One of the problems in dental implant treatment is the lack of periodontal ligament (PDL), which supports teeth, prevents infection, and transduces sensations such as chewiness. The objective of the ...present study was to develop a decellularized PDL for supporting an artificial tooth. To this end, we prepared mouse decellularized mandible bone with a PDL matrix by high hydrostatic pressure and DNase and detergent treatments and evaluated its reconstruction in vivo. After tooth extraction, the decellularized mandible bone with PDL matrix was implanted under the subrenal capsule in rat and observed that host cells migrated into the matrix and oriented along the PDL collagen fibers. The extracted decellularized tooth and de- and re-calcified teeth, which was used as an artificial tooth model, were re-inserted into the decellularized mandible bone and implanted under the subrenal capsule in rat. The reconstructed PDL matrix for the extracted decellularized tooth resembled the decellularized mandible bone without tooth extraction. This demonstrates that decellularized PDL matrix can reconstruct PDL tissue by controlling host cell migration, which could serve as a novel periodontal treatment approach.
Decellularized tissues, in which the extracellular matrix is isolated, have broad applications as implantable biomaterials and/or biological scaffolds for tissue repair, and show good clinical ...performance. Decellularized tissue characteristics, such as their shape, structure, mechanical properties, and biological activity, are strongly affected by the decellularization protocol. The orthotopic implantation of decellularized tissues, a common procedure, typically induces cell infiltration and extracellular matrix (ECM) reconstruction resulting in tissues that resemble the source tissues. The ectopic implantation of decellularized tissues results in reconstruction that is either adapted to the implantation site or to the decellularized tissue source. In this review, the differences between methods are discussed. In addition, new methods aimed at extending the applications of decellularized tissues are discussed, particularly methods that confer novel functions to decellularized tissues, such as devices that link native tissues with artificial materials using decellularized tissue as an intermediate.
Recent applications of decellularized tissue have included the use of hydrogels for injectable materials and three-dimensional (3D) bioprinting bioink for tissue regeneration. Microvascular formation ...is required for the delivery of oxygen and nutrients to support cell growth and regeneration in tissues and organs. The aim of the present study was to evaluate the formation of capillary networks in decellularized extracellular matrix (d-ECM) hydrogels. The d-ECM hydrogels were obtained from the small intestine submucosa (SIS) and the urinary bladder matrix (UBM) after decellularizing with sodium deoxycholate (SDC) and high hydrostatic pressure (HHP). The SDC d-ECM hydrogel gradually gelated, while the HHP d-ECM hydrogel immediately gelated. All d-ECM hydrogels had low matrix stiffness compared to that of the collagen hydrogel, according to a compression test. D-ECM hydrogels with various elastic moduli were obtained, irrespective of the decellularization method or tissue source. Microvascular-derived endothelial cells were seeded on d-ECM hydrogels. Few cells attached to the SDC d-ECM hydrogel with no network formation, while on the HHP d-ECM hydrogel, a capillary network structure formed between elongated cells. Long, branched networks formed on d-ECM hydrogels with lower matrix stiffness. This suggests that the capillary network structure that forms on d-ECM hydrogels is closely related to the matrix stiffness of the hydrogel.
Recent applications of decellularized tissues include the ectopic use of sheets and powders for three‐dimensional (3D) tissue reconstruction. Decellularized tissues are modified (or fabricated) with ...the desired functions for application to the target (transplanted or used) tissue, including soft–hard interregional tissues, such as ligaments, tendons, and periodontal ligaments. This study aimed to prepare a mineralized decellularized pericardium to construct a soft‐hard interregional tissue by 3D fabrication of decellularized pericardium, for example, rolling up to a cylindrical form. The decellularized pericardial tissue was prepared using the high hydrostatic pressurization (HHP) and surfactants method. The pericardium consisted of bundles of aligned fibers, and the bundles were slightly disordered when prepared with the surfactant decellularization method compared with that prepared using the HHP decellularization method. Mineralization of the decellularized pericardium was performed using an alternate soaking process with various cycles. The surface of the decellularized pericardium was covered with calcium phosphate precipitates, which accumulated on the surface with an increasing number of soaking cycles. The inside of the HHP decellularized pericardium was mineralized uniformly, whereas the mineralization of the decellularized pericardium decreased toward the interior. These findings suggest that the decellularization method strongly affects the structure and mineralized parts of the decellularized pericardium. The mineralized decellularized pericardium could be a candidate material for reconstructing alternative interregional tissues, such as ligaments and tendons.
Due to an increasing number of cardiovascular diseases, artificial heart valves and blood vessels have been developed. Although cardiovascular applications using decellularized tissue have been ...studied, the mechanisms of their functionality remain unknown. To determine the important factors for preparing decellularized cardiovascular prostheses that show good in vivo performance, the effects of the luminal surface structure of the decellularized aorta on thrombus formation and cell behavior were investigated. Various luminal surface structures of a decellularized aorta were prepared by heating, drying, and peeling. The luminal surface structure and collagen denaturation were evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining, and scanning electron microscopy (SEM) analysis. To evaluate the effects of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting tests and recellularization of endothelial cells and smooth muscle cells were performed. The results of the blood clotting test showed that the closer the luminal surface structure is to the native aorta, the higher the anti-coagulant property. The results of the cell seeding test suggest that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation, and functional expression accordingly. These results provide important factors for preparing decellularized cardiovascular prostheses and will lead to future developments in decellularized cardiovascular applications.
Dysferlinopathy is a group of autosomal recessive muscular dystrophies caused by variants in the dysferlin gene (DYSF), with variable proximal and distal muscle involvement. We performed DYSF gene ...analyses of 200 cases suspected of having dysferlinopathy (Cohort 1), and identified diagnostic variants in 129/200 cases, including 19 novel variants. To achieve a comprehensive genetic profile of dysferlinopathy, we analyzed the variant data from 209 affected cases from unrelated 209 families, including 80 previously diagnosed and 129 newly diagnosed cases (Cohort 2). Among the 90 types of variants identified in 209 cases, the NM_003494.3: c.2997G>T; p.Trp999Cys, was the most frequent (96/420; 22.9%), followed by c.1566C>G; p.Tyr522* (45/420; 10.7%) on an allele base. p.Trp999Cys was found in 70/209 cases (33.5%), including 20/104 cases (19.2%) with the Miyoshi muscular phenotype and 43/82 cases (52.4%) with the limb‐girdle phenotype. In the analysis of missense variants, p.Trp992Arg, p.Trp999Arg, p.Trp999Cys, p.Ser1000Phe, p.Arg1040Trp, and p.Arg1046His were located in the inner DysF domain, representing in 113/160 missense variants (70.6%). This large cohort highlighted the frequent missense variants located in the inner DysF domain as a hotspot for missense variants among our cohort of 209 cases (>95%, Japanese) and hinted at their potential as targets for future therapeutic strategies.
The DYSF variant data identified in 209 affected cases from unrelated families was analyzed. Among the 90 types of variants, the NM_003494.3: c.2997 G>T; p.Trp999Cys, was the most frequent (22.9%) on an allele base. The residual dysferlin expression on muscle fibers differed among cases with homozygous p. Trp999Cys. In the analysis of missense variants, p.Trp992Arg, p.Trp999Arg, p.Trp999Cys, p.Ser1000Phe, p.Arg1040Trp and p.Arg1046His were located in the IdysF domain, representing in 113/160 missense variants (70.6%). The IdysF domain was demonstrated as a hotspot for DYSF missense variants.
Menarche is delayed in patients with type 1 diabetic mellitus (T1DM) compared to non-diabetics. The purpose of this survey study was to define the age of onset of menarche in Japanese patients with ...T1DM, as well the secular trends in menarcheal age across the period of 1976–2020 and determine the effects of T1DM and disease management on that age. The study subjects (n = 155) were recruited from among Japanese T1DM patients who visited the outpatient clinic of the Department of Pediatrics, Osaka City University Hospital. The study subjects experienced menarche during 1976–2020. They were divided into the menarche-post-T1DM group (n = 117) and the menarche-pre-T1DM group (n = 38), in whom menarche occurred after or before the diagnosis of T1DM, respectively. The time of birth was also stratified into five decade/time bins extending from 1960s to 2000s. The subjects filled a questionnaire on menarche. Other clinical information was obtained from the medical records. The median age at menarche was 12.5 years (11.3–13.4) (25th–75th percentile) for the menarche-post-T1DM group and 11.8 years (10.9–13.0) for the menarche-pre-T1DM group (p = 0.024). Menarche occurred at a significantly younger age in recent years in the menarche-post-T1DM group (r = –0.209, p = 0.023), but no such trend was found in the control group. Analysis of data of subjects born after 1990 still showed significant delay associated with T1DM post-T1DM group: 12.3 years (11.3–13.2), pre-T1DM group: 11.8 years (11.0–12.2), p = 0.045. The results suggest that recent advances in insulin therapy seem to improve metabolism under T1DM but might have not enough impact on menarche in Japanese girls.
In this study, we designed and synthesized an implantable anti-CD25 antibody-immobilized polyethylene (CD25-PE) mesh to suppress tumor growth by removing regulatory T cells (Tregs). The PE mesh was ...graft-polymerized with poly(acrylic acid), and the anti-mouse CD25 antibody was then immobilized using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide reaction. Immobilization of the antibody on the PE mesh was confirmed by immunostaining. The CD25-PE mesh could effectively and selectively capture CD25-positive cells through antigen-antibody interactions when the CD25-PE mesh was incubated with a suspension of mouse spleen cells, including CD25-positive cells. In addition, implantation of the CD25-PE mesh into mice subcutaneously demonstrated the Treg-capturing ability of the CD25-PE mesh with only a weak inflammatory reaction. In tumor-bearing mice, tumor growth was suppressed by subcutaneous implantation of the CD25-PE mesh near the tumor for 1 week. These results suggested that the anti-CD25 antibody-immobilized material could capture Tregs in vivo and inhibit tumor proliferation in a limited tumor-bearing mouse model. Further research is needed to facilitate cancer immunotherapy using implantable anti-CD25 antibody-immobilized material as a Treg-capturing device.
Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of ...sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these “myotube” samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which “biopsy” and “myotube” samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.
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