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      The potential application of concentrated growth factor in pulp regeneration: an in vitro and in vivo study

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          Abstract

          Background

          Concentrated growth factor (CGF), as a natural biomaterial, is known to contain platelets, cytokines, and growth factors to facilitate the healing process, but there has been little information acquired in regenerative endodontics. The purpose of this study was to investigate the effects of CGF on proliferation, migration, and differentiation in human dental stem pulp cells (hDPSCs) exposed to lipopolysaccharide (LPS) in vitro and its potential role in pulp regeneration of the immature teeth in vivo .

          Methods

          In vitro experiments: CGF-conditioned medium were extracted by freeze-dried method. hDPSCs were isolated and identified. The proliferative potential of hDPSCs with different concentration of CGF and LPS was evaluated by Cell Counting Kit-8. Migration capacity was analyzed by Transwell assays, odonto/osteoblastic differentiation was determined by measuring alkaline phosphatase (ALP) activity using ALP staining, and the extent of mineralization was evaluated by using Alizarin red S staining. The mRNA expression level of DMP-1, DSPP, OPN, Runx2, and OCN were determined by quantitative polymerase chain reaction (qPCR).

          In vivo experiments: CGF were used as root canal filling agent of the immature single-rooted teeth in the beagle dogs. The teeth were then radiographed, extracted, fixed, demineralized, and subjected to histologic analyses at 8 weeks. The newly formed dentine-pulp complex and the development of apical foramen were evaluated by the hematoxylin-eosin (HE) and Masson trichrome technique. Soft tissues were analyzed by immunohistochemical staining of vascular endothelial growth factor (VEGF) and Nestin.

          Results

          In vitro experiments: The cultured cells exhibited the characteristics of mesenchymal stem cell. The treatment of LPS significantly increased the expression of TNF-α, IL-1β, IL-6, and IL-8 in hDPSCs, and CGF inhibited the mRNA expression of IL-8 in LPS-stimulated hDPSCs. The proliferation values of the CGF group in LPS-stimulated hDPSCs were significantly higher than that of the control group from day 3 to day 7 ( P < 0.05). In addition, the number of migratory cells of the CGF group was greater than that of the control group at 24 h with or without LPS treatment. ALP activities increased gradually in both groups from day 4 to day 7. The mineralized nodules and the expression of odontogenesis-related genes DMP-1 and DSPP, osteogenesis-related genes OPN, Runx2, and OCN were dramatically enhanced by CGF in LPS-stimulated hDPSCs at days 21 and 28.

          In vivo experiments: In CGF treated group, the results of radiograph, HE, and Masson trichrome staining showed a continuing developed tooth of the immature teeth in the beagle dogs (i.e., the ingrowth of soft tissues into the root canal, the thickened internal root dentin walls, and the closed apex), which resembled the normal tooth development in the positive control group. The immunohistochemical staining showed that VEGF and Nestin were both moderately expressed in the regenerated pulp-like tissues which indicating the vascularization and innervation.

          Conclusions

          CGF has a positive effect on the proliferation, migration, and differentiation of hDPSCs exposed to LPS in vitro, and it can also promote the regeneration of dentine-pulp complex of the immature teeth in the beagle dogs in vivo. Therefore, CGF could be a promising alternative biomaterial in regenerative endodontics.

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          Most cited references44

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          TNF-α signalling and inflammation: interactions between old acquaintances.

          Inflammation is a very important part of innate immunity and is regulated in many steps. One such regulating step is the cytokine network, where tumor necrosis factor α (TNF-α) plays one of the most important roles. A PubMed and Web of Science databases search was performed for studies providing evidences on the role of TNF-α in inflammation, apoptosis, and cancer. This review concisely summarizes the role of this pro-inflammatory cytokine during inflammation. It is focused mainly on TNF-α intracellular signaling and its influence on the typical inflammatory features in the organism. Being one of the most important pro-inflammatory cytokines, TNF-α participates in vasodilatation and edema formation, and leukocyte adhesion to epithelium through expression of adhesion molecules; it regulates blood coagulation, contributes to oxidative stress in sites of inflammation, and indirectly induces fever. The connection between TNF-α and cancer is mentioned as well.
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            Injury-Induced Senescence Enables In Vivo Reprogramming in Skeletal Muscle.

            In vivo reprogramming is a promising approach for tissue regeneration in response to injury. Several examples of in vivo reprogramming have been reported in a variety of lineages, but some including skeletal muscle have so far proven refractory. Here, we show that acute and chronic injury enables transcription-factor-mediated reprogramming in skeletal muscle. Lineage tracing indicates that this response frequently originates from Pax7+ muscle stem cells. Injury is associated with accumulation of senescent cells, and advanced aging or local irradiation further enhanced in vivo reprogramming, while selective elimination of senescent cells reduced reprogramming efficiency. The effect of senescence appears to be, at least in part, due to the release of interleukin 6 (IL-6), suggesting a potential link with the senescence-associated secretory phenotype. Collectively, our findings highlight a beneficial paracrine effect of injury-induced senescence on cellular plasticity, which will be important for devising strategies for reprogramming-based tissue repair.
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              Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF)

              Background The development of platelet-rich fibrin (PRF) drastically simplified the preparation procedure of platelet-concentrated biomaterials, such as platelet-rich plasma (PRP), and facilitated their clinical application. PRF’s clinical effectiveness has often been demonstrated in pre-clinical and clinical studies; however, it is still controversial whether growth factors are significantly concentrated in PRF preparations to facilitate wound healing and tissue regeneration. To address this matter, we performed a comparative study of growth factor contents in PRP and its derivatives, such as advanced PRF (A-PRF) and concentrated growth factors (CGF). Methods PRP and its derivatives were prepared from the same peripheral blood samples collected from healthy donors. A-PRF and CGF preparations were homogenized and centrifuged to produce extracts. Platelet and white blood cell counts in A-PRF and CGF preparations were determined by subtracting those counts in red blood cell fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts of whole blood samples. Concentrations of growth factors (TGF-β1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1β, IL-6) were determined using ELISA kits. Results Compared to PRP preparations, both A-PRF and CGF extracts contained compatible or higher levels of platelets and platelet-derived growth factors. In a cell proliferation assay, both A-PRF and CGF extracts significantly stimulated the proliferation of human periosteal cells without significant reduction at higher doses. Conclusions These data clearly demonstrate that both A-PRF and CGF preparations contain significant amounts of growth factors capable of stimulating periosteal cell proliferation, suggesting that A-PRF and CGF preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application.
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                Author and article information

                Contributors
                1632345@tongji.edu.cn
                qiaolujoyce@163.com
                yumeizhao@tongji.edu.cn
                461223781@qq.com
                hongshebin@163.com
                jing-pan@foxmail.com
                0086-21-66311625 , jiangbeizhan@tongji.edu.cn
                Journal
                Stem Cell Res Ther
                Stem Cell Res Ther
                Stem Cell Research & Therapy
                BioMed Central (London )
                1757-6512
                20 May 2019
                20 May 2019
                2019
                : 10
                : 134
                Affiliations
                [1 ]ISNI 0000000123704535, GRID grid.24516.34, Department of Operative Dentistry and Endodontics, School and Hospital of Stomatology, , Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, ; 399 Middle Yan Chang Road, Shanghai, 200072 China
                [2 ]ISNI 0000000123704535, GRID grid.24516.34, Department of Pediatric Dentistry, School and Hospital of Stomatology, , Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, ; Shanghai, 200072 China
                Author information
                http://orcid.org/0000-0002-0135-5917
                Article
                1247
                10.1186/s13287-019-1247-4
                6528367
                31109358
                4aacff93-16ea-411e-8134-c966cb79d1b3
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 7 January 2019
                : 15 April 2019
                : 29 April 2019
                Funding
                Funded by: Shanghai Science and Technology Commission Program
                Award ID: 18411969500
                Funded by: Shanghai Municipal Commission of Health and Family Planning Program
                Award ID: 201740223
                Categories
                Research
                Custom metadata
                © The Author(s) 2019

                Molecular medicine
                concentrated growth factor (cgf),human dental stem pulp cells (hdpscs),lipopolysaccharide (lps),inflammatory microenvironment,regenerative endodontic treatment (ret)

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