Periodontology 2000, Vol. 67, 2015, 251–267 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

PERIODONTOLOGY 2000

Mesenchymal stem cells from the oral cavity and their potential value in tissue engineering  & A L E J A N D R A P. C H A P A R R O A N T O N I O R. S A N Z , F L A V I O S. C A R R I ON

Periodontal disease is one of the most common conditions affecting humans, and the prevalence of advanced periodontitis in adults is about 15% (49). Periodontitis is a chronic inflammatory condition of the supporting tissues of the teeth, resulting in destruction of the attachment of the tooth to the surrounding bone. Untreated periodontitis may eventually lead to tooth loss. Fortunately, research has provided evidence that most types of periodontal disease can be successfully treated (63–110). Current treatment strategies focus on the removal of dental plaque and the long-term control of dental plaque accumulation, and these treatment strategies are generally successful in eliminating active disease and in promoting tissue repair. However, the complete regeneration of periodontal attachment lost to periodontal disease remains an elusive goal and a challenge (3); according to a position paper published by the American Academy of Periodontology in 2005 (3), the formation of new bone and cementum with supportive periodontal ligament is the ultimate objective that current periodontal-regeneration therapies are incapable of fulfilling. Regeneration is defined as the reproduction or reconstitution of a lost or injured part of the body, in such a way that the architecture and function of the tissues are completely restored (3). Thus, the aim of regenerative periodontal therapy is to restore the structure and function of the periodontium, which means regeneration of the supporting tissues, including alveolar bone, periodontal ligament and cementum, over a previously diseased root surface. Despite evidence that some regeneration can occur after therapy (3, 12, 20), this regeneration is usually only partial, in part because of the complexity of the biological events involved, such as signals for the synthesis of growth factors and the recruitment of specific cells for periodontal regeneration. Currently, bone grafts and

guided tissue regeneration are the two techniques for which vast histological documentation of periodontal regeneration is available (12, 76, 87). The periodontium is a highly complex organ consisting of epithelial tissue and soft and mineralized connective tissues, including gingiva, periodontal ligament, cementum and alveolar bone. The unique anatomy and composition of the periodontium makes periodontal wound healing a complex process because of the requirement for interaction of these three different tissues. Wound healing after conventional periodontal therapy, including surgical debridement, generally results in repair by the production of collagenous scar tissue accompanied by apical migration of gingival epithelium (11, 52, 83). In order for periodontal regeneration to occur, progenitor periodontal ligament cells must migrate to the denuded root surface, attach to it, proliferate and mature into an organized and functional fibrous attachment apparatus that inserts into a newly formed cementum. Similarly, progenitor bone cells must migrate, proliferate and mature in conjunction with the regenerating periodontal ligament (12, 58, 103). However, most of the current regenerative procedures, used either alone or in combination, have limitations in attaining complete regeneration, especially in deep periodontal defects (84, 87, 94, 106, 112). Wound healing, or the regenerative process of a specific tissue, requires a combination of fundamental events, such as appropriate levels and sequencing of regulatory signaling pathways, presence and number of progenitor cells responding to biological signals, appropriate extracellular matrix or carrier, and adequate blood supply (11, 52, 80). Based on tissue-engineering concepts, the healing/regeneration process of a tissue may be manipulated at one of the following points: regulation of molecules, extracellular matrix or scaffold, and cellular availability (50, 80, 84; Fig. 1).

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Following the identification of mesenchymal stem cells in bone marrow by Friedenstein et al. (34, 35), a new era in regenerative medicine began, and tissue engineering using mesenchymal stem cells has become a most interesting therapeutic option (6, 13, 82, 93, 105, 111), with several advantages, such as high-quality regeneration of damaged tissues without formation of a fibrous scar, minimal donor-site morbidity compared with autografts, and low risk of autoimmune rejection and disease transmission (17, 62, 68, 73, 91, 92, 98). The aim of this article was to describe the main sources of mesenchymal stem cells from tissues of the oral cavity and their potential in regenerative therapy. Special attention is placed on gingival tissue-derived mesenchymal stem cells because this is the most accessible source for stem cells in the oral cavity (68, 116).

Mesenchymal stem cells Adult stem cells, also known as somatic stem cells, are undifferentiated cells (found in small numbers in most adult tissues) that have the capacity to differentiate and expand into mature cells with specific functions (100, 108). The primary roles of adult stem cells are to maintain and repair tissues in which the cells are found, as well as to maintain the stem cell population. Stem cells can be isolated from tissues such as bone marrow, skeletal muscle, cartilage, dental organ, adipose tissue, synovium and cardiac tissue (61). The most extensively studied source of stem cells is the bone marrow, which contains hematopoietic stem cells and nonhematopoietic cells, referred to as mesenchymal stem cells (16, 40, 55). First identified in

1966 within the stromal compartment of bone marrow (34–36), mesenchymal stem cells include a unique population of multipotential cells that exhibit extensive proliferative ability and can differentiate along multiple tissue-specific lineages, such as osteoblasts, chondrocytes and adipocytes (78, 96). At present, there is no definitive marker for identifying mesenchymal stem cells, suggesting that the cell populations derived by the current methods are, in fact, heterogeneous. However, to define mesenchymal stem cells phenotypically, minimal criteria have been proposed by the International Society for Cellular Therapy. First, they must be plastic-adherent when maintained in standard culture conditions; second, they must express the surface markers CD73, CD90 and CD105 but not CD45, CD34, CD14 or CD11b, CD79a or CD19 and major histocompatibility complex class II surface molecules; and, third, they must retain the capacity to differentiate into osteoblasts, adipocytes and chondroblasts under standard in-vitro conditions (29). Another important characteristic of these cells is their fibroblast-like spindle shape in culture (9, 81). The presence of adult progenitor populations within various tissues, and their ability to adopt tissue-specific phenotypes, given the appropriate differentiation conditions, has led many investigators to suggest that the primary role of mesenchymal stem cells is to serve as cell replacement during the natural course of tissue turnover and homeostasis (14). In addition, mesenchymal stem cells may serve other important therapeutic roles because they appear to escape immune recognition, and exert anti-inflammatory and immune-modulatory effects via the suppression of T-, B-, natural killer and antigenpresenting cells, both in vitro and in vivo (102, 104). The immune-modulatory properties of these cells, and the ability to isolate and expand them in vitro without loss of their phenotype or multilineage potential, have generated great interest in using mesenchymal stem cells as a therapeutic modality for immunemediated diseases and tissue repair (27, 28, 100).

Mesenchymal stem cells from the oral cavity

Fig. 1. Levels of tissue engineering: molecules, scaffolds and cells.

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The use of stem cells for tissue engineering has great potential to solve clinical and surgical problems related to tissue loss and organ functional failures. Dental tissue-derived mesenchymal stem cell-like populations reside in specialized well-characterized tissues and may be used in regenerative medicine. The first type of dental stem cell was isolated from

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research group has corroborated those findings, as seen in Fig. 4A,B. Dental pulp stem cells, when seeded in a tridimensional scaffold of dentin, appear as odontoblast cells with prolongations of the cellular body into the structure of the dentin (44, 45). Their plasticity toward any specific cell lineage is defined by the components of the local microenvironment, such as growth factors, receptor molecules, signaling molecules, transcription factors and extracellular matrix protein. Dental pulp stem cells can differentiate into a diverse range of lineages, including odontoblasts, osteoblasts, chondrocytes, myocytes, neurocytes,

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Fig. 3. Flow cytometry and morphology of dental pulp stem cells. (A) Flow cytometry analysis of dental pulp stem cells. Histograms show passage 3 cells after staining with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies to the indicated cell-surface proteins (red histograms). (B, C) Morphology of dental pulp stem cells. Optical contrast phase microscopy shows (B) fibroblastoid cells adherent to plastic (103 magnification; scale bar = 200 lm) and (C) the fibroblast-like spindle shape of dental pulp stem cells (403 magnification; scale bar = 50 lm).

adipocytes, corneal epithelial cells, melanocytes and even induced pluripotent stem cells (5, 7, 15, 22, 114, 117). Odontoblastic differentiation and the formation of reparative dentin can occur as a result of the interaction of dental pulp stem cells with dentin matrix protein-1 [a noncollagen extracellular matrix protein (21)], transforming growth factor-b1 and fibroblast growth factor-2 (43). Figure 5 demonstrates three modes of differentiation of dental pulp stem cells (54). In immunocompromised mice, dental pulp stem cells have demonstrated the ability to generate functional dental tissue, and culture explants of dental

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research group has corroborated those findings, as seen in Fig. 4A,B. Dental pulp stem cells, when seeded in a tridimensional scaffold of dentin, appear as odontoblast cells with prolongations of the cellular body into the structure of the dentin (44, 45). Their plasticity toward any specific cell lineage is defined by the components of the local microenvironment, such as growth factors, receptor molecules, signaling molecules, transcription factors and extracellular matrix protein. Dental pulp stem cells can differentiate into a diverse range of lineages, including odontoblasts, osteoblasts, chondrocytes, myocytes, neurocytes,

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Fig. 3. Flow cytometry and morphology of dental pulp stem cells. (A) Flow cytometry analysis of dental pulp stem cells. Histograms show passage 3 cells after staining with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies to the indicated cell-surface proteins (red histograms). (B, C) Morphology of dental pulp stem cells. Optical contrast phase microscopy shows (B) fibroblastoid cells adherent to plastic (103 magnification; scale bar = 200 lm) and (C) the fibroblast-like spindle shape of dental pulp stem cells (403 magnification; scale bar = 50 lm).

adipocytes, corneal epithelial cells, melanocytes and even induced pluripotent stem cells (5, 7, 15, 22, 114, 117). Odontoblastic differentiation and the formation of reparative dentin can occur as a result of the interaction of dental pulp stem cells with dentin matrix protein-1 [a noncollagen extracellular matrix protein (21)], transforming growth factor-b1 and fibroblast growth factor-2 (43). Figure 5 demonstrates three modes of differentiation of dental pulp stem cells (54). In immunocompromised mice, dental pulp stem cells have demonstrated the ability to generate functional dental tissue, and culture explants of dental

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Fig. 4. Polarized microscopy of dental pulp stem cells showing mineralized nodules at (A) 103 magnification (scale bar = 200 lm) and (B) 403 magnification (scale bar = 50 lm).

pulp stem cells, mixed with hydroxyapatite/tricalcium phosphate, can form ectopic dentin/pulp-like complexes (41). These samples of heterogeneous populations of dental pulp stem cells form vascularized pulp-like tissue surrounded by a layer of odontoblast-like cells that express factors which produce dentin-containing tubules, similar to those found in natural dentin (8, 41). Huang et al. (46) reported that a dentin pulp-like complex, with wellestablished vascularity, can be regenerated de novo in empty root canal spaces by dental pulp stem cells, pro-

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viding a new potential alternative of biological treatment for endodontic diseases. Demarco et al. (24) investigated the effect of dentin, and a scaffold of polyL-lactic acid (PLLA Porogenâ; Porogen Corporation, Wobum, MA, USA), on the differentiation of human dental pulp stem cells, and found that dentin-related morphogens are important for the differentiation of dental pulp stem cells into odontoblasts and for the engineering of dental pulp-like tissues, suggesting that environmental cues influence the behavior and differentiation of dental pulp stem cells.

Osteogenic differentiation

Adipogenic differentiation

Chondrogenic differentiation

Fig. 5. Differentiation of dental pulp stem cells. (A, B) Osteogenic differentiation. (A) Undifferentiated group: negative staining with Alizarin Red (403 magnification; scale bar = 50 lm). (B) Osteoblastic differentiation: positive staining with Alizarin Red is visible as a brown color (403 magnification; scale bar = 50 lm). (C, D) Adipogenic differentiation. (C) Undifferentiated group: negative staining with Oil Red O (403 magnification; scale bar = 50 lm).

(D) Adipogenic differentiation: stained with Oil Red O. Drops of fat (stained red) are visible inside the cells (403 magnification; scale bar = 50 lm). (E, F) Chondrogenic differentiation. (E) Undifferentiated group: negative staining with Safranin O (403 magnification; scale bar =50 lm). (F) Differentiated chondrocytes: positive staining with Safranin O (403 magnification; scale bar = 50 lm).

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Furthermore, it is now recognized that dental pulp stem cells express neural markers and that they can differentiate into functionally active neurons (74). The dental pulp tissue, as mentioned before, is termed ‘ectomesenchymal’ because it derives from ectodermal cells that grow on the periphery of the neural tube during embryonic development, migrate into the oral region and differentiate into mesenchymal phenotypes; thus, dental pulp stem cells may originate from the neuroectoderm (97, 101). Dental pulp stem cells also serve as a cellular source for pulpal tissue renewal and regeneration, supporting the idea that the dental pulp contains a neural progenitor pool that has a high potential for neural stem cell therapy. In a recent study, dental pulp stem cells were transplanted into the cerebrospinal fluid of rats with induced cortical damage. Dental pulp stem cells migrated as single cells into the brain regions and were detected in the injured cortex expressing neuron-specific markers, suggesting that dental pulp stem cells can be considered as a source of neuroand gliogenesis in in-vivo damaged brain tissue (58, 59). An additional promising feature of dental pulp stem cells is their immune-modulatory action, as recent studies have shown their capacity to suppress the immune response (56, 77). This is a novel and exciting area of development and research.

Stem cells from human exfoliated deciduous teeth Stem cells may also be isolated from the pulp of human exfoliated deciduous teeth. These cells have the capacity of inducing bone formation, generating dentin and differentiating in vitro into other nondental, mesenchymal cell derivatives (70). The morphology of stem cells from human exfoliated deciduous teeth is similar to that of dental pulp stem cells, stem cells from apical papilla and dental follicle precursor stem cells (56), but they have a higher proliferation rate than do bone marrow mesenchymal stem cells and dental pulp stem cells. Stem cells from human exfoliated deciduous teeth express Oct4, CD13, CD29, CD44, CD73, CD90, CD105, CD146 and CD166, but not CD14, CD34 or CD45 (47, 79). Stem cells isolated from the pulp tissue of exfoliated deciduous teeth are capable of differentiating into a variety of cells, such as neural cells, osteoblasts, chondrocytes, adipocytes and myocytes (56, 70, 103). They form ectopic dentin-like tissue in vivo, but are unable to regenerate the dentin/pulp-like complex. The cells can differentiate into angiogenic endothelial cells and

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into odontoblasts capable of generating tubular dentin (86). Osteoinductive potential has also been described because they can repair critical-size calvarial defects in mice with substantial bone formation. Cultured stem cells from human exfoliated deciduous teeth also express neural and glial cell markers, which may be related to the neural-crest cell origin of the dental pulp (18). If stimulated with neurogenic medium, these stem cells show increased expression of bIII-tubulin, glutamate decarboxylase and neuronal nuclei protein, whereas other neural markers remain unchanged (70, 89).

Stem cells from the apical papilla The term apical papilla refers to the soft tissue at the apices of developing permanent teeth (91, 92). Apical papilla is a cell-rich zone, positioned between the apical papilla and the pulp (85). The distinction between the dental pulp and the apical papilla is that the apical papilla represents a precursor tissue for the radicular pulp. As stem cells from apical papilla (obtained from explant cultures or by enzymatic digestion of apical pulp tissue) are derived from a developing tissue, they may represent a more basic population of stem/progenitor cells. These cells express mesenchymal markers, such as CD13, CD24, CD29, CD44, CD73, CD90, CD105, CD106 and CD146, but not CD18, CD34, CD45 or CD150 (26, 47). In our own research, cultures from the apical papilla of third molars were obtained using the explant method, then isolated and characterized by flow cytometry. The mesenchymal markers CD105, CD90 and CD73 were expressed by >95% of the stem cell population, but the hematopoietic markers CD45, CD38 and CD34 were expressed by 90% positive) in contrast to hematopoietic markers (

Mesenchymal stem cells from the oral cavity and their potential value in tissue engineering.

Periodontal disease is one of the most common conditions affecting humans, and current treatment strategies, which focus on the removal and long-term ...
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