Evaluation of MC3T3-E1 Cell Osteogenesis in Different Cell Culture Media

Many biomaterials have been evaluated using cultured cells. In particular, osteoblast-like cells are often used to evaluate the osteocompatibility, hard-tissue-regeneration, osteoconductive, and osteoinductive characteristics of biomaterials. However, the evaluation of biomaterial osteogenesis-inducing capacity using osteoblast-like cells is not standardized; instead, it is performed under laboratory-specific culture conditions with different culture media. However, the effect of different media conditions on bone formation has not been investigated. Here, we aimed to evaluate the osteogenesis of MC3T3-E1 cells, one of the most commonly used osteoblast-like cell lines for osteogenesis evaluation, and assayed cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification under varying culture media conditions. Furthermore, the various media conditions were tested in uncoated plates and plates coated with collagen type I and poly-L-lysine, highly biocompatible molecules commonly used as pseudobiomaterials. We found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may affect biomaterial regeneration. We posit that an in vitro model that recapitulates in vivo bone formation should be established before evaluating biomaterials.ArticleInternational Journal of Molecular Sciences. 22(14):7752 (2021)journal articl

Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells

Background There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. Methods In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFN gamma for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry. Results We discovered that IFN gamma increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. Conclusions Here we clarify for the first time an additional mechanism of anti-tumour effect-as exerted by anti-PD-1 antibody decreasing Treg- we anticipate that our findings will lead to the development of new methods for cancer treatment.ArticleBMC CANCER. 20(1):25 (2020)journal articl

Antitumor Effect of Sclerostin against Osteosarcoma

Various risk factors and causative genes of osteosarcoma have been reported in the literature; however, its etiology remains largely unknown. Bone formation is a shared phenomenon in all types of osteosarcomas, and sclerostin is an extracellular soluble factor secreted by osteocytes that prevents bone formation by inhibiting the Wnt signaling pathway. We aimed to investigate the antitumor effect of sclerostin against osteosarcoma. Osteosarcoma model mice were prepared by transplantation into the dorsal region of C3H/He and BALB/c-nu/nu mice using osteosarcoma cell lines LM8 (murine) and 143B (human), respectively. Cell proliferations were evaluated by using alamarBlue and scratch assays. The migratory ability of the cells was evaluated using a migration assay. Sclerostin was injected intraperitoneally for 7 days to examine the suppression of tumor size and extension of survival. The administration of sclerostin to osteosarcoma cells significantly inhibited the growth and migratory ability of osteosarcoma cells. Kaplan–Meier curves and survival data demonstrated that sclerostin significantly inhibited tumor growth and improved survival. Sclerostin suppressed the proliferative capacity and migratory ability of osteosarcoma cells. Osteosarcoma model mice inhibited tumor growth and prolonged survival periods by the administration of sclerostin. The effect of existing anticancer drugs such as doxorubicin should be investigated for future clinical applications.ArticleCancers 13(23) : 6015(2021)journal articl

Bone-Regeneration Therapy Using Biodegradable Scaffolds: Calcium Phosphate Bioceramics and Biodegradable Polymers

Calcium phosphate-based synthetic bone is broadly used for the clinical treatment of bone defects caused by trauma and bone tumors. Synthetic bone is easy to use; however, its effects depend on the size and location of the bone defect. Many alternative treatment options are available, such as joint arthroplasty, autologous bone grafting, and allogeneic bone grafting. Although various biodegradable polymers are also being developed as synthetic bone material in scaffolds for regenerative medicine, the clinical application of commercial synthetic bone products with comparable performance to that of calcium phosphate bioceramics have yet to be realized. This review discusses the status quo of bone-regeneration therapy using artificial bone composed of calcium phosphate bioceramics such as β-tricalcium phosphate (βTCP), carbonate apatite, and hydroxyapatite (HA), in addition to the recent use of calcium phosphate bioceramics, biodegradable polymers, and their composites. New research has introduced potential materials such as octacalcium phosphate (OCP), biologically derived polymers, and synthetic biodegradable polymers. The performance of artificial bone is intricately related to conditions such as the intrinsic material, degradability, composite materials, manufacturing method, structure, and signaling molecules such as growth factors and cells. The development of new scaffold materials may offer more efficient bone regeneration

Decreased Lamin B1 Levels Affect Gene Positioning and Expression in Postmitotic Neurons

Gene expression programs and concomitant chromatin regulation change dramatically during the maturation of postmitotic neurons. Subnuclear positioning of gene loci is relevant to transcriptional regulation. However, little is known about subnuclear genome positioning in neuronal maturation. Using cultured murine hippocampal neurons, we found genomic locus 14qD2 to be enriched with genes that are upregulated during neuronal maturation. Reportedly, the locus is homologous to human 8p21.3, which has been extensively studied in neuropsychiatry and neurodegenerative diseases. Mapping of the 14qD2 locus in the nucleus revealed that it was relocated from the nuclear periphery to the interior. Moreover, we found a concomitant decrease in lamin B1 expression. Overexpression of lamin B1 in neurons using a lentiviral vector prevented the relocation of the 14qD2 locus and repressed the transcription of the Egr3 gene on this locus. Taken together, our results suggest that reduced lamin B1 expression during the maturation of neurons is important for appropriate subnuclear positioning of the genome and transcriptional programs.Published version is available for viewing only. (See "Related URI")「関連URI」より出版社版の閲覧専用ページへリン