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Stavroullakis AT, Goncalves LL, Levesque CM, Kishen A, Prakki A. Interaction of epigallocatechin-gallate and chlorhexidine with Streptococcus mutans stimulated odontoblast-like cells: Cytotoxicity, Interleukin-1β and co-species proteomic analyses. Arch Oral Biol 2021; 131:105268. [PMID: 34571395 DOI: 10.1016/j.archoralbio.2021.105268] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2021] [Revised: 08/10/2021] [Accepted: 09/15/2021] [Indexed: 10/20/2022]
Abstract
OBJECTIVES The dentin therapeutic agent chlorhexidine has inflammatory and cytotoxic characteristics urging investigation of alternatives like the natural compound epigallocatechin-gallate. The aim is to verify the effect of epigallocatechin-gallate and chlorhexidine on viability, interleukin-1β (IL-1β) and differential protein expression of MDPC-23 odontoblast-like cells stimulated by Streptococcus mutans. DESIGN Cells were stimulated with heat-killed S. mutans at multiplicity of infection (MOI) of 100-1000 and subsequently treated with 100-1 µM of epigallocatechin-gallate. Cells with no treatment or chlorhexidine were controls. Combined stimulated/treated cells were tested for cytotoxicity (Alamar-Blue, N = 3, n = 3), total protein (N = 3, n = 3), IL-1β (ELISA, N = 3, n = 3), and differential protein expression by liquid chromatography-tandem mass spectrometry (LC-MS/MS, n = 2). RESULTS Cells stimulated at MOI 100/1000 and treated with 10 µM epigallocatechin-gallate and chlorhexidine did not present cytotoxicity. IL-1β significantly increased in both un-stimulated and stimulated chlorhexidine 10 µM groups when compared to un-treated control (p < 0.05). MOI 100 chlorhexidine 10 µM group significantly increased IL-1β compared to un-stimulated chlorhexidine 10 µM and epigallocatechin-gallate 10 µM groups, as well as to MOI 100 epigallocatechin-gallate 10 µM group (p < 0.05). LC-MS/MS revealed S. mutans and mammalian proteins, with tooth-specific proteins exhibiting different abundance levels, depending on the tested condition. CONCLUSIONS Odontoblast-like cells stimulated with S. mutans at different MOI combined with epigallocatechin-gallate treatment did not cause cytotoxicity. S. mutans stimulation combined with chlorhexidine 100 µM treatment decreased cell viability, while treatment with chlorhexidine 10 µM concentration significantly increased IL-1β. S. mutans stimulation and treatment of cells resulted in varied protein expression.
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Affiliation(s)
- Alexander Terry Stavroullakis
- Department of Clinical Sciences - Restorative, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
| | - Lucelia Lemes Goncalves
- Department of Clinical Sciences - Restorative, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; Department of Restorative Dentistry, Institute of Science and Technology of São José dos Campos, Sao Paulo State University, São Paulo, Brazil
| | - Celine Marie Levesque
- Department of Biological and Diagnostic Sciences-Oral Microbiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
| | - Anil Kishen
- Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
| | - Anuradha Prakki
- Department of Clinical Sciences - Restorative, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
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Xiao X, Xin C, Zhang Y, Yan J, Chen Z, Xu H, Liang M, Wu B, Fang F, Qiu W. Characterization of Odontogenic Differentiation from Human Dental Pulp Stem Cells Using TMT-Based Proteomic Analysis. BIOMED RESEARCH INTERNATIONAL 2020; 2020:3871496. [PMID: 33490242 PMCID: PMC7789479 DOI: 10.1155/2020/3871496] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/24/2020] [Revised: 11/10/2020] [Accepted: 11/20/2020] [Indexed: 01/09/2023]
Abstract
BACKGROUND The repair of dental pulp injury relies on the odontogenic differentiation of dental pulp stem cells (DPSCs). To better understand the odontogenic differentiation of DPSCs and identify proteins involved in this process, tandem mass tags (TMTs) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to compare the proteomic profiles of induced and control DPSCs. METHODS The proteins expressed during osteogenic differentiation of human DPSCs were profiled using the TMT method combined with LC-MS/MS analysis. The identified proteins were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Then, a protein-protein interaction (PPI) network was constructed. Two selected proteins were confirmed by western blotting (WB) analysis. RESULTS A total of 223 proteins that were differentially expressed were identified. Among them, 152 proteins were significantly upregulated and 71 were downregulated in the odontogenic differentiation group compared with the control group. On the basis of biological processes in GO, the identified proteins were mainly involved in cellular processes, metabolic processes, and biological regulation, which are connected with the signaling pathways highlighted by KEGG pathway analysis. PPI networks showed that most of the differentially expressed proteins were implicated in physical or functional interaction. The protein expression levels of FBN1 and TGF-β2 validated by WB were consistent with the proteomic analysis. CONCLUSIONS This is the first proteomic analysis of human DPSC odontogenesis using a TMT method. We identified many new differentially expressed proteins that are potential targets for pulp-dentin complex regeneration and repair.
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Affiliation(s)
- Xijuan Xiao
- Yuncheng Stomatological Hospital, Yuncheng Stomatological Health School, South Section of Yuxi Road, Yuncheng 044000, China
| | - Caihong Xin
- Yuncheng Stomatological Hospital, Yuncheng Stomatological Health School, South Section of Yuxi Road, Yuncheng 044000, China
| | - Yuqin Zhang
- Yuncheng Stomatological Hospital, Yuncheng Stomatological Health School, South Section of Yuxi Road, Yuncheng 044000, China
| | - Jie Yan
- Yuncheng Stomatological Hospital, Yuncheng Stomatological Health School, South Section of Yuxi Road, Yuncheng 044000, China
| | - Zhao Chen
- Guangdong Provincial Key Laboratory of Oral Diseases, Guangzhou 510055, China
- Department of Stomatology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, China
| | - Huiyong Xu
- Department of Stomatology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, China
| | - Min Liang
- Department of Periodontology, Guanghua School and Hospital of Stomatology and Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China
| | - Buling Wu
- Department of Stomatology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, China
| | - Fuchun Fang
- Guangdong Provincial Key Laboratory of Oral Diseases, Guangzhou 510055, China
- Department of Stomatology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, China
| | - Wei Qiu
- Guangdong Provincial Key Laboratory of Oral Diseases, Guangzhou 510055, China
- Department of Stomatology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, China
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Perczel-Kovách K, Hegedűs O, Földes A, Sangngoen T, Kálló K, Steward MC, Varga G, Nagy KS. STRO-1 positive cell expansion during osteogenic differentiation: A comparative study of three mesenchymal stem cell types of dental origin. Arch Oral Biol 2020; 122:104995. [PMID: 33278647 DOI: 10.1016/j.archoralbio.2020.104995] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 10/31/2020] [Accepted: 11/16/2020] [Indexed: 02/08/2023]
Abstract
OBJECTIVE Although the osteogenic differentiation potential of mesenchymal stem cells of dental origin is well established, the roles of different marker proteins in this process remain to be clarified. Our aim was to compare the cellular and molecular changes, focusing in particular on mesenchymal stem cell markers, during in vitro osteogenesis in three dental stem cell types: dental follicle stem cells (DFSCs), periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs). DESIGN Human DFSCs, PDLSCs and DPSCs were isolated, cultured and their osteogenic differentiation was induced for 3 weeks. Mineralization was assessed by von Kossa staining and calcium concentration measurements. The expression of mesenchymal and osteogenic markers was studied by immunocytochemistry and qPCR techniques. Alkaline phosphatase (ALP) activity and the frequency of STRO-1 positive cells were also quantified. RESULTS The three cultures all showed abundant mineralization, with high calcium content by day 21. The expression of vimentin and nestin was sustained after osteogenic induction. The osteogenic medium induced a considerable elevation of STRO-1 positive cells. By day 7, the ALP mRNA level had increased more than 100-fold in DFSCs, PDLSCs, and DPSCs. Quantitative PCR results indicated dissimilarities of osteoblastic marker levels in the three dental stem cell cultures. CONCLUSIONS DFSCs, PDLSCs and DPSCs have similar functional osteogenic differentiation capacities although their expressional profiles of key osteogenic markers show considerable variations. The STRO-1 positive cell fraction expands during osteogenic differentiation while vimentin and nestin expression remain high. For identification of stemness, functional studies rather than marker expressions are needed.
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Affiliation(s)
- Katalin Perczel-Kovách
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary.
| | - Orsolya Hegedűs
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary.
| | - Anna Földes
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary.
| | - Thanyaporn Sangngoen
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary.
| | - Karola Kálló
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary
| | - Martin C Steward
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary; School of Medical Sciences, University of Manchester, Manchester, United Kingdom.
| | - Gábor Varga
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary.
| | - Krisztina S Nagy
- Department of Oral Biology, Semmelweis University, Nagyvárad Square 4. H-1089 Budapest, Hungary.
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Hosmani J, Assiri K, Almubarak HM, Mannakandath ML, Al-Hakami A, Patil S, Babji D, Sarode S, Devaraj A, Chandramoorthy HC. Proteomic profiling of various human dental stem cells - a systematic review. World J Stem Cells 2020; 12:1214-1236. [PMID: 33178402 PMCID: PMC7596439 DOI: 10.4252/wjsc.v12.i10.1214] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2020] [Revised: 08/06/2020] [Accepted: 09/01/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The proteomic signature or profile best describes the functional component of a cell during its routine metabolic and survival activities. Additional complexity in differentiation and maturation is observed in stem/progenitor cells. The role of functional proteins at the cellular level has long been attributed to anatomical niches, and stem cells do not deflect from this attribution. Human dental stem cells (hDSCs), on the whole, are a combination of mesenchymal and epithelial coordinates observed throughout craniofacial bones to pulp.
AIM To specify the proteomic profile and compare each type of hDSC with other mesenchymal stem cells (MSCs) of various niches. Furthermore, we analyzed the characteristics of the microenvironment and preconditioning changes associated with the proteomic profile of hDSCs and their influence on committed lineage differentiation.
METHODS Literature searches were performed in PubMed, EMBASE, Scopus, and Web of Science databases, from January 1990 to December 2018. An extra inquiry of the grey literature was completed on Google Scholar, ProQuest, and OpenGrey. Relevant MeSH terms (PubMed) and keywords related to dental stem cells were used independently and in combination.
RESULTS The initial search resulted in 134 articles. Of the 134 full-texts assessed, 96 articles were excluded and 38 articles that met the eligibility criteria were reviewed. The overall assessment of hDSCs and other MSCs suggests that differences in the proteomic profile can be due to stem cellular complexity acquired from varied tissue sources during embryonic development. However, our comparison of the proteomic profile suffered inconsistencies due to the heterogeneity of various hDSCs. We believe that the existence of a heterogeneous population of stem cells at a given niche determines the modalities of regeneration or tissue repair. Added prominences to the differences present between various hDSCs have been reasoned out.
CONCLUSION Systematic review on proteomic studies of various hDSCs are promising as an eye-opener for revisiting the proteomic profile and in-depth analysis to elucidate more refined mechanisms of hDSC functionalities.
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Affiliation(s)
- Jagadish Hosmani
- Diagnostic Dental Sciences, College of Dentistry, King Khalid University, Abha 61471, Asir, Saudi Arabia
| | - Khalil Assiri
- Diagnostic Dental Sciences, King Khalid University, Abha 61471, Asir, Saudi Arabia
| | | | | | - Ahmed Al-Hakami
- Center for Stem Cell Research and Department of Microbiology and Clinical Parasitology, King Khalid University, Abha 61421, Asir, Saudi Arabia
| | - Shankargouda Patil
- Maxillofacial Surgery and Diagnostic Sciences, Division of oral Pathology, Jazan 45142, Jazan, Saudi Arabia
| | - Deepa Babji
- Department of Oral Pathology and Microbiology, Maratha Mandal's NG Halgekar Institute of Dental Sciences and Research Centre, Belgaun 590 010, Karnataka, India
| | - Sachin Sarode
- Department of Oral Pathology, Y Patil Dental College and Hospital, Pune 411018, Maharashtra, India
| | - Anantharam Devaraj
- Center for Stem Cell Research and Department of Microbiology and Clinical Parasitology, King Khalid University, Abha 61421, Asir, Saudi Arabia
| | - Harish C Chandramoorthy
- Center for Stem Cell Research and Department of Microbiology and Clinical Parasitology, King Khalid University, Abha 61421, Asir, Saudi Arabia
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Yue W, Kim S, Jung HS, Lee JM, Lee S, Kim E. Differential Protein Expression in Human Dental Pulp: Comparison of Healthy, Inflamed, and Traumatic Pulp. J Clin Med 2019; 8:jcm8081234. [PMID: 31426363 PMCID: PMC6723928 DOI: 10.3390/jcm8081234] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2019] [Accepted: 08/14/2019] [Indexed: 12/19/2022] Open
Abstract
Trauma or injury to the dental pulp causes inflammation. This study compared the proteome of healthy pulp with inflamed pulp and traumatic pulp to identify the differentially expressed proteins in the diseased state. Five participants were grouped based on the pulpal status of the teeth: healthy, inflamed, or traumatic pulp. Pulp was extirpated and stored immediately in liquid nitrogen. Pulp tissues were subjected to 2-dimensional gel electrophoresis, and spot selection was performed. The selected spots were analyzed using liquid chromatography-tandem mass spectrometry and identified by correlating mass spectra to the proteomic databases. Fifteen spots showed increased expression in the inflamed and traumatic pulp. Annexin V, type II keratin, and hemoglobin levels were increased two-fold in the inflamed and traumatic pulp group and annexin V, mutant beta-actin, and hemoglobin were increased by ten-fold in the inflamed or traumatic pulp group, compared to levels in the healthy pulp group. Annexin V constituted two out of fifteen protein spots, and seemed to play a critical role in inhibiting inflammation and promoting the immune reaction. Further studies on this protein concerning its role in pulp repair are necessary to elucidate the underlying mechanisms.
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Affiliation(s)
- Wonyoung Yue
- Microscope Center, Department of Conservative Dentistry and Oral Science Research Center, College of Dentistry, Yonsei University, Seoul 03722, Korea
| | - Sunil Kim
- Microscope Center, Department of Conservative Dentistry and Oral Science Research Center, College of Dentistry, Yonsei University, Seoul 03722, Korea
| | - Han-Sung Jung
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Korea
| | - Jong-Min Lee
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Korea
| | - Sukjoon Lee
- Department of Applied Life Science, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Korea
| | - Euiseong Kim
- Microscope Center, Department of Conservative Dentistry and Oral Science Research Center, College of Dentistry, Yonsei University, Seoul 03722, Korea.
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Liao C, Ou Y, Wu Y, Zhou Y, Liang S, Wang Y. Sclerostin inhibits odontogenic differentiation of human pulp‐derived odontoblast‐like cells under mechanical stress. J Cell Physiol 2019; 234:20779-20789. [PMID: 31025337 DOI: 10.1002/jcp.28684] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2019] [Revised: 04/02/2019] [Accepted: 04/05/2019] [Indexed: 01/13/2023]
Affiliation(s)
- Chufang Liao
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) & Key Laboratory of Oral Biomedicine Ministry of Education School & Hospital of Stomatology, Wuhan University Wuhan China
| | - Yanjing Ou
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) & Key Laboratory of Oral Biomedicine Ministry of Education School & Hospital of Stomatology, Wuhan University Wuhan China
| | - Yun Wu
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) & Key Laboratory of Oral Biomedicine Ministry of Education School & Hospital of Stomatology, Wuhan University Wuhan China
| | - Yi Zhou
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) & Key Laboratory of Oral Biomedicine Ministry of Education School & Hospital of Stomatology, Wuhan University Wuhan China
- Department of Prosthodontics Hospital of Stomatology, Wuhan University Wuhan China
| | - Shanshan Liang
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) & Key Laboratory of Oral Biomedicine Ministry of Education School & Hospital of Stomatology, Wuhan University Wuhan China
- Department of Prosthodontics Hospital of Stomatology, Wuhan University Wuhan China
| | - Yining Wang
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) & Key Laboratory of Oral Biomedicine Ministry of Education School & Hospital of Stomatology, Wuhan University Wuhan China
- Department of Prosthodontics Hospital of Stomatology, Wuhan University Wuhan China
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Wang H, Ma D, Zhang X, Xu S, Ning T, Wu B. Comparative proteomic profiling of human dental pulp stem cells and periodontal ligament stem cells under in vitro osteogenic induction. Arch Oral Biol 2018; 89:9-19. [PMID: 29407636 DOI: 10.1016/j.archoralbio.2018.01.015] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2017] [Revised: 01/16/2018] [Accepted: 01/23/2018] [Indexed: 12/14/2022]
Abstract
OBJECTIVE This study aimed to compare the proteomic profiling of human dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) under in vitro osteogenic induction, which imitates the microenvironment during osteo-/odontogenesis of DPSCs and PDLSCs. DESIGN The proteomic profiles of osteoinduced DPSCs and PDLSCs from a single donor were compared using the isobaric tag for relative and absolute quantitation (iTRAQ) technique and subsequent bioinformatics analysis. RESULTS A total of 159 differentially expressed proteins in PDLSCs and DPSCs were identified, 82 of which had a higher expression level in PDLSCs, while 77 were more highly expressed in DPSCs. Among these enriched proteins, certain members from the collagen, heat shock protein and protein S100 families may distinguish osteoinduced PDLSCs and DPSCs. Gene ontology (GO) classification revealed that a large number of the enriched terms distinguishing PDLSCs and DPSCs are involved in catalytic activity, protein binding, regulation of protein metabolic processes and response to stimulus. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated several involved pathways, including the fatty acid biosynthesis pathway, pantothenate and CoA biosynthesis pathway, arachidonic acid metabolism pathway and PPAR signaling pathway. Further verification showed that the mineralization and migration capacities of PDLSCs were greater than those of DPSCs, in which heat shock protein beta-1, Protein S100-A10 and S100-A11 may play a part. CONCLUSIONS Less than 5% of the differentially expressed proteins make up the comparative proteomic profile between osteoinduced PDLSCs and DPSCs. This study helps to characterize the differences between osteoinduced PDLSCs and DPSCs in vitro.
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Affiliation(s)
- He Wang
- Department of Stomatology, Nanfang Hospital, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China; College of Stomatology, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China
| | - Dandan Ma
- Department of Stomatology, Nanfang Hospital, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China; College of Stomatology, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China
| | - Xiaoyi Zhang
- Department of Stomatology, Nanfang Hospital, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China; College of Stomatology, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China
| | - Shuaimei Xu
- Department of Endodontics and Operative Dentistry, Stomatological Hospital, Southern Medical University, No. 366 South Jiangnan Avenue, Guangzhou, 510280, China
| | - Tingting Ning
- Department of Stomatology, Nanfang Hospital, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China; College of Stomatology, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China
| | - Buling Wu
- Department of Stomatology, Nanfang Hospital, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China; College of Stomatology, Southern Medical University, No. 1838 North Guangzhou Avenue, Guangzhou, 510515, China.
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Li J, Tian W, Song J. Proteomics Applications in Dental Derived Stem Cells. J Cell Physiol 2017; 232:1602-1610. [PMID: 27791269 DOI: 10.1002/jcp.25667] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2016] [Accepted: 10/26/2016] [Indexed: 02/05/2023]
Affiliation(s)
- Jie Li
- College of Stomatology; Chongqing Medical University; Chongqing China
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences; Chongqing China
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education; Chongqing China
| | - Weidong Tian
- National Engineering Laboratory for Oral Regenerative Medicine; West China Hospital of Stomatology; Sichuan University; Chengdu China
| | - Jinlin Song
- College of Stomatology; Chongqing Medical University; Chongqing China
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences; Chongqing China
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education; Chongqing China
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Zhang X, Du Y, Ling J, Li W, Liao Y, Wei X. Dickkopf-related protein 3 negatively regulates the osteogenic differentiation of rat dental follicle cells. Mol Med Rep 2017; 15:1673-1681. [PMID: 28259940 PMCID: PMC5364975 DOI: 10.3892/mmr.2017.6165] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2015] [Accepted: 12/15/2016] [Indexed: 01/05/2023] Open
Abstract
The present study aimed to investigate the effect of Dickkopf-related protein 3 (DKK3) on osteogenic differentiation of rat dental follicle cells (DFCs). A PCR array analysis of Wnt pathway activation in DFCs identified genes dysregulated by mineral induction. Among them, DKK3expression levels were decreased, and further experiments were conducted to investigate its role in DFC osteogenesis. By comparing DFCs grown in normal growth and mineral-induction media for 4 weeks, the present study confirmed that DKK3 was a potential target gene of osteogenesis through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB). A short hairpin RNA (shRNA) was introduced into DFCs using a lentiviral vector to inhibit DKK3 expression. An alkaline phosphatase (ALP) activity assay and Alizarin Red staining were performed to observe the DKK3-shRNA DFCs. In addition, the osteogenic differentiation of DKK3-shRNA DFCs was analyzed by RT-qPCR and WB. In vivo, DKK3-shRNA DFCs seeded on hydroxyapatite/β-tricalcium phosphate (HA/TCP) scaffolds were transplanted into the subcutaneous tissue of mice with severe combined immunodeficiency, followed by hematoxylin-eosin and Masson staining. The results confirmed that DKK3 expression was downregulated during mineral induction in rat DFCs. Lentivirus-mediated expression of DKK3 shRNA in DFCs promoted calcified-nodule formation, ALP activity and the expression of β-catenin, runt-related transcription factor 2 and osteocalcin, compared with control cells. In vivo, the implanted section presented the majority of newly formed osteoid matrices and collagen, with limited space between the HA/TCP scaffolds and matrices. In conclusion, DKK3 expression negatively regulates the osteogenic differentiation of DFCs and, conversely, downregulation of DKK3 may enhance DFC osteogenesis.
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Affiliation(s)
- Xinchun Zhang
- Department of Prosthodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Yu Du
- Department of Operative Dentistry and Endodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Junqi Ling
- Department of Operative Dentistry and Endodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Weiqiang Li
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China
| | - Yan Liao
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China
| | - Xi Wei
- Department of Operative Dentistry and Endodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
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Karamzadeh R, Baghaban Eslaminejad M, Sharifi-Zarchi A. Comparative In Vitro Evaluation of Human Dental Pulp and Follicle Stem Cell Commitment. CELL JOURNAL 2016; 18:609-618. [PMID: 28042545 PMCID: PMC5086339 DOI: 10.22074/cellj.2016.4727] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Accepted: 02/22/2016] [Indexed: 01/09/2023]
Abstract
Objective Pulp and periodontal tissues are well-known sources of mesenchymal stem cells
(MSCs) that provide a promising place in tissue engineering and regenerative medicine. The
molecular mechanisms underlying commitment and differentiation of dental stem cells that originate from different dental tissues are not fully understood. In this study, we have compared the
expression levels of pluripotency factors along with immunological and developmentally-related
markers in the culture of human dental pulp stem cells (hDPSCs), human dental follicle stem
cells (hDFSCs), and human embryonic stem cells (hESCs).
Materials and Methods In this experimental study, isolated human dental stem cells
were investigated using quantitative polymerase chain reaction (qPCR), immunostaining,
and fluorescence-activated cell sorting (FACS). Additionally, we conducted gene ontology
(GO) analysis of differentially expressed genes and compared them between dental stem
cells and pluripotent stem cells.
Results The results demonstrated that pluripotency (OCT4 and SOX2) and immunological
(IL-6 and TLR4) factors had higher expressions in hDFSCs, with the exception of the JAGGED-1/NOTCH1 ratio, c-MYC and NESTIN which expressed more in hDPSCs. Immunostaining of
OCT4, SOX2 and c-MYC showed cytoplasmic and nucleus localization in both groups at
similar passages. GO analysis showed that the majority of hDFSCs and hDPSCs populations
were in the synthesis (S) and mitosis (M) phases of the cell cycle, respectively.
Conclusion This study showed different status of heterogeneous hDPSCs and hDFSCs
in terms of stemness, differentiation fate, and cell cycle phases. Therefore, the different
behaviors of dental stem cells should be considered based on clinical treatment variations.
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Affiliation(s)
- Razieh Karamzadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Mohamadreza Baghaban Eslaminejad
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Ali Sharifi-Zarchi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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Uzawa K, Kasamatsu A, Saito T, Takahara T, Minakawa Y, Koike K, Yamatoji M, Nakashima D, Higo M, Sakamoto Y, Shiiba M, Tanzawa H. Long-term culture of human odontoma-derived cells with a Rho kinase inhibitor. Exp Cell Res 2016; 347:232-240. [PMID: 27514999 DOI: 10.1016/j.yexcr.2016.08.005] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Revised: 07/27/2016] [Accepted: 08/07/2016] [Indexed: 10/21/2022]
Abstract
Because of cellular senescence/apoptosis, no effective culture systems are available to maintain replication of cells from odontogenic tumors especially for odontoma, and, thus, the ability to isolate human odontoma-derived cells (hODCs) for functional studies is needed. The current study was undertaken to develop an approach to isolate hODCs and fully characterize the cells in vitro. The hODCs were cultured successfully with a Rho-associated protein kinase inhibitor (Y-27632) for an extended period with stabilized lengths of the telomeres to sustain a similar phenotype/property as the primary tumoral cells. While the hODCs showed stable long-term expansion with expression of major dental epithelial markers including dentin sialophosphoprotein (DSPP) even in the three-dimensional microenvironment, they lack the specific markers for the characteristics of stem cells. Moreover, cells from dental pulp showed significant up-regulation of DSPP when co-cultured with the hODCs, while control fibroblasts with the hODCs did not. Taken together, we propose that the hODCs can be isolated and expanded over the long term with Y-27632 to investigate not only the development of the hODCs but also other types of benign human tumors.
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Affiliation(s)
- Katsuhiro Uzawa
- Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8670, Japan; Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan.
| | - Atsushi Kasamatsu
- Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan
| | - Tomoaki Saito
- Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8670, Japan
| | - Toshikazu Takahara
- Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8670, Japan
| | - Yasuyuki Minakawa
- Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8670, Japan
| | - Kazuyuki Koike
- Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan
| | - Masanobu Yamatoji
- Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan
| | - Dai Nakashima
- Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan
| | - Morihiro Higo
- Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan
| | - Yosuke Sakamoto
- Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan
| | - Masashi Shiiba
- Department of Medical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8670, Japan
| | - Hideki Tanzawa
- Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8670, Japan; Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba 260-8677, Japan
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Proteomic analysis of human tooth pulp proteomes – Comparison of caries-resistant and caries-susceptible persons. J Proteomics 2016; 145:127-136. [DOI: 10.1016/j.jprot.2016.04.022] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2015] [Revised: 04/08/2016] [Accepted: 04/17/2016] [Indexed: 01/13/2023]
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13
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Chen C, Xie N, Ling J, Du Y, Gu H. Proteomic analysis of the effects of CSF-1 and IL-1α on dental follicle cells. Mol Med Rep 2016; 14:2405-14. [PMID: 27484316 DOI: 10.3892/mmr.2016.5567] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2015] [Accepted: 05/23/2016] [Indexed: 11/06/2022] Open
Abstract
Tooth eruption is a complex physiological process involving both osteogenesis and bone resorption. Signals from the dental follicle (DF) regulate bone remodeling during tooth eruption. Interleukin-1α (IL-1α) may be the initial promoter of tooth eruption, whereas colony‑stimulating factor‑1 (CSF‑1) may attract monocytes into the DF and stimulate osteoclast differentiation. In the present study, differential proteomics was employed to explore protein changes in rat DF cells (DFCs) under the effects of CSF‑1 and IL‑1α. A total of 47 protein spots were differentially expressed in rat DFCs, and 40 protein spots were identified by MALDI‑TOF‑MS. The identified proteins were grouped into functional categories including cytoskeletal proteins, metal‑binding proteins, proteins involved in secretion and degradation, cell cycle proteins and stress proteins. In IL‑1α‑induced rat DFCs, 31 proteins were upregulated compared with the control and included heat shock protein β‑1 (HSP25, also known as HSP27/HSPβ1), vimentin, TMEM43, the GTP‑binding protein Rab‑3D, 6‑pyruvoyl tetrahydrobiopterin synthase and actin. In total, 7 proteins were downregulated, including serum albumin, GIPC1, DNA primase large subunit, cullin‑5 and cyclin‑G1. In CSF‑1‑induced rat DFCs, 3 proteins were upregulated and 7 proteins were downregulated when compared with the controls. The upregulated proteins included the GTP‑binding protein Rab‑3D and α‑actin. The downregulated proteins included cullin‑5, serum albumin, PDZ domain‑containing protein and cyclin‑G1. The differential expression of vimentin, actin, HSP25 and Rab‑3D was verified by western blotting and reverse transcription‑quantitative polymerase chain reaction analyses. The present findings provide an insight into the mechanisms involved in tooth eruption.
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Affiliation(s)
- Chanchan Chen
- Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Research Institute of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Nan Xie
- Department of Oral Pathology, Guanghua School of Stomatology, Research Institute of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Junqi Ling
- Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Research Institute of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Yu Du
- Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Research Institute of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Haijing Gu
- Department of Pediatric Dentistry, Guanghua School of Stomatology, Research Institute of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
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14
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Advances of Proteomic Sciences in Dentistry. Int J Mol Sci 2016; 17:ijms17050728. [PMID: 27187379 PMCID: PMC4881550 DOI: 10.3390/ijms17050728] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2016] [Revised: 05/01/2016] [Accepted: 05/09/2016] [Indexed: 12/13/2022] Open
Abstract
Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed.
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15
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Li X, Jiang H, Huang Y, Gong Q, Wang J, Ling J. Expression and Function of the Actin-severing Protein Adseverin in the Proliferation, Migration, and Differentiation of Dental Pulp Cells. J Endod 2015; 41:493-500. [DOI: 10.1016/j.joen.2014.11.030] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2014] [Revised: 11/29/2014] [Accepted: 11/30/2014] [Indexed: 12/18/2022]
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16
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Huang Y, Jiang H, Gong Q, Li X, Ling J. Lipopolysaccharide stimulation improves the odontoblastic differentiation of human dental pulp cells. Mol Med Rep 2014; 11:3547-52. [PMID: 25528991 DOI: 10.3892/mmr.2014.3120] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Accepted: 11/20/2014] [Indexed: 11/05/2022] Open
Abstract
Lipopolysaccharide (LPS) is one of the causative agents of pulpitis and previous studies have demonstrated that the LPS stimulation of human aortic valve interstitial cells induces inflammatory mediators and the gene expression of osteogenic factors. Therefore, in the present study, it was hypothesized that LPS affects the odontoblastic differentiation of human dental pulp cells (hDPCs). In order to investigate this, an in vitro study using hDPCs was performed. Increased alkaline phosphatase (ALP) activity was observed in the hDPCs treated with LPS, which was more marked when the cells were costimulated with odontogenic induction medium (OM). LPS also appeared to increase the gene expression levels of dentin sialophosphoprotein and dentin matrix protein‑1 and the protein expression level of dental sialoprotein in the hDPCs, particularly in combination with OM. In addition, the size and the number of nodules formed in the hDPCs exposed to OM and LPS were increased compared to those stimulated by OM alone. To determine the role of nuclear factor κB (NF‑κB) during the LPS‑induced odontoblastic differentiation of hDPCs, immunofluorescence was performed. The nuclear translocation of NF‑κB, induced by LPS was confirmed, suggesting its involvement in the LPS‑induced increase in odontoblastic differentiation of hDPCs. In conclusion, there may be an association between LPS stimulation, with or without OM, and odontoblastic differentiation.
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Affiliation(s)
- Yihua Huang
- Guangdong Provincial Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Hongwei Jiang
- Guangdong Provincial Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Qimei Gong
- Guangdong Provincial Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Xuyan Li
- Guangdong Provincial Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
| | - Junqi Ling
- Guangdong Provincial Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
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17
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Eckhardt A, Jágr M, Pataridis S, Mikšík I. Proteomic Analysis of Human Tooth Pulp: Proteomics of Human Tooth. J Endod 2014; 40:1961-6. [DOI: 10.1016/j.joen.2014.07.001] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2014] [Revised: 06/25/2014] [Accepted: 07/03/2014] [Indexed: 01/17/2023]
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18
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Liu HJ, Wang T, Li QM, Guan XY, Xu Q. Knock-down of p300 decreases the proliferation and odontogenic differentiation potentiality of HDPCs. Int Endod J 2014; 48:976-85. [PMID: 25288362 DOI: 10.1111/iej.12392] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2014] [Accepted: 10/02/2014] [Indexed: 01/17/2023]
Abstract
AIM To investigate the role of p300 in the regulation of proliferation and odontogenic differentiation of human dental pulp cells (HDPCs). METHODOLOGY The recombinant lentiviral vector pshRNA-copGFP was used to knock-down p300 expression in HDPCs. Protein level of acetylated H3 was detected. The proliferation of HDPCs was measured using the CCK8 assay. The cell cycle and apoptosis were analysed using flow cytometry and TUNEL staining, respectively. The expression levels of Cdc25A, p21(waf1) and the cleaved products of caspase 3 and caspase 7 were determined utilizing real-time quantitative polymerase chain reaction and Western blotting analysis. The alkaline phosphatase (ALP) activity was measured, and the formation of mineralized nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression levels of the odontogenic differentiation markers DMP-1, DSPP and DSP were detected utilizing real-time quantitative polymerase chain reaction and Western blotting analysis. RESULTS After p300 was knocked down in HDPCs, p300 was significantly down-regulated at both the mRNA and protein levels, and histone H3 acetylation was reduced. The proliferation capacity of HDPCs was suppressed in p300 knock-down groups. The cells were arrested in the G0/G1 phase of the cell cycle, and cell apoptosis was triggered. ALP activity, the formation of mineralized nodules and the expression levels of DMP-1, DSPP and DSP were all decreased in p300-knock-down HDPCs undergoing odontogenic differentiation. CONCLUSION Knocking down p300 restrains the proliferation and odontogenic differentiation potentiality of HDPCs.
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Affiliation(s)
- H J Liu
- Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China
| | - T Wang
- Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China
| | - Q M Li
- Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China
| | - X Y Guan
- Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China
| | - Q Xu
- Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China
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19
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Zhang W, Zhang X, Ling J, Liu W, Zhang X, Ma J, Zheng J. Proliferation and odontogenic differentiation of BMP2 gene‑transfected stem cells from human tooth apical papilla: an in vitro study. Int J Mol Med 2014; 34:1004-12. [PMID: 25070743 PMCID: PMC4152145 DOI: 10.3892/ijmm.2014.1862] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2014] [Accepted: 07/09/2014] [Indexed: 01/09/2023] Open
Abstract
Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue-derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral-mediated BMP2 gene-transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector-transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK-8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: i) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ii) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; iii) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro.
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Affiliation(s)
- Wen Zhang
- Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology and Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510080, P.R. China
| | - Xiaolei Zhang
- Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology and Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510080, P.R. China
| | - Junqi Ling
- Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology and Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510080, P.R. China
| | - Wei Liu
- Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology and Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510080, P.R. China
| | - Xinchun Zhang
- Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology and Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510080, P.R. China
| | - Jinglei Ma
- Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology and Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510080, P.R. China
| | - Jianmao Zheng
- Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology and Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510080, P.R. China
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JÁGR M, ECKHARDT A, PATARIDIS S, BROUKAL Z, DUŠKOVÁ J, MIKŠÍK I. Proteomics of Human Teeth and Saliva. Physiol Res 2014; 63:S141-54. [DOI: 10.33549/physiolres.932702] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Teeth have been a focus of interest for many centuries – due to medical problems with them. They are the hardest part of the human body and are composed of three mineralized parts – enamel, dentin and cementum, together with the soft pulp. However, saliva also has a significant impact on tooth quality. Proteomic research of human teeth is now accelerating, and it includes all parts of the tooth. Some methodological problems still need to be overcome in this research field – mainly connected with calcified tissues. This review will provide an overview of the current state of research with focus on the individual parts of the tooth and pellicle layer as well as saliva. These proteomic results can help not only stomatology in terms of early diagnosis, identifying risk factors, and systematic control.
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Affiliation(s)
| | | | | | | | | | - I. MIKŠÍK
- Department of Analysis of Biologically Important Compounds, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic
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21
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Rezende TMB, Lima SMF, Petriz BA, Silva ON, Freire MS, Franco OL. Dentistry proteomics: From laboratory development to clinical practice. J Cell Physiol 2013; 228:2271-84. [DOI: 10.1002/jcp.24410] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2013] [Accepted: 05/21/2013] [Indexed: 12/12/2022]
Affiliation(s)
- Taia M. B. Rezende
- Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
- Curso de Odontologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
| | - Stella M. F. Lima
- Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
- Curso de Odontologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
| | - Bernardo A. Petriz
- Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
| | - Osmar N. Silva
- Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
| | - Mirna S. Freire
- Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
| | - Octávio L. Franco
- Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia; Universidade Católica de Brasília; Brasília Distrito Federal Brazil
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The Effect of Matrix Extracellular Phosphoglycoprotein and Its Downstream Osteogenesis-related Gene Expression on the Proliferation and Differentiation of Human Dental Pulp Cells. J Endod 2012; 38:330-8. [DOI: 10.1016/j.joen.2011.10.015] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2011] [Revised: 10/14/2011] [Accepted: 10/16/2011] [Indexed: 12/28/2022]
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Xiao Y, Chen J. Proteomics approaches in the identification of molecular signatures of mesenchymal stem cells. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2012; 129:153-76. [PMID: 22790357 DOI: 10.1007/10_2012_143] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Mesenchymal stem cells (MSCs) are undifferentiated, multi-potent stem cells with the ability to renew. They can differentiate into many types of terminal cells, such as osteoblasts, chondrocytes, adipocytes, myocytes, and neurons. These cells have been applied in tissue engineering as the main cell type to regenerate new tissues. However, a number of issues remain concerning the use of MSCs, such as cell surface markers, the determining factors responsible for their differentiation to terminal cells, and the mechanisms whereby growth factors stimulate MSCs. In this chapter, we will discuss how proteomic techniques have contributed to our current knowledge and how they can be used to address issues currently facing MSC research. The application of proteomics has led to the identification of a special pattern of cell surface protein expression of MSCs. The technique has also contributed to the study of a regulatory network of MSC differentiation to terminal differentiated cells, including osteocytes, chondrocytes, adipocytes, neurons, cardiomyocytes, hepatocytes, and pancreatic islet cells. It has also helped elucidate mechanisms for growth factor-stimulated differentiation of MSCs. Proteomics can, however, not reveal the accurate role of a special pathway and must therefore be combined with other approaches for this purpose. A new generation of proteomic techniques have recently been developed, which will enable a more comprehensive study of MSCs.
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Affiliation(s)
- Yin Xiao
- Institute of Health and Biomedical Innovation Queensland University of Technology, 60 Musk Avenue, Kelvin Grove Brisbane, QLD, 4059, Australia,
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24
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Chen C, Wei X, Ling J, Xie N. Expression of matrilin-2 and -4 in human dental pulps during dentin-pulp complex wound healing. J Endod 2011; 37:642-9. [PMID: 21496664 DOI: 10.1016/j.joen.2011.02.018] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2010] [Revised: 02/01/2011] [Accepted: 02/03/2011] [Indexed: 01/09/2023]
Abstract
INTRODUCTION Matrilin-2 and matrilin-4 are members of the matrilin family displaying broad tissue distribution. We recently reported that matrilin-2 showed significant down-regulation during the odontogenic differentiation of dental pulp cells (DPCs). It is reported that matrilin-4 was the only extracellular matrix biogenesis and organization-related gene detected in odontoblasts but not DPCs. However, the exact role of matrlin-2 and -4 in dental pulps remains unclear. The aim of our study was to analyze the expression of matrilin-2 and -4 in human dental pulps and their relation to dentin-pulp complex wound healing. METHODS Immunohistology was performed on the paraffin-embedded tissue sections of human dental pulps from sound and deep carious teeth. Matrilin-2 and -4 messenger RNAs were detected by quantitative real-time reverse-transcription polymerase chain reaction, and the proteins were shown by immunofluorescence and Western blot during odontogenic differentiation of the DPCs. RESULTS In the sound dental pulp, matrilin-2 immunoreactivity was observed throughout the pulp, whereas matrilin-4 was observed only in the odontoblast layer. In deep carious dental pulp, matrilin-2 protein was weakly stained, whereas matrilin-4 was detected in the pulp under the carious lesion. During odontogenic differentiation of DPCs, the expression of matrilin-2 messenger RNA was down-regulated within 14 days followed by a statistical increase on day 21, and the matrilin-2 protein level was down-regulated within the 3 weeks, whereas the messenger RNA and protein expressions of matrilin-4 increased in a time-dependent manner. CONCLUSIONS Matrilin-2 and matrilin-4 have been shown in human dental pulps and might be involved in the dentin-pulp complex wound-healing process.
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Affiliation(s)
- Chanchan Chen
- Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China
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Expression pattern of Oct-4, Sox2, and c-Myc in the primary culture of human dental pulp derived cells. J Endod 2011; 37:466-72. [PMID: 21419292 DOI: 10.1016/j.joen.2010.12.012] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2010] [Revised: 12/16/2010] [Accepted: 12/20/2010] [Indexed: 01/09/2023]
Abstract
Dental pulp cells (DPCs) have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. Therefore, it is interesting to know whether the replicative senescence during the culture of dental pulp cells is related to the diminishing of the expression of these transcription factors. In this study, we investigated the expression of the reprogramming markers Oct-4, Sox2, and c-Myc in the in vitro explant cultured dental pulp tissues and explant cultured dental pulp cells (DPCs) at various passages by immunofluorescence staining and real-time polymerase chain reaction analysis. Our results demonstrated that Oct-4, Sox2, and c-Myc translocated from nucleus in the first 2 passages to cytoplasm after the third passage in explant cultured DPCs. The mRNA expression of Oct-4, Sox2, and c-Myc elevated significantly over the first 2 passages, peaked at second passage (P < .05), and then decreased along the number of passages afterwards (P < .05). For the first time we demonstrated that the expression of reprogramming markers Oct-4, Sox2, and c-Myc was detectable in the early passaged DPCs, and the sequential loss of these markers in the nucleus during DPC cultures might be related to the cell fate of dental pulp derived cells during the long-term in vitro cultivation under current culture conditions.
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Abstract
Ectomesenchymal dental stem cells could be feasible tools for dental tissue engineering. Dental follicle cells are a promising example, since they are capable of differentiation into various dental tissue cells, such as osteoblasts or cementoblasts. However, cellular mechanisms of cell proliferation and differentiation are not understood in detail. Basic knowledge of these molecular processes may shorten the time before ectomesenchymal dental stem cells can be exploited for bone augmentation in regenerative medicine. Recent developments in proteomics and transcriptomics have made information about genome-wide expression profiles accessible, which can aid in clarifying molecular mechanisms of cells. This review describes the transcriptomes and proteomes of dental follicle cells before and after differentiation, and compares them with differentially expressed populations from dental tissue or bone marrow.
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Affiliation(s)
- C Morsczeck
- Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
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Liu L, Ling J, Wei X, Wu L, Xiao Y. Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation. J Endod 2009; 35:1368-76. [PMID: 19801232 DOI: 10.1016/j.joen.2009.07.005] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2009] [Revised: 07/02/2009] [Accepted: 07/03/2009] [Indexed: 12/28/2022]
Abstract
INTRODUCTION During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. METHODS In this study, we investigated the differential expression of 84 stem cell-related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. RESULTS Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. CONCLUSIONS This study has generated an overview of stem cell-related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-beta/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.
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Affiliation(s)
- Lu Liu
- Department of Operative Dentistry, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China
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