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Astudillo-Ortiz E, Babo PS, Sunde PT, Galler KM, Gomez-Florit M, Gomes ME. Endodontic Tissue Regeneration: A Review for Tissue Engineers and Dentists. TISSUE ENGINEERING. PART B, REVIEWS 2023; 29:491-513. [PMID: 37051704 DOI: 10.1089/ten.teb.2022.0211] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/14/2023]
Abstract
The paradigm shift in the endodontic field from replacement toward regenerative therapies has witnessed the ever-growing research in tissue engineering and regenerative medicine targeting pulp-dentin complex in the past few years. Abundant literature on the subject that has been produced, however, is scattered over diverse areas of knowledge. Moreover, the terminology and concepts are not always consensual, reflecting the range of research fields addressing this subject, from endodontics to biology, genetics, and engineering, among others. This fact triggered some misinterpretations, mainly when the denominations of different approaches were used as synonyms. The evaluation of results is not precise, leading to biased conjectures. Therefore, this literature review aims to conceptualize the commonly used terminology, summarize the main research areas on pulp regeneration, identify future trends, and ultimately clarify whether we are really on the edge of a paradigm shift in contemporary endodontics toward pulp regeneration.
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Affiliation(s)
- Esteban Astudillo-Ortiz
- 3B's Research Group, I3Bs-Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Braga, Portugal
- ICVS/3B's-PT Government Associate Laboratory, Braga, Portugal
- Department of Endodontics, School of Dentistry, University of Cuenca, Cuenca, Ecuador
| | - Pedro S Babo
- 3B's Research Group, I3Bs-Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Braga, Portugal
- ICVS/3B's-PT Government Associate Laboratory, Braga, Portugal
| | - Pia T Sunde
- Department of Endodontics, Institute of Clinical Dentistry, Faculty of Dentistry, University of Oslo, Oslo, Norway
| | - Kerstin M Galler
- Department of Operative Dentistry and Periodontology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
| | | | - Manuela E Gomes
- 3B's Research Group, I3Bs-Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Braga, Portugal
- ICVS/3B's-PT Government Associate Laboratory, Braga, Portugal
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Yang C, Du XY, Luo W. Clinical application prospects and transformation value of dental follicle stem cells in oral and neurological diseases. World J Stem Cells 2023; 15:136-149. [PMID: 37181000 PMCID: PMC10173814 DOI: 10.4252/wjsc.v15.i4.136] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 01/18/2023] [Accepted: 03/21/2023] [Indexed: 04/26/2023] Open
Abstract
Since dental pulp stem cells (DPSCs) were first reported, six types of dental SCs (DSCs) have been isolated and identified. DSCs originating from the craniofacial neural crest exhibit dental-like tissue differentiation potential and neuro-ectodermal features. As a member of DSCs, dental follicle SCs (DFSCs) are the only cell type obtained at the early developing stage of the tooth prior to eruption. Dental follicle tissue has the distinct advantage of large tissue volume compared with other dental tissues, which is a prerequisite for obtaining a sufficient number of cells to meet the needs of clinical applications. Furthermore, DFSCs exhibit a significantly higher cell proliferation rate, higher colony-formation capacity, and more primitive and better anti-inflammatory effects than other DSCs. In this respect, DFSCs have the potential to be of great clinical significance and translational value in oral and neurological diseases, with natural advantages based on their origin. Lastly, cryopreservation preserves the biological properties of DFSCs and enables them to be used as off-shelf products for clinical applications. This review summarizes and comments on the properties, application potential, and clinical transformation value of DFSCs, thereby inspiring novel perspectives in the future treatment of oral and neurological diseases.
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Affiliation(s)
- Chao Yang
- Research and Development Department, Shenzhen Uni-medica Technology Co., Ltd, Shenzhen 518051, Guangdong Province, China
- Department of Stomatology, The People’s Hospital of Longhua, Shenzhen 518109, Guangdong Province, China
| | - Xin-Ya Du
- Department of Stomatology, The People’s Hospital of Longhua, Shenzhen 518109, Guangdong Province, China
| | - Wen Luo
- Department of Stomatology, The First Affiliated Hospital of Hainan Medical University, Haikou 570102, Hainan Province, China
- School of Stomatology, Hainan Medical University, Haikou 571199, Hainan Province, China
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Meng L, Wei Y, Liang Y, Hu Q, Xie H. Stem cell homing in periodontal tissue regeneration. Front Bioeng Biotechnol 2022; 10:1017613. [PMID: 36312531 PMCID: PMC9607953 DOI: 10.3389/fbioe.2022.1017613] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Accepted: 10/03/2022] [Indexed: 11/29/2022] Open
Abstract
The destruction of periodontal tissue is a crucial problem faced by oral diseases, such as periodontitis and tooth avulsion. However, regenerating periodontal tissue is a huge clinical challenge because of the structural complexity and the poor self-healing capability of periodontal tissue. Tissue engineering has led to advances in periodontal regeneration, however, the source of exogenous seed cells is still a major obstacle. With the improvement of in situ tissue engineering and the exploration of stem cell niches, the homing of endogenous stem cells may bring promising treatment strategies in the future. In recent years, the applications of endogenous cell homing have been widely reported in clinical tissue repair, periodontal regeneration, and cell therapy prospects. Stimulating strategies have also been widely studied, such as the combination of cytokines and chemokines, and the implantation of tissue-engineered scaffolds. In the future, more research needs to be done to improve the efficiency of endogenous cell homing and expand the range of clinical applications.
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Affiliation(s)
- Lingxi Meng
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Yige Wei
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Yaxian Liang
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Qin Hu
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China
| | - Huixu Xie
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
- *Correspondence: Huixu Xie,
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Function of Dental Follicle Progenitor/Stem Cells and Their Potential in Regenerative Medicine: From Mechanisms to Applications. Biomolecules 2021; 11:biom11070997. [PMID: 34356621 PMCID: PMC8301812 DOI: 10.3390/biom11070997] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2021] [Revised: 07/04/2021] [Accepted: 07/04/2021] [Indexed: 02/06/2023] Open
Abstract
Dental follicle progenitor/stem cells (DFPCs) are a group of dental mesenchyme stem cells that lie in the dental follicle and play a critical role in tooth development and maintaining function. Originating from neural crest, DFPCs harbor a multipotential differentiation capacity. More importantly, they have superiorities, including the easy accessibility and abundant sources, active self-renewal ability and noncontroversial sources compared with other stem cells, making them an attractive candidate in the field of tissue engineering. Recent advances highlight the excellent properties of DFPCs in regeneration of orofacial tissues, including alveolar bone repair, periodontium regeneration and bio-root complex formation. Furthermore, they play a unique role in maintaining a favorable microenvironment for stem cells, immunomodulation and nervous related tissue regeneration. This review is intended to summarize the current knowledge of DFPCs, including their stem cell properties, physiological functions and clinical application potential. A deep understanding of DFPCs can thus inspire novel perspectives in regenerative medicine in the future.
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Nizami MZI, Nishina Y. Recent Advances in Stem Cells for Dental Tissue Engineering. ENGINEERING MATERIALS FOR STEM CELL REGENERATION 2021:281-324. [DOI: 10.1007/978-981-16-4420-7_12] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Chen J, Li W, Li Q, Wang Y, Zhao B, Han X, Deng J, Liu Y. The composite sandwich structure of dNCPs polyelectrolyte multilayers induced the osteogenic differentiation of PDLSCs in vitro. J Appl Biomater Funct Mater 2020; 18:2280800020942719. [PMID: 33176539 DOI: 10.1177/2280800020942719] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
This study reported about the fabrication of dentin non-collagenous proteins (dNCPs) polyelectrolyte multilayers and evaluated its osteogenic potential. The composite sandwich structure of dNCPs polyelectrolyte multilayers was generated on the surface of polycaprolactone electrospinning membranes by the Layer-by-Layer self-assembly technique. The dNCPs-coated membranes comprised the experimental group and the non-coated membranes acted as the control. Nanofiber morphologies of both membranes were observed under scanning electron microscope. The release of dNCPs was evaluated by ELISA kit. Periodontal ligament stem cells (PDLSCs) were seeded on both membranes. The morphology changes and proliferation of cells were tested. The expressions of osteogenic-related genes and proteins were evaluated by RT-PCR, alkaline phosphatase (ALP) activity assay, and immunofluorescence staining. dNCPs-coated membranes displayed significantly different fiber morphology than the non-coated membranes. A stable release of dentin phosphoprotein was maintained from day 4 to day 15 in the experimental group. Cells on dNCPs-coated membranes were found to have cuboidal or polygonal shapes. The proliferative rate of cells was significantly lower in the experimental group from day 4 to day 9 (p<0.05). However, cells on the dNCPs-coated membranes demonstrated a significantly higher ALP content and expression levels of osteogenic gene and proteins than the controls (p<0.05). These results indicated that dNCPs polyelectrolyte multilayers could induce the osteogenic differentiation of PDLSCs in vitro.
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Affiliation(s)
- Jing Chen
- Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China.,Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China
| | - Wenxing Li
- Chengdu Zhuoyue dental clinic, Chengdu, P.R. China
| | - Qiang Li
- Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China.,Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China
| | - Yuhui Wang
- Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China.,Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China
| | - Bingjiao Zhao
- Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China.,Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China
| | - Xinxin Han
- Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China
| | - Jiajia Deng
- Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China.,Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China
| | - Yuehua Liu
- Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China.,Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University, Shanghai, P.R. China
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Zhang S, Zhang W, Li Y, Ren L, Deng H, Yin X, Gao X, Pan S, Niu Y. Human Umbilical Cord Mesenchymal Stem Cell Differentiation Into Odontoblast-Like Cells and Endothelial Cells: A Potential Cell Source for Dental Pulp Tissue Engineering. Front Physiol 2020; 11:593. [PMID: 32714196 PMCID: PMC7344301 DOI: 10.3389/fphys.2020.00593] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Accepted: 05/11/2020] [Indexed: 12/19/2022] Open
Abstract
OBJECTIVES Dental pulp regeneration is considered an ideal approach for treating dental pulp disease. Because pulp is composed of various cells, determining the proper seed cells is critical. We explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration. METHODS Liquid extract of human treated dentin matrix (LE-TDM) was acquired to culture hUCMSCs. Odontoblast-specific markers were detected by western blot, qRT-PCR, and immunofluorescence assays. Endothelial differentiation of hUCMSCs was examined according to VEGF induction by western blot, qRT-PCR, and Matrigel assays. hUCMSCs and VEGF-induced hUCMSCs (V-hUCMSCs) were also cocultured in vivo for the Matrigel plug assay and in vitro for RNA-sequencing (RNA-seq). Finally, encapsulated mono-cultured hUCMSCs or cocultured hUCMSCs and V-hUCMSCs in scaffolds were injected into the root segments and transplanted into immunodeficient mice for dental pulp regeneration. RESULTS Under LE-TDM induction, hUCMSCs expressed specific odontoblast markers (DSPP, DMP-1, DSP). Under VEGF induction, hUCMSCs expressed functional endothelial markers (CD31, eNOs, vWF). In vivo, the Matrigel plug assay indicated that cocultured hUCMSCs and V-hUCMSCs formed extensive vessel-like structures. RNA-seq results indicated that cocultured V-hUCMSCs exhibited high Hif-1 signaling pathway activity. Both the hUCMSCs mono-culture and coculture groups showed pulp-like tissue regeneration. The cocultured group showed more extracellular matrix and vascularization than the mono-cultured group in vivo. CONCLUSION hUCMSCs can differentiate into odontoblast-like cells and functional endothelial cells. Cocultured hUCMSCs and V-hUCMSCs formed vessel-like structures and regenerated dental pulp-like tissue. Therefore, hUCMSCs can be used as an alternative seed cell source for angiogenesis and dental pulp regeneration.
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Affiliation(s)
- Shuang Zhang
- Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Endodontics, School of Stomatology, Harbin Medical University, Harbin, China
| | - Weiwei Zhang
- Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Endodontics, School of Stomatology, Harbin Medical University, Harbin, China
| | - Yanping Li
- Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Endodontics, School of Stomatology, Harbin Medical University, Harbin, China
| | - Liping Ren
- Department of Prosthodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Haotian Deng
- Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Endodontics, School of Stomatology, Harbin Medical University, Harbin, China
| | - Xiaowei Yin
- Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Endodontics, School of Stomatology, Harbin Medical University, Harbin, China
| | - Xu Gao
- Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, China
| | - Shuang Pan
- Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Endodontics, School of Stomatology, Harbin Medical University, Harbin, China
| | - Yumei Niu
- Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Endodontics, School of Stomatology, Harbin Medical University, Harbin, China
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Dental Follicle Cells: Roles in Development and Beyond. Stem Cells Int 2019; 2019:9159605. [PMID: 31636679 PMCID: PMC6766151 DOI: 10.1155/2019/9159605] [Citation(s) in RCA: 63] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2019] [Accepted: 08/16/2019] [Indexed: 02/05/2023] Open
Abstract
Dental follicle cells (DFCs) are a group of mesenchymal progenitor cells surrounding the tooth germ, responsible for cementum, periodontal ligament, and alveolar bone formation in tooth development. Cascades of signaling pathways and transcriptional factors in DFCs are involved in directing tooth eruption and tooth root morphogenesis. Substantial researches have been made to decipher multiple aspects of DFCs, including multilineage differentiation, senescence, and immunomodulatory ability. DFCs were proved to be multipotent progenitors with decent amplification, immunosuppressed and acquisition ability. They are able to differentiate into osteoblasts/cementoblasts, adipocytes, neuron-like cells, and so forth. The excellent properties of DFCs facilitated clinical application, as exemplified by bone tissue engineering, tooth root regeneration, and periodontium regeneration. Except for the oral and maxillofacial regeneration, DFCs were also expected to be applied in other tissues such as spinal cord defects (SCD), cardiomyocyte destruction. This article reviewed roles of DFCs in tooth development, their properties, and clinical application potentials, thus providing a novel guidance for tissue engineering.
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Zhai Q, Dong Z, Wang W, Li B, Jin Y. Dental stem cell and dental tissue regeneration. Front Med 2018; 13:152-159. [PMID: 29971640 DOI: 10.1007/s11684-018-0628-x] [Citation(s) in RCA: 122] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2017] [Accepted: 12/14/2017] [Indexed: 12/22/2022]
Abstract
The teeth are highly differentiated chewing organs formed by the development of tooth germ tissue located in the jaw and consist of the enamel, dentin, cementum, pulp, and periodontal tissue. Moreover, the teeth have a complicated regulatory mechanism, special histologic origin, diverse structure, and important function in mastication, articulation, and aesthetics. These characteristics, to a certain extent, greatly complicate the research in tooth regeneration. Recently, new ideas for tooth and tissue regeneration have begun to appear with rapid developments in the theories and technologies in tissue engineering. Numerous types of stem cells have been isolated from dental tissue, such as dental pulp stem cells (DPSCs), stem cells isolated from human pulp of exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAPs), and dental follicle cells (DFCs). All these cells can regenerate the tissue of tooth. This review outlines the cell types and strategies of stem cell therapy applied in tooth regeneration, in order to provide theoretical basis for clinical treatments.
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Affiliation(s)
- Qiming Zhai
- State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Center for Tissue Engineering, Fourth Military Medical University, Xi'an, 710032, China
| | - Zhiwei Dong
- Department of Oral and Maxillofacial Surgery, General Hospital of Shenyang Military Area Command, Shenyang, 110840, China
| | - Wei Wang
- State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, School of Stomatology, Fourth Military Medical University, Xi'an, 710032, China
| | - Bei Li
- State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Center for Tissue Engineering, Fourth Military Medical University, Xi'an, 710032, China. .,Xi'an Institute of Tissue Engineering and Regenerative Medicine, Xi'an, 710032, China.
| | - Yan Jin
- State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Center for Tissue Engineering, Fourth Military Medical University, Xi'an, 710032, China. .,Xi'an Institute of Tissue Engineering and Regenerative Medicine, Xi'an, 710032, China.
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Meng Z, Chen G, Chen J, Yang B, Yu M, Feng L, Jiang Z, Guo W, Tian W. Tumorigenicity analysis of heterogeneous dental stem cells and its self-modification for chromosome instability. Cell Cycle 2016; 14:3396-407. [PMID: 26322910 DOI: 10.1080/15384101.2015.1036204] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Heterogeneity demonstrates that stem cells are constituted by several sub-clones in various differentiation states. The heterogeneous state is maintained by cross-talk among sub-clones, thereby ensuring stem cell adaption. In this study, we investigated the roles of heterogeneity on genetic stability. Three sub-clones (DF2, DF8 and DF18) were isolated from heterogeneous dental stem cells (DSCs), and were proved to be chromosome instability (CIN) after long term expansion. Cell apoptosis were not detected in sub-clones, which exhibited strong tumorigenesis tendency, coupled with weak expression of p53 and aberrant ultra-structure. However, 3 sub-clones did not overexpress tumor related markers or induce tumorigenesis in vivo. The mixed-culture study suggested that 3-clone-mixed culturing cells (DF1) presented apparent decrease in the ratio of aneuploidy. The screening experiment further proved that 3 sub-clones functioned separately in this modification procedure but only mixed culturing all 3 sub-clones, simulated heterogeneous microenvironment, could achieve complete modification. Additionally, osteogenesis capability of 3 sub-clones was partially influenced by CIN while DSCs still kept stronger osteogenesis than sub-clones. These results suggested aberrant sub-clones isolated from heterogeneous DSCs were not tumorigenesis and could modify CIN by cross-talk among themselves, indicating that the heterogeneity played a key role in maintaining genetic stability and differentiation capability in dental stem cells.
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Affiliation(s)
- Zhaosong Meng
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,c Department of Oral and Maxillofacial Surgery ; West China School of Stomatology; Sichuan University ; Chengdu , China
| | - Guoqing Chen
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China
| | - Jinlong Chen
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,c Department of Oral and Maxillofacial Surgery ; West China School of Stomatology; Sichuan University ; Chengdu , China
| | - Bo Yang
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,c Department of Oral and Maxillofacial Surgery ; West China School of Stomatology; Sichuan University ; Chengdu , China
| | - Mei Yu
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China
| | - Lian Feng
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China
| | - Zongting Jiang
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China
| | - Weihua Guo
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,d Department of Pedodontics ; West China School of Stomatology; Sichuan University ; Chengdu , China
| | - Weidong Tian
- a National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,b State Key Laboratory of Oral Diseases; West China Hospital of Stomatology; Sichuan University ; Chengdu , China.,c Department of Oral and Maxillofacial Surgery ; West China School of Stomatology; Sichuan University ; Chengdu , China
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Tabatabaei FS, Torshabi M. Effects of Non-Collagenous Proteins, TGF-β1, and PDGF-BB on Viability and Proliferation of Dental Pulp Stem Cells. EJOURNAL OF ORAL MAXILLOFACIAL RESEARCH 2016; 7:e4. [PMID: 27099698 PMCID: PMC4837608 DOI: 10.5037/jomr.2016.7104] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/12/2016] [Accepted: 03/22/2016] [Indexed: 12/17/2022]
Abstract
Objectives The dentin matrix servers as a reservoir of growth factors, sequestered during dentinogenesis. The aim of this study was to assess the viability and proliferation of dental pulp stem cells in the presence of dentin matrix-derived non-collagenous proteins and two growth factors; platelet-derived growth factor BB and transforming growth factor beta 1. Material and Methods The dental pulp cells were isolated and cultured. The dentin proteins were extracted and purified. The MTT assay was performed for assessment of cell viability and proliferation in the presence of different concentrations of dentin proteins and growth factors during 24 - 72 h post-treatment. Results The cells treated with 250 ng/mL dentin proteins had the best viability and proliferation ability in comparison with other concentrations (P < 0.05). The MTT assay demonstrated that cells cultured with 5 ng/mL platelet-derived growth factor BB had the highest viability at each time point as compared to other groups (P < 0.05). However, in presence of platelet-derived growth factor BB alone and in combination with transforming growth factor beta 1 and dentin proteins (10 ng/mL), significant higher viability was seen at all time points (P < 0.05). The least viability and proliferation at each growth factor concentration was seen in cells treated with combination of transforming growth factor beta 1 and dentin proteins at 72 h (P < 0.05). Conclusions The results indicated that the triple combination of growth factors and matrix-derived non-collagenous proteins (especially at 10 ng/mL concentration) has mitogenic effect on dental pulp stem cells.
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Affiliation(s)
- Fahimeh Sadat Tabatabaei
- Department of Dental Biomaterials, School of Dentistry, Shahid Beheshti University of Medical sciencesIran.; Department of Tissue Engineering, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical SciencesIran
| | - Maryam Torshabi
- Department of Dental Biomaterials, School of Dentistry, Shahid Beheshti University of Medical sciencesIran.; Department of Tissue Engineering, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical SciencesIran
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12
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Zheng W, Wang S, Wang J, Jin F. Periodontitis promotes the proliferation and suppresses the differentiation potential of human periodontal ligament stem cells. Int J Mol Med 2015; 36:915-22. [PMID: 26310866 PMCID: PMC4564090 DOI: 10.3892/ijmm.2015.2314] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Accepted: 05/15/2015] [Indexed: 12/21/2022] Open
Abstract
The aim of the present study was to investigate the periodontitis-associated changes in the number, proliferation and differentiation potential of human periodontal ligament stem cells (PDLSCs). Cultures of human periodontal ligament cells (PDLCs) were established from healthy donors and donors with periodontitis. The numbers of stem cell were characterized using flow cytometry. PDLSCs were isolated from the PDLCs by immunomagnetic bead selection. Colony-forming abilities, osteogenic and adipogenic potential, gene expression of cementoblast phenotype, alkaline phosphatase activity and in vivo differentiation capacities were then evaluated. Periodontitis caused an increase in the proliferation of PDLSCs and a decrease in the commitment to the osteoblast lineage. This is reflected by changes in the expression of osteoblast markers. When transplanted into immunocompromised mice, PDLSCs from the healthy donors exhibited the capacity to produce cementum PDL-like structures, whereas, the inflammatory PDLSCs transplants predominantly formed connective tissues. In conclusion, the data from the present study suggest that periodontitis affects the proliferation and differentiation potential of human PDLSCs in vitro and in vivo.
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Affiliation(s)
- Wei Zheng
- Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041, P.R. China
| | - Shi Wang
- Department of Stomatology, The Third Central Hospital, Tianjin 300041, P.R. China
| | - Jianguo Wang
- Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041, P.R. China
| | - Fang Jin
- Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
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13
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Wen X, Liu L, Deng M, Liu R, Zhang L, Nie X. In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75(+) stem cells with dental follicle cell conditioned medium. Exp Cell Res 2015; 337:76-86. [PMID: 26165934 DOI: 10.1016/j.yexcr.2015.07.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2015] [Revised: 06/28/2015] [Accepted: 07/06/2015] [Indexed: 12/17/2022]
Abstract
Cranial neural crest-derived cells (CNCCs) play important role in epithelial-mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75(+)) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75(+) CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features to cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75(+) cells, suggesting their differentiation along cementoblast-like lineage. p75(+) stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial-mesenchymal interactions in tooth morphogenesis.
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Affiliation(s)
- Xiujie Wen
- Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042, China
| | - Luchuan Liu
- Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042, China
| | - Manjing Deng
- Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042, China
| | - Rui Liu
- Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042, China
| | - Li Zhang
- Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042, China
| | - Xin Nie
- Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042, China.
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14
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Osteogenic differentiation and gene expression profile of human dental follicle cells induced by human dental pulp cells. J Mol Histol 2014; 46:93-106. [PMID: 25520056 DOI: 10.1007/s10735-014-9604-1] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Accepted: 12/15/2014] [Indexed: 12/20/2022]
Abstract
Dental follicle cells (DFCs) differentiate into cementoblasts or osteoblasts under appropriate triggering. However, the mechanism(s) for osteogenic differentiation of DFCs are still unclear. The purpose of this study was to examine the effects of dental papilla-derived human dental pulp cells (hDPCs) on osteogenic differentiation of human DFCs (hDFCs) in vitro and in vivo and to compare gene expression in hDFCs in the presence or absence of hDPCs. To evaluate the osteogenic differentiation of hDFCs induced by hDPCs, hDFCs were cultured in osteogenic medium with or without hDPCs-conditioned medium (CM) in vitro and the cells transplanted into the subcutaneous tissue of immunodeficient mice in vivo. The hDPCs-CM enhanced alkaline phosphatase promoter activity of hDFCs in osteogenic culture. The expression of several osteoblast marker genes was increased in hDFCs treated with hDPCs-CM compared to hDFCs in normal medium. The hDFCs induced by hDPCs-CM also produced more calcified nodules than hDFCs in normal medium. In transplantation experiments, hDPCs-CM promoted the osteogenic induction and bone formation of hDFCs. Microarray analysis and quantitative real-time PCR showed that osteogenesis-related genes including WNT2, VCAN, OSR2, FOSB, and POSTN in hDFCs were significantly upregulated after induction by hDPCs-CM compared to hDFCs in normal medium. These findings indicate that hDPCs could increase the expression of osteogenic genes in hDFCs and stimulate their osteogenesis and could be a cellular resource for bone regeneration therapy when induced by hDPCs-derived factors.
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15
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Ji B, Sheng L, Chen G, Guo S, Xie L, Yang B, Guo W, Tian W. The combination use of platelet-rich fibrin and treated dentin matrix for tooth root regeneration by cell homing. Tissue Eng Part A 2014; 21:26-34. [PMID: 25111570 DOI: 10.1089/ten.tea.2014.0043] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Endogenous regeneration through cell homing provides an alternative approach for tissue regeneration, except cell transplantation, especially considering clinical translation. However, tooth root regeneration through cell homing remains a provocative approach in need of intensive study. Both platelet-rich fibrin (PRF) and treated dentin matrix (TDM) are warehouses of various growth factors, which can promote cell homing. We hypothesized that endogenous stem cells are able to sense biological cues from PRF membrane and TDM, and contribute to the regeneration of tooth root, including soft and hard periodontal tissues. Therefore, the biological effects of canine PRF and TDM on periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMSCs) were evaluated respectively in vitro. Beagle dogs were used as orthotopic transplantation model. It was found that PRF significantly recruited and stimulated the proliferation of PDLSCs and BMSCs in vitro. Together, PRF and TDM induced cell differentiation by upregulating the mineralization-related gene expression of bone sialoprotein (BSP) and osteopotin (OPN) after 7 days coculture. In vivo, transplantation of autologous PRF and allogeneic TDM into fresh tooth extraction socket achieved successful root regeneration 3 months postsurgery, characterized by the regeneration of cementum and periodontal ligament (PDL)-like tissues with orientated fibers, indicative of functional restoration. The results suggest that tooth root connected to the alveolar bone by cementum-PDL complex can be regenerated through the implantation of PRF and TDM in a tooth socket microenvironment, probably by homing of BMSCs and PDLSCs. Furthermore, bioactive cues and inductive microenvironment are key factors for endogenous regeneration. This approach provides a tangible pathway toward clinical translation.
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Affiliation(s)
- Baohui Ji
- 1 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University , Chengdu, People's Republic of China
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16
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Fan JZ, Yang L, Meng GL, Lin YS, Wei BY, Fan J, Hu HM, Liu YW, Chen S, Zhang JK, He QZ, Luo ZJ, Liu J. Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway. Mol Cell Biochem 2014; 392:85-93. [PMID: 24752351 PMCID: PMC4053611 DOI: 10.1007/s11010-014-2021-7] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2013] [Accepted: 03/05/2014] [Indexed: 12/22/2022]
Abstract
Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5'-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.
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Affiliation(s)
- Jin-Zhu Fan
- Institute of Orthopedic Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, People's Republic of China
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Li Y, Li SQ, Gao YM, Li J, Zhang B. Crucial role of Notch signaling in osteogenic differentiation of periodontal ligament stem cells in osteoporotic rats. Cell Biol Int 2014; 38:729-36. [PMID: 24677709 DOI: 10.1002/cbin.10257] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2013] [Accepted: 01/20/2014] [Indexed: 01/08/2023]
Abstract
Estrogen deficiency-induced osteoporosis typically occurs in postmenopausal women and has been strongly associated with periodontal diseases. Periodontal ligament stem cells (PDLSCs) isolated from the periodontal ligament can differentiate into many types of specialized cells, including osteoblast-like cells that contribute to periodontal tissue repair. The Notch signaling pathway is highly conserved and associated with self-renewal potential and cell-fate determination. Recently, several studies have focused on the relationship between Notch signaling and osteogenic differentiation. However, the precise mechanisms underlying this relationship are largely unknown. We have successfully isolated PDLSCs from both ovariectomized (OVX) and sham-operated rats. Both the mRNA and protein levels of Notch1 and Jagged1 were upregulated when PDLSCs were cultured in osteogenic induction media. Mineralization assays showed decreased calcium deposits in OVX-PDLSCs treated with a γ-secretase inhibitor compared with control cells. Thus Notch signaling is important in maintaining the osteogenic differentiation of PDLSCs in osteoporotic rats, which help in the development of a potential therapeutic strategy for periodontal disease in postmenopausal women.
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Affiliation(s)
- Ying Li
- Department of Stomatology, General Hospital of the Air Force of Chinese PLA, Beijing, 100853, China
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18
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Lei G, Yu Y, Jiang Y, Wang S, Yan M, Smith AJ, Smith G, Cooper PR, Tang C, Zhang G, Yu J. Differentiation of BMMSCs into odontoblast-like cells induced by natural dentine matrix. Arch Oral Biol 2013; 58:862-70. [PMID: 23414708 DOI: 10.1016/j.archoralbio.2013.01.002] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2012] [Revised: 12/02/2012] [Accepted: 01/06/2013] [Indexed: 01/09/2023]
Abstract
OBJECTIVE To assess the odontogenic potential of bone marrow mesenchymal stem cells (BMMSCs) to differentiate into odontoblast-like cells under the morphogenetic influence of dentine matrix as a possible basis for new stem cell-mediated therapeutic approaches to pulp diseases. DESIGN BMMSCs were harvested from the whole bone marrow and cells at passages 3-5 were used for subsequent experiments. For in vitro studies, 1×10(4) cells were seeded on the surface of dentine slabs and co-cultured for 2 weeks in 24-well plates, then fixed, decalcified, embedded in paraffin and serial sections were processed for analyses. Haematoxylin-eosin (HE) staining was used for the morphological analysis of BMMSCs on the dentine slabs. The protein expression of dentine sialoprotein (DSP) in co-cultured BMMSCs was detected by immunohistochemical (IHC) staining. For in vivo studies, 5×10(6) cells were collected as cell pellets, seeded onto dentine slices and transplanted into renal capsules for 6 weeks. Histological analyses of harvested tissues were performed as described for the in vitro studies. Total RNA and protein were extracted from harvested tissues and Dspp/DSP expression was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. RESULTS After 2 weeks of co-culture with dentine slabs, BMMSCs demonstrated good viability in terms of morphological appearance and some showed polarization and extension of their cytoplasmic processes into dentine tubules with DSP expression. In vivo study demonstrated similar morphological changes and DSP expression in cells adjacent to dentine. RT-PCR and Western blot also demonstrated that the expression of Dspp/DSP in the co-cultured BMMSCs groups was higher than in the control groups. CONCLUSION Dentine matrix can signal morphogenic induction of differentiation of BMMSCs into odontoblast-like cells in vivo and in vitro.
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Affiliation(s)
- Gang Lei
- Institute of Stomatology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, China
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19
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Aonuma H, Ogura N, Takahashi K, Fujimoto Y, Iwai S, Hashimoto H, Ito K, Kamino Y, Kondoh T. Characteristics and osteogenic differentiation of stem/progenitor cells in the human dental follicle analyzed by gene expression profiling. Cell Tissue Res 2012; 350:317-31. [PMID: 22890370 DOI: 10.1007/s00441-012-1477-6] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2011] [Accepted: 06/25/2012] [Indexed: 12/11/2022]
Abstract
The dental follicle is an ectomesenchymal tissue that surrounds developing tooth germ and that contains osteoblastic-lineage-committed stem/progenitor cells. We examined the osteogenic potential of human dental follicle cells (hDFC) by microarray analysis. We first compared the characteristics of hDFC with those of human bone marrow mesenchymal stem cells (hMSC). Like hMSC, hDFC expressed stem cell markers such as STRO-1 and Notch-1 and differentiated not only into the osteoblastic lineage, but also into the adipogenic lineage. We analyzed the gene expression profiles of hDFC and hMSC that were not differentiated toward the osteogenic lineage. The expression of cell markers and growth factor receptors by hDFC and hMSC was similar, whereas the expression pattern of homeobox genes differed between hDFC and hMSC. Next, we investigated gene expression in hDFC during osteogenic differentiation. Gene expression profiles were analyzed in hDFC cultured in osteogenic induction medium (OIM) or in growth medium (GM) for 3 and 10 days. Many genes whose expression was regulated under these conditions were functionally categorized as "transcription" genes. Osteogenic markers were up-regulated in hDFC during osteogenic differentiation, whereas neurogenic markers were down-regulated. The genes whose expression was regulated in hDFC during osteogenic differentiation were further analyzed by ingenuity pathway analysis and real-time polymerase chain reaction. Bone morphogenetic protein and transforming growth factor-β signaling pathways were activated in hDFC cultured in OIM for 3 days. This study indicates that the dental follicle contains stem cells and/or osteoblastic progenitor cells and is a potential cellular resource for bone regeneration therapy.
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Affiliation(s)
- H Aonuma
- Department of Maxillofacial Surgery, Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba, 271-8587, Japan
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20
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Guo W, Chen L, Gong K, Ding B, Duan Y, Jin Y. Heterogeneous dental follicle cells and the regeneration of complex periodontal tissues. Tissue Eng Part A 2012; 18:459-70. [PMID: 21919800 DOI: 10.1089/ten.tea.2011.0261] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Dental follicle cells (DFCs) are a heterogeneous population that exhibit a variety of phenotypes. However, it remains unclear whether DFCs can maintain stem cell characteristics, or mediate tissue-regeneration to form single or complex tissues in the periodontium, after long-term culturing. Therefore, DFCs were isolated from human impacted molars (HIM-DFCs), passaged >30 times, and then evaluated for their heterogeneity and multipotential differentiation. Morphology, proliferation, epitope profile, and mineralization characteristics of clones derived from single HIM-DFCs in vitro were also assayed. HIM-DFCs (passage #30) were found to be positive for the heterogeneous markers, Notch-1, stro-1, alkaline phosphomonoesterase (ALP), type I collagen (COL-I), type III collagen (COL-III), and osteocalcine. Moreover, passage #30 of the HDF1, 2, and 3 subclone classes identified in this study were found to express high levels of the mesenchymal stem cells markers, CD146 and Stro1. HDF3 subclones were also associated with the strongest ALP staining detected, and strongly expressed osteoblast and cementoblast markers, including COL-I, COL-III, bone sialoprotein (BSP), and Runx2. In contrast, HDF1 subclone analyzed strongly expressed COL-I and COL-III, yet weakly expressed BSP and Runx2. The HDF2 subclone was associated with the strongest proliferative capacity. To evaluate differentiation characteristics in vivo, these various cell populations were combined with ceramic bovine bone and implanted into subcutaneous pockets of nude mice. The 30th passage of subclone HDF1 and 3 were observed to contribute to fiber collagens and the mineralized matrix present, respectively, whereas HDF2 subclones were found to have a minimal role in these formations. The formation of a cementum-periodontal ligament (PDL) complex was observed 6 weeks after HIM-DFCs (passage #30) were implanted in vivo, thus suggesting that these cells maintain stem cell characteristics. Therefore, subclone HDF1-3 may be related to the differentiation of fibroblasts in the PDL, undifferentiated cells, and osteoblasts and cementoblasts, respectively. Overall, this study is the first to amplify HIM-DFCs and associated subclones with the goal of reconstructing complex or single periodontium. Moreover, our results demonstrate the potential for this treatment approach to address periodontal defects that result from periodontitis, or for the regeneration of teeth.
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Affiliation(s)
- Weihua Guo
- Research and Development Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi'an, People's Republic of China
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21
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Li C, Yang X, He Y, Ye G, Li X, Zhang X, Zhou L, Deng F. Bone morphogenetic protein-9 induces osteogenic differentiation of rat dental follicle stem cells in P38 and ERK1/2 MAPK dependent manner. Int J Med Sci 2012; 9:862-71. [PMID: 23155360 PMCID: PMC3498751 DOI: 10.7150/ijms.5027] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/12/2012] [Accepted: 09/17/2012] [Indexed: 11/28/2022] Open
Abstract
Dental follicle stem cells are a group of cells possessing osteogenic, adipogenetic and neurogenic differentiations, but the specific mechanism underlying the multilineage differentiation remains still unclear. Great attention has been paid to bone morphogenetic protein-9 (BMP-9) due to its potent osteogenic activity. In the present study, rat dental follicle stem cells were isolated and purified, and cells of passage 3 underwent adenovirus mediated BMP-9 gene transfection to prepare dental follicle stem cells with stable BMP-9 expression. Detection of alkaline phosphatase (ALP) and calcium deposition showed dental follicle stem cells transfected with BMP-9 gene could significantly promote the osteogenesis. In addition, SB203580 and PD98059 were employed to block the p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2), respectively. Detection of ALP and calcium deposition revealed the BMP-9 induced osteogenic differentiation of dental follicle stem cells depended on MAPK signaling pathway.
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Affiliation(s)
- Conghua Li
- Department of Stomatology, Research Center for Oral Diseases and Biomedicine in Chongqing, Affiliated Stomatological Hospital, Chongqing Medical University, China
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22
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Zhou Y, Yan Z, Zhang H, Lu W, Liu S, Huang X, Luo H, Jin Y. Expansion and Delivery of Adipose-Derived Mesenchymal Stem Cells on Three Microcarriers for Soft Tissue Regeneration. Tissue Eng Part A 2011; 17:2981-97. [DOI: 10.1089/ten.tea.2010.0707] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Yalei Zhou
- Department of Oral Histology and Pathology, School of Stomatology, The Fourth Military Medical University, Xi'an, P.R. China
- Research and Development Center for Tissue Engineering, The Fourth Military Medical University, Xi'an, P.R. China
| | - Zhiwei Yan
- Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University, Xi'an, P.R. China
| | - Hongmei Zhang
- Department of Oral Histology and Pathology, School of Stomatology, The Fourth Military Medical University, Xi'an, P.R. China
- Research and Development Center for Tissue Engineering, The Fourth Military Medical University, Xi'an, P.R. China
| | - Wei Lu
- Department of Oral Histology and Pathology, School of Stomatology, The Fourth Military Medical University, Xi'an, P.R. China
- Research and Development Center for Tissue Engineering, The Fourth Military Medical University, Xi'an, P.R. China
| | - Shiyu Liu
- Department of Oral Histology and Pathology, School of Stomatology, The Fourth Military Medical University, Xi'an, P.R. China
- Research and Development Center for Tissue Engineering, The Fourth Military Medical University, Xi'an, P.R. China
| | - Xinhui Huang
- Research and Development Center for Tissue Engineering, The Fourth Military Medical University, Xi'an, P.R. China
| | - Hailang Luo
- Research and Development Center for Tissue Engineering, The Fourth Military Medical University, Xi'an, P.R. China
| | - Yan Jin
- Department of Oral Histology and Pathology, School of Stomatology, The Fourth Military Medical University, Xi'an, P.R. China
- Research and Development Center for Tissue Engineering, The Fourth Military Medical University, Xi'an, P.R. China
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23
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Wen X, Nie X, Zhang L, Liu L, Deng M. Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro. Biochem Biophys Res Commun 2011; 409:583-9. [PMID: 21619870 DOI: 10.1016/j.bbrc.2011.05.067] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2011] [Accepted: 05/11/2011] [Indexed: 12/16/2022]
Abstract
Adipose tissue-derived stem cells (ADSCs), which are easily harvested and show excellent pluripotency potential, have generated considerable interest in regenerative medicine. In this study, the differentiation of ADSCs was assessed after treatment with dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs). ADSCs exhibited a fibroblast-like morphology and high proliferative capacity. However, after treatment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and in vitro mineralization behaviour of ADSCs were significantly enhanced. Mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype. These results demonstrate that ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment and could be a potential source of cementogenic cells for periodontal regeneration.
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Affiliation(s)
- Xiujie Wen
- Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042, China
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24
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Zhang B, Li Y, Zhou Q, Ding Y. Estrogen Deficiency Leads to Impaired Osteogenic Differentiation of Periodontal Ligament Stem Cells in Rats. TOHOKU J EXP MED 2011; 223:177-86. [DOI: 10.1620/tjem.223.177] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Affiliation(s)
- Bin Zhang
- Department of Orthodontics, School of Stomatology, The Fourth Military Medical University
| | - Ying Li
- Institute of Stomatology, General Hospital of Chinese PLA
| | - Qiang Zhou
- Department of General Dentistry and Emergency, School of Stomatology, The Fourth Military Medical University
| | - Yin Ding
- Department of Orthodontics, School of Stomatology, The Fourth Military Medical University
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25
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Sun HH, Jin T, Yu Q, Chen FM. Biological approaches toward dental pulp regeneration by tissue engineering. J Tissue Eng Regen Med 2010; 5:e1-16. [PMID: 21413154 DOI: 10.1002/term.369] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2010] [Accepted: 08/31/2010] [Indexed: 01/07/2023]
Abstract
Root canal therapy has been the predominant approach in endodontic treatment, wherein the entire pulp is cleaned out and replaced with a gutta-percha filling. However, living pulp is critical for the maintenance of tooth homeostasis and essential for tooth longevity. An ideal form of therapy, therefore, might consist of regenerative approaches in which diseased/necrotic pulp tissues are removed and replaced with regenerated pulp tissues to revitalize the teeth. Dental pulp regeneration presents one of the most challenging issues in regenerative dentistry due to the poor intrinsic ability of pulp tissues for self-healing and regrowth. With the advent of modern tissue engineering and the discovery of dental stem cells, biological therapies have paved the way to utilize stem cells, delivered or internally recruited, to generate dental pulp tissues, where growth factors and a series of dentine extracellular matrix molecules are key mediators that regulate the complex cascade of regeneration events to be faithfully fulfilled.
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Affiliation(s)
- Hai-Hua Sun
- Department of Operative Dentistry and Endodontics, School of Stomatology, Fourth Military Medical University, Xi'an 710032, Shaanxi, People's Republic of China
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26
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Wang YX, Ma ZF, Huo N, Tang L, Han C, Duan YZ, Jin Y. Porcine tooth germ cell conditioned medium can induce odontogenic differentiation of human dental pulp stem cells. J Tissue Eng Regen Med 2010; 5:354-62. [PMID: 20799278 DOI: 10.1002/term.321] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2010] [Accepted: 04/16/2010] [Indexed: 12/15/2022]
Abstract
It is suggested that the differentiation of tooth-derived stem cells is modulated by the local microenvironment in which they reside. Previous studies have indicated that tooth germ cell-conditioned medium (TGC-CM) holds the potential to induce dental pulp stem cells (DPSCs) to differentiate into the odontogenic lineage. Nevertheless, human TGC-CM (hTGC-CM) is not feasible in practical application, so we conjectured that xenogenic TGC-CM might exert a similar influence on human dental stem cells. In this study, we chose swine as the xenogenic origin and compared the effect of porcine tooth germ cell-conditioned medium (pTGC-CM) with its human counterpart on human DPSCs. Morphological appearance, colony-forming assay, in vitro multipotential ability, protein and gene expression of the odontogenic phenotype and the in vivo differentiation capacity of DPSCs were evaluated. The results showed that pTGC-CM exerted a similar effect to hTGC-CM in inducing human DPSCs to present odontogenic changes, which were indicated by remarkable morphological changes, higher multipotential capability and the expression of some odontogenic markers in gene and protein levels. Besides, the in vivo results showed that pTGC-CM-treated DPSCs, similar to hTGC-CM-treated DPSCs, could form a more regular dentine-pulp complex. Our data provided the first evidence that pTGC-CM is able to exert almost the same effect on DPSCs with hTGC-CM. The observations suggest that the application of xenogenic TGC-CM may facilitate generating bioengineered teeth from tooth-derived stem cells in future.
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Affiliation(s)
- Yin-Xiong Wang
- Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi 710032, People's Republic of China
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27
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Chen FM, Jin Y. Periodontal tissue engineering and regeneration: current approaches and expanding opportunities. TISSUE ENGINEERING PART B-REVIEWS 2010; 16:219-55. [PMID: 19860551 DOI: 10.1089/ten.teb.2009.0562] [Citation(s) in RCA: 226] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The management of periodontal tissue defects that result from periodontitis represents a medical and socioeconomic challenge. Concerted efforts have been and still are being made to accelerate and augment periodontal tissue and bone regeneration, including a range of regenerative surgical procedures, the development of a variety of grafting materials, and the use of recombinant growth factors. More recently, tissue-engineering strategies, including new cell- and/or matrix-based dimensions, are also being developed, analyzed, and employed for periodontal regenerative therapies. Tissue engineering in periodontology applies the principles of engineering and life sciences toward the development of biological techniques that can restore lost alveolar bone, periodontal ligament, and root cementum. It is based on an understanding of the role of periodontal formation and aims to grow new functional tissues rather than to build new replacements of periodontium. Although tissue engineering has merged to create more opportunities for predictable and optimal periodontal tissue regeneration, the technique and design for preclinical and clinical studies remain in their early stages. To date, the reconstruction of small- to moderate-sized periodontal bone defects using engineered cell-scaffold constructs is technically feasible, and some of the currently developed concepts may represent alternatives for certain ideal clinical scenarios. However, the predictable reconstruction of the normal structure and functionality of a tooth-supporting apparatus remains challenging. This review summarizes current regenerative procedures for periodontal healing and regeneration and explores their progress and difficulties in clinical practice, with particular emphasis placed upon current challenges and future possibilities associated with tissue-engineering strategies in periodontal regenerative medicine.
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Affiliation(s)
- Fa-Ming Chen
- Department of Periodontology and Oral Medicine, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi, P.R. China
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Yang ZH, Jin F, Zhang XJ, Liu X, Zhang YF, Liu JQ, Duan YZ, Jin Y. A Novel Possible Strategy Based on Self-Assembly Approach to Achieve Complete Periodontal Regeneration. Artif Organs 2010; 34:603-9. [DOI: 10.1111/j.1525-1594.2009.00991.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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Guo W, He Y, Zhang X, Lu W, Wang C, Yu H, Liu Y, Li Y, Zhou Y, Zhou J, Zhang M, Deng Z, Jin Y. The use of dentin matrix scaffold and dental follicle cells for dentin regeneration. Biomaterials 2009; 30:6708-23. [DOI: 10.1016/j.biomaterials.2009.08.034] [Citation(s) in RCA: 128] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2009] [Accepted: 08/27/2009] [Indexed: 01/09/2023]
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Li S, Ma Z, Niu Z, Qian H, Xuan D, Hou R, Ni L. NASA-Approved Rotary Bioreactor Enhances Proliferation and Osteogenesis of Human Periodontal Ligament Stem Cells. Stem Cells Dev 2009; 18:1273-82. [PMID: 19327006 DOI: 10.1089/scd.2008.0371] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Shi Li
- Department of Endodontics, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
- Center of Oral Disease, 306th Hospital, Beijing, People’s Republic of China
- Department of Orthodontics, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
| | - Zhaofeng Ma
- Department of Oral and Maxillofacial Surgery, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
- Department of Implantology, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
| | - Zhongying Niu
- Department of Endodontics, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
- Center of Oral Disease, 306th Hospital, Beijing, People’s Republic of China
| | - Hong Qian
- Department of Orthodontics, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
| | - Dongying Xuan
- Department of Periodontology, Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou, People’s Republic of China
| | - Rui Hou
- Department of Oral and Maxillofacial Surgery, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
| | - Longxing Ni
- Department of Endodontics, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China
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Ma D, Ma Z, Zhang X, Wang W, Yang Z, Zhang M, Wu G, Lu W, Deng Z, Jin Y. Effect of age and extrinsic microenvironment on the proliferation and osteogenic differentiation of rat dental pulp stem cells in vitro. J Endod 2009; 35:1546-53. [PMID: 19840645 DOI: 10.1016/j.joen.2009.07.016] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2009] [Revised: 07/26/2009] [Accepted: 07/27/2009] [Indexed: 01/09/2023]
Abstract
INTRODUCTION It is suggested that dental pulp stem cells (DPSCs) possess pluripotent differentiation and self-renewal capacity and play a crucial role in maintaining dental pulp homeostasis. However, little is known about the age-related changes of DPSCs, and whether aging and its microenvironment are associated with DPSCs remains a question. In this study, age-related changes in proliferation and osteogenic differentiation ability of rat DPSCs were assessed. METHODS To examine the influence of microenvironment factors on different ages of DPSCs, we exposed adult rat DPSCs to juvenile rat dental pulp cell-conditioned medium (DPC-CM), and juvenile DPSCs were exposed to adult DPC-CM. Morphologic appearance, colony-forming assay, cell cycle analysis, 3-(4,5-dimethyl-thyazol-2-yl)-2,5-diphenyltetrazolium, gene expression, and mineralization assay after osteogenic induction of DPSCs were evaluated. RESULTS DPSCs isolated from the juvenile donors displayed increased proliferation and decreased osteogenic differentiation ability compared with the adult DPSCs. Interestingly, adult DPSCs induced by juvenile DPC-CM demonstrated enhanced proliferation but decreased osteogenic differentiation ability, whereas DPSCs from juvenile donors induced by adult DPC-CM showed decreased proliferation but enhanced osteogenic differentiation ability. CONCLUSIONS Our data suggest that age-related changes of DPSCs should be taken into account when DPSCs are intended to be used for investigations and application. Furthermore, the activity of DPSCs can be modulated by the extrinsic microenvironment.
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Affiliation(s)
- Dandan Ma
- Department of Oral Histology and Pathology, School of Stomatology, Fourth Military Medical University, Shaanxi, China
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Ma Z, Li S, Song Y, Tang L, Ma D, Liu B, Jin Y. The biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro and in vivo. Tissue Eng Part A 2009; 14:2059-68. [PMID: 18939934 DOI: 10.1089/ten.tea.2008.0021] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
It was recognized that periodontal progenitor cells penetrate disintegrated Hertwig's epithelial root sheath, and contact with root dentin give rise to periodontium formation. Clinically, direct contact of the conditioned or denuded root surfaces with periodontal cells seems to be a prerequisite for periodontal regeneration. In this study, we investigated the biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro and in vivo. Chemical-conditioned root dentin (CCRD) was prepared by process of partly demineralization and deproteinization. Treated HPDLSCs with DNCPs showed increased proliferation and adhesion ability. Induced HPDLSCs presented several features of cementoblast differentiation, as indicated by morphologic changes, enhanced alkaline phosphatase (ALP) activity, increased matrix mineralization, and upregulated expression of mineralization-associated genes. Incubation of treated HPDLSC aggregate in vivo revealed that cementum-like tissues formed along the CCRD surface with fibrous tissue adjacent to or inserted into it, but untreated HPDLSCs cannot form similar structure. To our knowledge, this is the first study to apply active proteins derived from dentin with periodontal stem cells to construct periodontal structure, which may shed light on human periodontal tissue regeneration.
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Affiliation(s)
- Zhaofeng Ma
- Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi, China
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Jin ZL, Zhang YK, Sun HY, Lin Z, Bi YC, Duan YZ, Ding Y. Osteogenic-related gene expression profiles of human dental follicle cells induced by dexamethasone. Acta Pharmacol Sin 2008; 29:1013-20. [PMID: 18718169 DOI: 10.1111/j.1745-7254.2008.00834.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
AIM Human dental follicle cells (hDFC) have the ability to differentiate into mineralized tissue-forming cells during root and periodontal development or osteogenic induction in vitro. The present study aimed to validate the osteogenic induction of hDFC by dexamethasone (DEX) and to explore the changes of related genes responsible for the osteogenic differentiation process. METHODS Passage-cultured hDFC were induced by DEX and analyzed for mineralization activity by morphological observation, alkaline phosphatase (ALP) activity, and alizarin red S staining. GEArray Q series human osteogenesis gene array was used to describe large-scale gene expression in treated hDFC compared to the control group. Quantitative real-time RT-PCR was performed to confirm the microarray data by analyzing the expression of 7 critical transcripts. RESULTS Osteogenic differentiation of hDFC was confirmed by morphological change, elevated ALP activity and calcified nodules. In 96 genes investigated through the microarray analysis, 20 genes were upregulated and 8 genes were downregulated more than 2-fold. The results of the real-time RT-PCR correlated with the microarray analysis. The expression of the transforming growth factor-beta superfamily showed varying degrees of increase, and fibroblast growth factors exhibited a differential changing trend of expression. The expression of most types of collagen genes representative of extracellular matrixes increased under DEX treatment while small mothers against decapentaplegic 6 and 7 expressions significantly decreased. CONCLUSION Our results demonstrated that hDFC displayed osteoblastic features in both phenotypic and genotypic traits induced by DEX in vitro.
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Affiliation(s)
- Zuo-lin Jin
- Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi'an 710032, China.
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Wu J, Li W, Dai J, Duan Y, Jin Y. Dentin elasticity may contribute to the differentiation of dental follicle cells into cementoblast lineages. ACTA ACUST UNITED AC 2008. [DOI: 10.1016/j.bihy.2008.01.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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