Development of a Self-Assembled Dermal Substitute from Human Fibroblasts Using Long-term Three-Dimensional Culture.

Skin substitutes have emerged as an alternative to autografts for the treatment of skin defects. Among them, scaffold-based dermal substitutes have been extensively studied; however, they have certain limitations, such as delayed vascularization, limited elasticity, and the inability to achieve permanent engraftment. Self-assembled, cell-based dermal substitutes are a promising alternative that may overcome these shortcomings but have not yet been developed. In this study, we successfully developed a cell-based dermal substitute (cultured dermis) through the long-term culture of human dermal fibroblasts using the net-mold method, which enables three-dimensional cell culture without the use of a scaffold. Spheroids prepared from human dermal fibroblasts were poured into a net-shaped mold and cultured for 2, 4, or 6 months. The dry weight, tensile strength, collagen and glycosaminoglycan levels, and cell proliferation capacity were assessed and compared among the 2-, 4-, and 6-month culture periods. We found that collagen and glycosaminoglycan levels decreased over time, while the dry weight remained unchanged. Tensile strength increased at 4 months, suggesting that remodeling had progressed. In addition, the cell proliferation capacity was maintained, even after a 6-month culture period. Unexpectedly, the internal part of the cultured dermis became fragile, resulting in the division of the cultured dermis into two collagen-rich tissues, each of which had a thickness of 400 µm and sufficient strength to be sutured during in vivo analysis. The divided 4-month cultured dermis was transplanted to skin defects of immunocompromised mice and its wound healing effects were compared to those of a clinically available collagen-based artificial dermis. The cultured dermis promoted epithelialization and angiogenesis more effectively than the collagen-based artificial dermis. Although further improvements are needed, such as the shortening of the culture period and increasing the size of the cultured dermis, we believe that the cultured dermis presented in this study has the potential to be an innovative material for permanent skin coverage.

Sex-Specific Differences in the Transcriptome of the Human Dorsolateral Prefrontal Cortex in Schizophrenia.

Schizophrenia presents clinical and biological differences between males and females. This study investigated transcriptional profiles in the dorsolateral prefrontal cortex (DLPFC) using postmortem data from the largest RNA-sequencing (RNA-seq) database on schizophrenic cases and controls. Data for 154 male and 113 female controls and 160 male and 93 female schizophrenic cases were obtained from the CommonMind Consortium. In the RNA-seq database, the principal component analysis showed that sex effects were small in schizophrenia. After we analyzed the impact of sex-specific differences on gene expression, the female group showed more significantly changed genes compared with the male group. Based on the gene ontology analysis, the female sex-specific genes that changed were overrepresented in the mitochondrion, ATP (phosphocreatine and adenosine triphosphate)-, and metal ion-binding relevant biological processes. An ingenuity pathway analysis revealed that the differentially expressed genes related to schizophrenia in the female group were involved in midbrain dopaminergic and γ-aminobutyric acid (GABA)-ergic neurons and microglia. We used methylated DNA-binding domain-sequencing analyses and microarray to investigate the DNA methylation that potentially impacts the sex differences in gene transcription using a maternal immune activation (MIA) murine model. Among the sex-specific positional genes related to schizophrenia in the PFC of female offspring from MIA, the changes in the methylation and transcriptional expression of loci ACSBG1 were validated in the females with schizophrenia in independent postmortem samples by real-time PCR and pyrosequencing. Our results reveal potential genetic risks in the DLPFC for the sex-dependent prevalence and symptomology of schizophrenia.

Bioengineering of a scaffold-less three-dimensional tissue using net mould.

Tissue engineering has attracted attention worldwide because of its application in regenerative medicine, drug screening, and cultured meat. Numerous biofabrication techniques for producing tissues have been developed, including various scaffold and printing methods. Here, we have proposed a novel tissue engineering method using a net metal mould without the use of a scaffold. Briefly, normal human dermal fibroblasts seeded on a dimple plate were subjected to static culture technique for several days to form spheroids. Spheroids of diameter ⩾200µm were poured into a net-shaped mould of gap ⩽100µm and subjected to shake-cultivation for several weeks, facilitating their fusion to form a three-dimensional (3D) tissue. Through this study, we successfully constructed a scaffold-free 3D tissue having strength that can be easily manipulated, which was difficult to construct using conventional tissue engineering methods. We also investigated the viability of the 3D tissue and found that the condition of the tissues was completely different depending on the culture media used. Collectively, this method allows scaffold-free culture of 3D tissues of unprecedented thickness, and may contribute largely to next-generation tissue engineering products.

Three-Dimensional Multilayered Microstructure Using Needle Array Bioprinting System.

Tissue engineering is an essential component of developing effective regenerative therapies. In this study, we introduce a promising method to create scaffold-free three-dimensional (3D) tissue engineered multilayered microstructures from cultured cells using the "3D tissue fabrication system" (Regenova®; Cyfuse, Tokyo, Japan). This technique utilizes the adhesive nature of cells. When cells are cultured in nonadhesive wells, they tend to aggregate and form a spheroidal structure. The advantage of this approach is that cellular components can be mixed into one spheroid, thereby promoting the formation of extracellular matrices, such as collagen and elastin. This system enables one to create a predesigned 3D structure composed of cultured cells. We found that the advantages of this system to be (1) the length, size, and shape of the structure that were designable and highly reproducible because of the computer controlled robotics system, (2) the graftable structure could be created within a reasonable period (8 days), and (3) the constructed tissue did not contain any foreign material, which may avoid the potential issues of contamination, biotoxicity, and allergy. The utilization of this robotic system enabled the creation of a 3D multilayered microstructure made of cell-based spheres with a satisfactory mechanical properties and abundant extracellular matrix during a short period of time. These results suggest that this new technology will represent a promising, attractive, and practical strategy in the field of tissue engineering. Impact statement The utilization of the "three dimensional tissue fabrication system" enabled the creation of a three-dimensional (3D) multilayered microstructure made of cell-based spheres with a satisfactory mechanical properties and abundant extracellular matrix during a short period of time. These results suggest that this new technology will represent a promising, attractive, and practical strategy in the field of tissue engineering.

Subcortical band heterotopia (SBH) in males: clinical, imaging and genetic findings in comparison with females.

Subcortical band heterotopia (SBH) or double cortex syndrome is a neuronal migration disorder, which occurs very rarely in males: to date, at least 110 females but only 11 in males have been reported. The syndrome is usually associated with mutations in the doublecortin (DCX) (Xq22.3-q23) gene, and much less frequently in the LIS1 (17p13.3) gene. To determine whether the phenotypic spectrum, the genetic basis and genotype-phenotype correlations of SBH in males are similar to those in females, we compared the clinical, imaging and molecular features in 30 personally evaluated males and 60 previously reported females with SBH. Based on the MRI findings, we defined the following band subtypes: partial, involving one or two cerebral lobes; intermediate, involving two lobes and a portion of a third; diffuse, with substantial involvement of three or more lobes; and pachygyria-SBH, in which posterior SBH merges with anterior pachygyria. Karyo typing and mutation analysis of DCX and/or LIS1 were performed in 23 and 24 patients, respectively. The range of clinical phenotypes in males with SBH greatly overlapped that in females. MRI studies revealed that some anatomical subtypes of SBH, such as partial and intermediate posterior, pachygyria-SBH and diffuse bands with posterior predominance, were more frequently or exclusively present in males. Conversely, classical diffuse SBH and diffuse bands with anterior predominance were more frequent in females. Males had either mild or the most severe band subtypes, and these correlated with the over-representation of normal/borderline intelligence and severe mental retardation, respectively. Conversely, females who had predominantly diffuse bands exhibited mostly mild or moderate mental retardation. Seven patients (29%) had missense mutations in DCX; in four, these were germline mutations, whereas in three there was evidence for somatic mosaicism. A germline missense mutation of LIS1 and a partial trisomy of chromosome 9p were identified in one patient (4%) each. One male each had a possible pathogenic intronic base change in both DCX and LIS1 genes. Our study shows that SBH in males is a clinically heterogeneous syndrome, mostly occurring sporadically. The clinical spectrum is similar to that of females with SBH. However, the greater cognitive and neuroradiological heterogeneity and the small number of mutations identified to date in the coding sequences of the DCX and LIS1 genes in males differ from the findings in females. This suggests other genetic mechanisms such as mutations in the non-coding regions of the DCX or LIS1 genes, gonadal or somatic mosaicism, and finally mutations of other genes.

Induction of hematopoietic prostaglandin D synthase in hyalinated necrotic muscle fibers: its implication in grouped necrosis.

Prostaglandin (PG) D(2) exerts pro-inflammatory and anti-inflammatory functions under various pathological conditions. We found that the immunoreactivity of hematopoietic PGD synthase (HPGDS) appeared in necrotic muscle fibers, mainly in foci of grouped necrosis, in Duchenne's muscular dystrophy (DMD) and polymyositis (PM), but not in Becker's muscular dystrophy or in Fukuyama-type congenital muscular dystrophy. The HPGDS expression in DMD and PM was observed to be transient in hyalinated fibers at the early necrotic stage but not detected in opaque fibers, in fibers infiltrated by monocytes/lymphocyte, or in regenerating fibers. The immunoreactivities of a cytosolic form of phospholipase A(2) and cyclooxygenase-2, the upstream enzymes of the arachnoid acid cascade, were similarly observed in the HPGDS-positive fibers, suggesting that PGD(2) was produced in situ in those necrotic muscle fibers.

Human herpesvirus-6 infection in neonates: Not protected by only humoral immunity.

BACKGROUND: Infants are usually protected from various viral infections, including human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7) infections, during the early infantile period by antibodies transferred from their mothers. However, rare cases of exanthem subitum (ES) in neonates have been described in published reports. METHODS: From the infantile patients of febrile illness, HHV-6 and HHV-7 DNA were examined by the polymerase chain reaction method. Antibodies to HHV-6 and HHV-7 were detected by indirect immuno-fluorescence assay and neutralization test. Viral isolation was attempted from the patient's peripheral blood mononuclear cells (PBMC) during the acute phase of febrile illness. RESULTS: Human herpesvirus-6 was verified virologically in two neonates who were clinically diagnosed as ES within the first month of life. Although high copies of HHV-6 DNA were detected in their PBMC during the acute phase, the isolation of HHV-6 from their PBMC was not successful. Neutralizing antibodies to HHV-6 were detected in sera of the acute phase, and those antibodies were considered to be transferred from their mothers. Antibody titers showed fourfold elevation in sera of the convalescent phase. The HHV-6 infection occurred despite the presence of pre-existing maternal antibody. Human herpesvirus-7 and HHV-7 DNA were not detected from their clinical samples. CONCLUSIONS: This observation suggests that HHV-6 infection could not be protected by only humoral immunity.

Unverricht-Lundborg disease with cystatin B gene abnormalities.

The clinical, neurophysiologic, and genetic findings in two Japanese patients with the Unverricht-Lundborg type of progressive myoclonus epilepsy are described. The cystatin B gene of Patient 1 exhibited expansion of the dodecamer (12-mer) repeat located in the 5' region and a point mutation (G-->A mutation) in exon 2. The cystatin B gene of Patient 2 exhibited homozygous expansion of the dodecamer repeat. Both parents of Patient 2 were heterozygous carriers. The two patients had a similar clinical course, and their symptoms were similar to those of previously reported patients in Finland. They both had a good response to zonisamide and low-dose primidone. We recommend that zonisamide and low-dose primidone should be introduced as the first drugs of choice for the treatment of patients with the Unverricht-Lundborg type of progressive myoclonus epilepsy.