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      Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro

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          Abstract

          Introduction

          Maintaining or regenerating a vital pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell aggregates (spheres) were applied onto bovine and human root canal models to evaluate their potential use as pre-differentiated tissue units for dental pulp tissue regeneration.

          Methods

          Human dental pulp cells (DPC) were derived from wisdom teeth, cultivated into three-dimensional cell spheres and seeded onto bovine and into human root canals. Sphere formation, tissue-like and mineralization properties as well as growth behavior of cells on dentin structure were evaluated by light microscopy (LM), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX).

          Results

          Spheres and outgrown cells showed tissue-like properties, the ability to merge with other cell spheres and extra cellular matrix formation; CLSM investigation revealed a dense network of actin and focal adhesion contacts (FAC) inside the spheres and a pronounced actin structure of cells outgrown from the spheres. A dentin-structure-orientated migration of the cells was shown by SEM investigation. Besides the direct extension of the cells into dentinal tubules, the coverage of the tubular walls with cell matrix was detected. Moreover, an emulation of dentin-like structures with tubuli-like and biomineral formation was detected by SEM- and EDX-investigation.

          Conclusions

          The results of the present study show tissue-like behavior, the replication of tubular structures and the mineralization of human dental pulp spheres when colonized on root dentin. The application of cells in form of pulp spheres on root dentin reveals their beneficial potential for dental tissue regeneration.

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          Most cited references36

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          Stem cell properties of human dental pulp stem cells.

          In this study, we characterized the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of human dental pulp stem cells (DPSCs). DPSCs were capable of forming ectopic dentin and associated pulp tissue in vivo. Stromal-like cells were reestablished in culture from primary DPSC transplants and re-transplanted into immunocompromised mice to generate a dentin-pulp-like tissue, demonstrating their self-renewal capability. DPSCs were also found to be capable of differentiating into adipocytes and neural-like cells. The odontogenic potential of 12 individual single-colony-derived DPSC strains was determined. Two-thirds of the single-colony-derived DPSC strains generated abundant ectopic dentin in vivo, while only a limited amount of dentin was detected in the remaining one-third. These results indicate that single-colony-derived DPSC strains differ from each other with respect to their rate of odontogenesis. Taken together, these results demonstrate that DPSCs possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation.
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            Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP).

            The aim of this study was to compare the in vitro osteo/odontogenic differentiation potential of mesenchymal stem cells (MSCs) derived from the dental pulp (dental pulp stem cells - DPSCs) or the apical papilla (stem cells from the apical papilla - SCAP) of permanent developing teeth. DPSCs and SCAP cultures were established from impacted third molars of young healthy donors at the stage of root development. Cultures were analysed for stem cell markers, including STRO-1, CD146, CD34 and CD45 using flow cytometry. Cells were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4) and β-glycerophosphate. Cultures were analysed for morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers (dentine sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN, alkaline phosphatase-ALP), using immunocytochemistry and reverse transcriptase-polymerase chain reaction. All DPSCs and SCAP cultures were positive for STRO-1, CD146 and CD34, in percentages varying according to cell type and donor, but negative for CD45. Both types of MSCs displayed an active potential for cellular migration, organization and mineralization, producing 3D mineralized structures. These structures progressively expressed differentiation markers, including DSPP, BSP, OCN, ALP, having the characteristics of osteodentin. SCAP, however, showed a significantly higher proliferation rate and mineralization potential, which might be of significance for their use in bone/dental tissue engineering. This study provides evidence that different types of dental MSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source for osteo/odontogenic differentiation and biomineralization that could be further applied for stem cell-based clinical therapies. Copyright © 2010 Elsevier Ltd. All rights reserved.
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              The application of tissue engineering to regeneration of pulp and dentin in endodontics.

              Caries, pulpitis, and apical periodontitis increase health care costs and attendant loss of economic productivity. They ultimately result in premature tooth loss and therefore diminishing the quality of life. Advances in vital pulp therapy with pulp stem/progenitor cells might give impetus to regenerate dentin-pulp complex without the removal of the whole pulp. Tissue engineering is the science of design and manufacture of new tissues to replace lost parts because of diseases including cancer and trauma. The three key ingredients for tissue engineering are signals for morphogenesis, stem cells for responding to morphogens and the scaffold of extracellular matrix. In preclinical studies cell therapy and gene therapy have been developed for many tissues and organs such as bone, heart, liver, and kidney as a means of delivering growth factors, cytokines, or morphogens with stem/progenitor cells in a scaffold to the sites of tissue injury to accelerate and/or induce a natural biological regeneration. The pulp tissue contains stem/progenitor cells that potentially differentiate into odontoblasts in response to bone morphogenetic proteins (BMPs). There are two strategies to regenerate dentin. First, is in vivo therapy, where BMP proteins or BMP genes are directly applied to the exposed or amputated pulp. Second is ex vivo therapy and consists of isolation of stem/progenitor cells from pulp tissue, differentiation into odontoblasts with recombinant BMPs or BMP genes and finally transplanted autogenously to regenerate dentin. This review is focused on the recent progress in this area and discusses the barriers and challenges for clinical utility in endodontics.
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                Author and article information

                Contributors
                Journal
                Head Face Med
                Head Face Med
                Head & Face Medicine
                BioMed Central
                1746-160X
                2014
                20 June 2014
                : 10
                : 25
                Affiliations
                [1 ]Technische Universität Dresden, Institute of Material Science, Chair for Biomaterials, Budapester Strasse 27, D-01069 Dresden, Germany
                [2 ]Department of Restorative and Pediatric Dentistry, University Hospital Carl Gustav Carus, Fetscherstrasse 74, D-01307 Dresden, Germany
                [3 ]Department of Oral and Maxillofacial Surgery, University Hospital Carl Gustav Carus, Fetscherstrasse 74, D-01307 Dresden, Germany
                Article
                1746-160X-10-25
                10.1186/1746-160X-10-25
                4074584
                24946771
                f5de0740-88b2-4809-b83a-e1ca83df027c
                Copyright © 2014 Neunzehn et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 6 April 2014
                : 12 June 2014
                Categories
                Research

                Orthopedics
                biomineralization,dental pulp cells,tissue formation,pulp spheres,pulp tissue regeneration

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