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      Exploring the impact of culture techniques and patient demographics on the success rate of primary culture of human periodontal ligament stem cells

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          Abstract

          Background/purpose

          Periodontal ligament stem cells (PDLSCs) have the potential for regenerating periodontal tissue. The study aims to investigate the impact of demographics (ages, gender, disease) and culture techniques (shipping storage time and culture method) on the success of primary culture.

          Materials and methods

          PDLSCs were collected from 51 teeth of 26 patients and cultured via outgrowth (OG) and enzymatic digestion (ED) methods. Cells characteristics were confirmed by flow cytometry, MTT, and ARS. The primary culture success rate was evaluated with a serial chi-square test to determine the relationship with culture technique (ED/OG and ≤4 h/prolonged culture) and patient demographics (Young/Old, Female/Male, and Health/Periodontitis).

          Results

          The overall success rate of Health group (69.7%) was higher than Periodontitis (38.9%). Culturing within 4 h possessed a higher success rate (71.8%) than prolonged group (16.7%) regardless of patient demographics, and using OG method (81.5%) revealed more promising. Subgroup analysis of 39 cases (culture within 4 h) found that the success rate of OG was higher than ED in the Old group (87.5%–25.0%) and in the Periodontitis group (83.3%–25.0%).

          Conclusion

          Primary culturing of PDLSCs within 4 h and using the outgrowth method led to higher success rates regardless of patient demographics. It can achieve successful PDLSCs culture of older patients or patients with periodontal disease by appropriate culture technique.

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          Most cited references39

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          VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.

          Cytokine-activated human endothelial cells express vascular cell adhesion molecule-1 (VCAM-1), which binds lymphocytes. We now identify the integrin VLA-4 as a receptor for VCAM-1 because VLA-4 surface expression on K-562 cells (following transfection of the VLA alpha 4 subunit cDNA) resulted in specific cell adhesion to VCAM-1, and anti-VLA-4 antibodies completely inhibited VCAM-1-dependent cell-cell attachment. In addition, VLA-4 expression allowed K-562 cells to attach to the heparin II binding region (FN-40) of fibronectin. However, VLA-4/VCAM-1 and VLA-4/FN-40 interactions are readily distinguishable: only the former was inhibited by the anti-VLA-4 monoclonal antibody HP1/3, and only the latter was inhibited by soluble FN-40. The VCAM-1/VLA-4 ligand-receptor pair may play a major role in the recruitment of mononuclear leukocytes to inflammatory sites in vivo.
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            Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S.

            M. McLeod (1980)
            The procedure described by Inouye ('76) for the staining of full-term mouse fetal skeletons has been adapted for use with mouse embryos and fetuses of days 14-18 of gestation. The main adaptations for younger specimens involve a longer time in acetone, in lieu of skinning, and omission of the aqueous KOH step. These adaptations require more time but result in consistently good staining of intact specimens.
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              Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model.

              Periodontal regeneration has been challenged with chemical reagents and/or biological approaches, however, there is still no sufficient technique that can regenerate complete periodontium, including alveolar bone, cementum, and well-oriented collagen fibers. The purpose of this study was to examine multi-layered sheets of periodontal ligament (PDL)-derived cells for periodontal regeneration. Canine PDL cells were isolated enzymatically and expanded in vitro. The cell population contained cells capable of making single cell-derived colonies at an approximately 20% frequency. Expression of mRNA of periodontal marker genes, S100 calcium binding protein A4 and periostin, was observed. Alkaline phosphatase activity and gene expression of both osteoblastic/cementoblastic and periodontal markers were upregulated by osteoinductive medium. Then, three-layered PDL cell sheets supported with woven polyglycolic acid were transplanted to dental root surfaces having three-wall periodontal defects in an autologous manner, and bone defects were filled with porous beta-tricalcium phosphate. Cell sheet transplantation regenerated both new bone and cementum connecting with well-oriented collagen fibers, while only limited bone regeneration was observed in control group where cell sheet transplantation was eliminated. These results suggest that PDL cells have multiple differentiation properties to regenerate periodontal tissues comprising hard and soft tissues. PDL cell sheet transplantation should prove useful for periodontal regeneration in clinical settings.
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                Author and article information

                Contributors
                Journal
                J Dent Sci
                J Dent Sci
                Journal of Dental Sciences
                Association for Dental Sciences of the Republic of China
                1991-7902
                2213-8862
                01 August 2023
                April 2024
                01 August 2023
                : 19
                : 2
                : 961-970
                Affiliations
                [a ]Graduate Institute of Clinical Medical Science, School of Medicine, China Medical University, Taichung, Taiwan
                [b ]School of Dentistry, College of Dentistry, China Medical University, Taichung, Taiwan
                [c ]Department of Orthodontics, China Medical University Hospital Medical Center, Taichung, Taiwan
                [d ]Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan
                [e ]Graduate Institute of Basic Medical Science, School of Medicine, China Medical University, Taichung, Taiwan
                [f ]Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan
                [g ]Department of Post-Baccalaureate Veterinary Medicine, Asia University, Taichung, Taiwan
                [h ]Department of Biomedical Engineering, College of Biomedical Engineering, China Medical University, Taichung, Taiwan
                Author notes
                []Corresponding author. School of Dentistry, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. kenkoyu@ 123456mail.cmu.edu.tw
                [∗∗ ]Corresponding author. Department of Biomedical Engineering, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. djlin@ 123456mail.cmu.edu.tw
                Article
                S1991-7902(23)00228-3
                10.1016/j.jds.2023.07.023
                11010602
                af1b7a52-6e1d-41e3-835c-e68863565089
                © 2023 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 14 June 2023
                : 18 July 2023
                Categories
                Original Article

                primary culture,outgrowth,enzymatic digestion,pdlscs,cell banking

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