous scaphoid screw fixation is a popular treatment for acute scaphoid fractures with no or minimal displacement. For treating scaphoid nonunions, however, open reduction and internal fixation with bone grafting is still the most popular treatment. Percutaneous fixation with bone grafting through the screw insertion hole has received little attention, although it minimizes damage to the surrounding tissues. We report excellent results of six scaphoid nonunions treated by retrograde percutaneous fixation with curettage and bone grafting through the distal insertion hole of a fully threaded headless screw., Methods: Six scaphoid nonunions with substantial bone loss were treated, including one revision case. All nonunions were located at the middle third of the scaphoid. The mean patient age at operation was 26 years, and the mean interval between fracture and surgery was 7 months. In the revision case, the interval between the primary and revision surgery was 6 months. In one case, curettage alone was performed before retrograde insertion of the headless screw. In the other cases including the revision, curettage and bone grafting with a bone biopsy needle was required through a distal insertion hole., Results: The mean follow-up was 11.3 months. Radiologically, union was achieved at averaged 12 weeks postoperatively. At the final follow-up, there was significant improvement in the wrist extension range of movement (from 65.8° to 80.8°) and grip strength (from 69.5% to 93.0% of the unaffected side). Five patients were free of pain, and one experienced mild pain only during heavy manual labor. The mean VAS, DASH, and Cooney wrist scores were 0.3, 1 and 99, respectively. All patients returned to their work or athletic activities., Conclusions: Retrograde percutaneous fixation with bone grafting through the distal insertion hole of a fully threaded headless screw is a promising option for surgical treatment of scaphoid nonunions.