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

Mesenchymal stem cells from theoral cavity and their potentialvalue in tissue engineeringANTONIO R. SANZ, FLAVIO S. CARRI �ON & ALEJANDRA P. CHAPARRO

Periodontal disease is one of the most commonconditions affecting humans, and the prevalence ofadvanced periodontitis in adults is about 15% (49).Periodontitis is a chronic inflammatory condition ofthe supporting tissues of the teeth, resulting indestruction of the attachment of the tooth to thesurrounding bone. Untreated periodontitis mayeventually lead to tooth loss. Fortunately, researchhas provided evidence that most types of periodon-tal disease can be successfully treated (63–110). Cur-rent treatment strategies focus on the removal ofdental plaque and the long-term control of dentalplaque accumulation, and these treatment strategiesare generally successful in eliminating active diseaseand in promoting tissue repair. However, the com-plete regeneration of periodontal attachment lost toperiodontal disease remains an elusive goal and achallenge (3); according to a position paper pub-lished by the American Academy of Periodontologyin 2005 (3), the formation of new bone and cemen-tum with supportive periodontal ligament is theultimate objective that current periodontal-regener-ation therapies are incapable of fulfilling. Regenera-tion is defined as the reproduction or reconstitutionof a lost or injured part of the body, in such a waythat the architecture and function of the tissues arecompletely restored (3). Thus, the aim of regenera-tive periodontal therapy is to restore the structureand function of the periodontium, which meansregeneration of the supporting tissues, including alve-olar bone, periodontal ligament and cementum, overa previously diseased root surface. Despite evidencethat some regeneration can occur after therapy (3, 12,20), this regeneration is usually only partial, in partbecause of the complexity of the biological eventsinvolved, such as signals for the synthesis of growthfactors and the recruitment of specific cells for peri-odontal regeneration. Currently, bone grafts and

guided tissue regeneration are the two techniques forwhich vast histological documentation of periodontalregeneration is available (12, 76, 87).

The periodontium is a highly complex organ con-sisting of epithelial tissue and soft and mineralizedconnective tissues, including gingiva, periodontalligament, cementum and alveolar bone. The uniqueanatomy and composition of the periodontiummakes periodontal wound healing a complex pro-cess because of the requirement for interaction ofthese three different tissues. Wound healing afterconventional periodontal therapy, including surgicaldebridement, generally results in repair by the pro-duction of collagenous scar tissue accompanied byapical migration of gingival epithelium (11, 52, 83).In order for periodontal regeneration to occur, pro-genitor periodontal ligament cells must migrate tothe denuded root surface, attach to it, proliferateand mature into an organized and functionalfibrous attachment apparatus that inserts into anewly formed cementum. Similarly, progenitor bonecells must migrate, proliferate and mature in con-junction with the regenerating periodontal ligament(12, 58, 103). However, most of the current regenerativeprocedures, used either alone or in combination, havelimitations in attaining complete regeneration, espe-cially in deep periodontal defects (84, 87, 94, 106, 112).

Wound healing, or the regenerative process of a spe-cific tissue, requires a combination of fundamentalevents, such as appropriate levels and sequencing ofregulatory signaling pathways, presence and numberof progenitor cells responding to biological signals,appropriate extracellular matrix or carrier, and ade-quate blood supply (11, 52, 80). Based on tissue-engi-neering concepts, the healing/regeneration process ofa tissue may be manipulated at one of the followingpoints: regulation of molecules, extracellular matrix orscaffold, and cellular availability (50, 80, 84; Fig. 1).

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Periodontology 2000, Vol. 67, 2015, 251–267 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Following the identification of mesenchymal stemcells in bone marrow by Friedenstein et al. (34, 35), anew era in regenerative medicine began, and tissueengineering using mesenchymal stem cells hasbecome a most interesting therapeutic option (6, 13,82, 93, 105, 111), with several advantages, such ashigh-quality regeneration of damaged tissues withoutformation of a fibrous scar, minimal donor-site mor-bidity compared with autografts, and low risk of auto-immune rejection and disease transmission (17, 62,68, 73, 91, 92, 98). The aim of this article was todescribe the main sources of mesenchymal stem cellsfrom tissues of the oral cavity and their potential inregenerative therapy. Special attention is placed ongingival tissue-derived mesenchymal stem cellsbecause this is the most accessible source for stemcells 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 inmost adult tissues) that have the capacity to differen-tiate and expand into mature cells with specific func-tions (100, 108). The primary roles of adult stem cellsare to maintain and repair tissues in which the cellsare found, as well as to maintain the stem cell popula-tion. Stem cells can be isolated from tissues such asbone marrow, skeletal muscle, cartilage, dental organ,adipose tissue, synovium and cardiac tissue (61). Themost extensively studied source of stem cells is thebone marrow, which contains hematopoietic stemcells and nonhematopoietic cells, referred to as mes-enchymal stem cells (16, 40, 55). First identified in

1966 within the stromal compartment of bone mar-row (34–36), mesenchymal stem cells include aunique population of multipotential cells that exhibitextensive proliferative ability and can differentiatealong multiple tissue-specific lineages, such as osteo-blasts, chondrocytes and adipocytes (78, 96). At pres-ent, there is no definitive marker for identifyingmesenchymal stem cells, suggesting that the cell pop-ulations derived by the current methods are, in fact,heterogeneous. However, to define mesenchymalstem cells phenotypically, minimal criteria have beenproposed by the International Society for CellularTherapy. First, they must be plastic-adherent whenmaintained in standard culture conditions; second,they must express the surface markers CD73, CD90and CD105 but not CD45, CD34, CD14 or CD11b,CD79a or CD19 and major histocompatibility complexclass II surface molecules; and, third, they must retainthe capacity to differentiate into osteoblasts, adipocytesand chondroblasts under standard in-vitro conditions(29). Another important characteristic of these cells istheir fibroblast-like spindle shape in culture (9, 81).

The presence of adult progenitor populationswithin various tissues, and their ability to adopt tis-sue-specific phenotypes, given the appropriate differ-entiation conditions, has led many investigators tosuggest that the primary role of mesenchymal stemcells is to serve as cell replacement during the naturalcourse of tissue turnover and homeostasis (14). Inaddition, mesenchymal stem cells may serve otherimportant therapeutic roles because they appear toescape immune recognition, and exert anti-inflam-matory and immune-modulatory effects via the sup-pression of T-, B-, natural killer and antigen-presenting 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 vitrowithout loss of their phenotype or multilineage poten-tial, have generated great interest in using mesenchy-mal stem cells as a therapeutic modality for immune-mediated diseases and tissue repair (27, 28, 100).

Mesenchymal stem cells from theoral cavity

The use of stem cells for tissue engineering has greatpotential to solve clinical and surgical problemsrelated to tissue loss and organ functional failures.Dental tissue-derived mesenchymal stem cell-likepopulations reside in specialized well-characterizedtissues and may be used in regenerative medicine.The first type of dental stem cell was isolated from

Fig. 1. Levels of tissue engineering: molecules, scaffoldsand cells.

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the human pulp tissue and termed ‘postnatal dentalpulp stem cells’ (42). Subsequently, four other typesof dental-mesenchymal stem cell-like populations,from exfoliated deciduous teeth (70), periodontal liga-ment (88), apical papilla of developing teeth (91, 92)and dental follicle precursor cells (72), were isolatedand characterized (31). Our research group has iden-tified another source of mesenchymal stem cells inthe oral cavity, namely the mesenchymal stem cellpopulation from human gingival tissue (68). The mes-enchymal stem cells of the oral cavity seem to beresponsible for the maintenance and repair of theirassociated tissues (68, 115, 116). The oral stem cellsshow characteristics similar to those described byPittenger et al. (78) and Noth et al. (75) for bonemarrow mesenchymal stem cells (i.e. they can, usingspecific differentiation media and conditions, differ-entiate to osteogenic, chondrogenic and adipogeniclineages); however, differences have been notedbetween the dental stem cell and bone marrow mes-enchymal stem cell populations. Dental stem cellsappear to be more committed to odontogenic devel-opment than to osteogenic development (60), andthey also exhibit the potential to differentiate intoneurogenic linages (53, 95). Dental mesenchymaltissue is termed ‘ectomesenchyme’ as a result of itsinitial interaction with the neural crest, a feature thatmay differentiate ectomesenchymal-derived stemcells and bone marrow-derived stem cells (47).Greater insights into the biology of dental stem cellpopulations are a prerequisite for understandingthe potential of these cells in regenerative medicine(71, 91).

Embryogenesis of the tooth andsupporting structures

The dentition is a complex and unique structure ofthe body consisting of hard, soft and neural tissues.The development of teeth and tooth-supportingstructures is initiated early during embryonic humandevelopment (103). Neural crest cells migrate into thefirst facial arc forming a layer of ectomesenchymeunder the ectodermal epithelium of the primitive sto-modeum (4, 23, 103). Molecular interaction betweenthe two primary tissues triggers a series of events,which lead eventually to tooth development (4, 48,103). At about the sixth intrauterine week, two thick-enings of epithelium invade the underlying mesen-chymal tissue (4, 10), the buccal plate and the dentalplate (4, 10). The first gives rise to the oral buccal tis-sue through the apoptosis of epithelial cells of the

middle and surface strata (10); and the second givesrise to primary and permanent teeth and their attach-ment apparatus (4, 10, 103). The development of theenamel organ is derived from the dental plate, whichgoes through four sequential stages: sprout; cap; earlybell; and late bell (103). The enamel organ is formedin the cap stage of the dental plate. Simultaneously,ectomesenchymal cells give rise to the dental papillacomplex and the dental follicle (also called the dentalsac; 4, 23, 30, 47, 48, 103); these two structures,respectively, produce pulp dentin and the periodon-tium (4, 23, 103; Fig. 2).

Dental pulp stem cells

Dental pulp stem cells have been obtained by enzy-matic digestion of pulp tissue from permanent thirdmolars (30, 42, 47, 53, 69). They are multipotent cellsthat proliferate extensively, can be safely cryopre-served, possess immunosuppressive properties andexpress markers such as CD13, CD29, CD44, CD59,CD73, CD90, CD105, CD146 and STRO-1, but notCD14, CD24, CD34, CD45, CD19 and HLA-DR (47, 73,101). Figure 3A shows flow cytometry analyses ofdental pulp stem cells. The cells obtained from ex-plants of third molar pulps showed fibroblastic prop-erties (Fig. 3B,C). A principal feature of dental pulpstem cells is their potential to differentiate into odon-toblastic lineages (47), characterized by polarized cel-lular bodies and mineralized nodules (2, 21, 99). Our

Fig. 2. Tooth embryogenesis: dental lamina, enamel retic-ulum, dental papilla and dental sac.

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research group has corroborated those findings, asseen in Fig. 4A,B.

Dental pulp stem cells, when seeded in a tridi-mensional scaffold of dentin, appear as odontoblastcells with prolongations of the cellular body into thestructure of the dentin (44, 45). Their plasticitytoward any specific cell lineage is defined by thecomponents of the local microenvironment, such asgrowth factors, receptor molecules, signaling mole-cules, transcription factors and extracellular matrixprotein. Dental pulp stem cells can differentiate intoa diverse range of lineages, including odontoblasts,osteoblasts, chondrocytes, myocytes, neurocytes,

adipocytes, corneal epithelial cells, melanocytes andeven induced pluripotent stem cells (5, 7, 15, 22,114, 117). Odontoblastic differentiation and the for-mation of reparative dentin can occur as a result ofthe interaction of dental pulp stem cells with dentinmatrix protein-1 [a noncollagen extracellular matrixprotein (21)], transforming growth factor-b1 andfibroblast growth factor-2 (43). Figure 5 demon-strates three modes of differentiation of dental pulpstem cells (54).

In immunocompromised mice, dental pulp stemcells have demonstrated the ability to generate func-tional dental tissue, and culture explants of dental

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Fig. 3. Flow cytometry and morphol-ogy of dental pulp stem cells. (A)Flow cytometry analysis of dentalpulp stem cells. Histograms showpassage 3 cells after staining withfluorescein isothiocyanate (FITC)- orphycoerythrin (PE)-conjugated anti-bodies to the indicated cell-surfaceproteins (red histograms). (B, C)Morphology of dental pulp stemcells. Optical contrast phase micros-copy shows (B) fibroblastoid cellsadherent to plastic (103 magnifica-tion; scale bar = 200 lm) and (C) thefibroblast-like spindle shape of den-tal pulp stem cells (403 magnifica-tion; scale bar = 50 lm).

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pulp stem cells, mixed with hydroxyapatite/trical-cium phosphate, can form ectopic dentin/pulp-likecomplexes (41). These samples of heterogeneouspopulations of dental pulp stem cells formvascularized pulp-like tissue surrounded by a layer ofodontoblast-like cells that express factors which pro-duce dentin-containing tubules, similar to thosefound in natural dentin (8, 41). Huang et al. (46)reported that a dentin pulp-like complex, with well-established vascularity, can be regenerated de novo inempty root canal spaces by dental pulp stem cells, pro-

viding a new potential alternative of biological treat-ment for endodontic diseases. Demarco et al. (24)investigated the effect of dentin, and a scaffold of poly-L-lactic acid (PLLA Porogen�; Porogen Corporation,Wobum, MA, USA), on the differentiation of humandental pulp stem cells, and found that dentin-relatedmorphogens are important for the differentiation ofdental pulp stem cells into odontoblasts and for theengineering of dental pulp-like tissues, suggesting thatenvironmental cues influence the behavior and differ-entiation of dental pulp stem cells.

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Osteogenic differentiation

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Adipogenic differentiationE F

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: posi-tive staining with Alizarin Red is visible as a brown color(403 magnification; scale bar = 50 lm). (C, D) Adipogenicdifferentiation. (C) Undifferentiated group: negative stain-ing with Oil Red O (403 magnification; scale bar = 50 lm).

(D) Adipogenic differentiation: stained with Oil RedO. Drops of fat (stained red) are visible inside the cells(403 magnification; scale bar = 50 lm). (E, F) Chondro-genic differentiation. (E) Undifferentiated group: negativestaining with Safranin O (403 magnification; scalebar =50 lm). (F) Differentiated chondrocytes: positivestaining with Safranin O (403 magnification; scalebar = 50 lm).

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Fig. 4. Polarized microscopy of den-tal pulp stem cells showing mineral-ized nodules at (A) 103magnification (scale bar = 200 lm)and (B) 403 magnification (scalebar = 50 lm).


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Furthermore, it is now recognized that dental pulpstem cells express neural markers and that they candifferentiate into functionally active neurons (74). Thedental pulp tissue, as mentioned before, is termed‘ectomesenchymal’ because it derives from ectoder-mal cells that grow on the periphery of the neuraltube during embryonic development, migrate intothe oral region and differentiate into mesenchymalphenotypes; thus, dental pulp stem cells may origi-nate from the neuroectoderm (97, 101). Dental pulpstem cells also serve as a cellular source for pulpal tis-sue renewal and regeneration, supporting the ideathat the dental pulp contains a neural progenitor poolthat has a high potential for neural stem cell therapy.In a recent study, dental pulp stem cells were trans-planted into the cerebrospinal fluid of rats withinduced cortical damage. Dental pulp stem cellsmigrated as single cells into the brain regions andwere detected in the injured cortex expressing neu-ron-specific markers, suggesting that dental pulpstem cells can be considered as a source of neuro-and gliogenesis in in-vivo damaged brain tissue (58,59). An additional promising feature of dental pulpstem cells is their immune-modulatory action, asrecent studies have shown their capacity to suppressthe immune response (56, 77). This is a novel andexciting area of development and research.

Stem cells from human exfoliateddeciduous teeth

Stem cells may also be isolated from the pulp ofhuman exfoliated deciduous teeth. These cellshave the capacity of inducing bone formation, gener-ating dentin and differentiating in vitro into othernondental, mesenchymal cell derivatives (70). Themorphology of stem cells from human exfoliateddeciduous teeth is similar to that of dental pulp stemcells, stem cells from apical papilla and dental follicleprecursor stem cells (56), but they have a higher pro-liferation rate than do bone marrow mesenchymalstem cells and dental pulp stem cells. Stem cells fromhuman exfoliated deciduous teeth express Oct4,CD13, CD29, CD44, CD73, CD90, CD105, CD146 andCD166, but not CD14, CD34 or CD45 (47, 79). Stemcells isolated from the pulp tissue of exfoliated decid-uous teeth are capable of differentiating into a varietyof cells, such as neural cells, osteoblasts, chondro-cytes, adipocytes and myocytes (56, 70, 103). Theyform ectopic dentin-like tissue in vivo, but are unableto regenerate the dentin/pulp-like complex. The cellscan differentiate into angiogenic endothelial cells and

into odontoblasts capable of generating tubular den-tin (86). Osteoinductive potential has also beendescribed because they can repair critical-size calvari-al defects in mice with substantial bone formation.Cultured stem cells from human exfoliated deciduousteeth also express neural and glial cell markers, whichmay be related to the neural-crest cell origin of thedental pulp (18). If stimulated with neurogenic med-ium, these stem cells show increased expression ofbIII-tubulin, glutamate decarboxylase and neuronalnuclei protein, whereas other neural markers remainunchanged (70, 89).

Stem cells from the apical papilla

The term apical papilla refers to the soft tissue atthe apices of developing permanent teeth (91, 92).Apical papilla is a cell-rich zone, positionedbetween the apical papilla and the pulp (85). Thedistinction between the dental pulp and the apicalpapilla is that the apical papilla represents a pre-cursor tissue for the radicular pulp. As stem cellsfrom apical papilla (obtained from explant culturesor by enzymatic digestion of apical pulp tissue)are derived from a developing tissue, they mayrepresent a more basic population of stem/progen-itor cells. These cells express mesenchymal mark-ers, such as CD13, CD24, CD29, CD44, CD73,CD90, CD105, CD106 and CD146, but not CD18,CD34, CD45 or CD150 (26, 47). In our ownresearch, cultures from the apical papilla of thirdmolars were obtained using the explant method,then isolated and characterized by flow cytometry.The mesenchymal markers CD105, CD90 andCD73 were expressed by >95% of the stem cellpopulation, but the hematopoietic markers CD45,CD38 and CD34 were expressed by <1% of stemcells (54; Fig. 6A–C) (data not shown for CD105and CD 38).

Stem cells from the apical papilla also have thecapacity to undergo osteogenic/dentinogenic, neuro-genic and adipogenic differentiation; in fact, theydisplay an expression pattern of osteogenic/dentino-genic markers and growth factor receptors similar tothat observed in dental pulp stem cells, but withlower expression levels (1, 91). Stem cells from apicalpapilla also have the ability to induce root formation(37, 47, 48). In our own research, stem cells obtainedfrom retained third molars, at the third passage in adifferentiation medium, were induced to differenti-ate into osteogenic, chondrogenic and adipogeniccell lineages (54; Fig. 7).

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Stem cells from the periodontalligament

The periodontal ligament is unique in the bodybecause it resides between two different hard tissues,namely bone and cementum. The presence ofmultiple cell types within the periodontal ligamentsuggests that this tissue contains progenitor cells ableto maintain tissue homeostasis and regeneration ofperiodontal tissues. Previous evidence has shown thatthe periodontal ligament contains cell populationsthat can differentiate into cementum-forming cells,bone-forming cells and collagen-forming cells (38, 51,67, 113). Stem cells from the periodontal ligament

express the mesenchymal stem cell markers CD10,CD13, CD29, CD44, CD59, CD73, CD90 and CD105but do not express CD14, CD34, CD45, CD38, CD54or HLA-DR (32, 33, 47, 64, 90). They demonstrate, asfound for other mesenchymal stem cells, a fibroblast-like spindle shape (33; Fig. 8A–C).

Similarly to other dental stem cells, stem cellsfrom the periodontal ligament have the potential todifferentiate in vitro into adipogenic, osteogenic andchondrogenic cells (33, 50, 53); however, formationof calcified nodules is less prominent than thatobserved for stem cells from the dental pulpor from exfoliated deciduous teeth (38, 64, 113).Stem cells from the periodontal ligament have the

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Fig. 6. Flow cytometry and morphol-ogy of apical papilla stem cells. (A)Flow cytometry analysis of apicalpapilla stem cells. Histograms ofcells after staining with fluores-cein isothiocyanate (FITC)- orphycoerythrin (PE)-conjugated anti-bodies to the indicated cell-surfaceproteins (green histograms). (B, C)Morphology of apical papilla stemcells. A fibroblast-like spindle shapewas observed. 80% confluence wasobserved after 17–19 days of culture.(B) 103 magnification (scalebar = 200 lm). (C) 403 magnifica-tion (scale bar = 50 lm).


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A B

Osteogenic differentiationC D

Adipogenic differentiationE F

Chondrogenic differentiation

Fig. 7. Differentiation of stem cells from the apicalpapilla. (A, B) Osteogenic differentiation. (A) Undifferenti-ated control: negative staining with Alzarin Red (403magnification; scale bar = 50 lm). (B) Differentiated tothe osteoblast lineage. Positive staining with Alizarin Red(403 magnification; scale bar = 50 lm). (C, D) Adipogenicdifferentiation. (C) Undifferentiated control: negativestaining with Oil Red O (403 magnification; scalebar = 50 lm). (D) Differentiated to the adipogenic

lineage. Positive staining with Oil Red O. Drops of fat(stained red) are visible inside the cells (403 magnifica-tion; scale bar = 50 lm). (E, F) Chondrogenic differentia-tion. (E) Undifferentiated control: negative staining withSafranin O (403 magnification; scale bar = 50 lm). (F)Differentiated to the chondrogenic lineage. Positive stain-ing with Safranin O (403 magnification; scale bar =50 lm). The results are representative of five independentexperiments.

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Fig. 8. Flow cytometry and morphol-ogy of stem cells from the periodon-tal ligament. (A) Flow cytometryanalyses show that stem cells fromthe periodontal ligament expressmesenchymal stem cell markerssuch as express mesenchymal stemcells markers such as CD13, CD90,CD73, CD105, CD44 but do notexpress CD34, CD45, CD38 andCD54. FITC, fluorescein isothiocya-nate; PE, phycoerythrin; Tri Color,TC. (B, C) Contrast phase micros-copy showing the morphology ofstem cells from the periodontal liga-ment at (B) passage 4 (103 magnifi-cation) and (C) passage 9 (103magnification) of culture.

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capacity to differentiate in vivo into functional ce-mentoblasts and to form collagen fibers embeddedin cementum-like tissue when implanted subcuta-neously in immune-compromised mice, suggestingpotential to regenerate the cementum/periodontalligament-like complex in vivo (88). Furthermore,when transplanted into periodontal defects inimmune-compromised mice, stem cells from theperiodontal ligament regenerated a periodontal liga-ment-like tissue and were also associated with thetrabecular bone in the regenerated periodontium.This finding suggests that stem cells from the peri-odontal ligament can also participate in alveolarbone regeneration (88).

Stem cells from human gingival tissue

The presence of mesenchymal stem cells in humangingival connective tissue was described by ourresearch group in 2010 (68). Gingival connective tis-sue samples were obtained from healthy subjects andde-epithelialized, leaving only connective tissue. Theexplants were minced, cultured for 3–4 weeks andcharacterized by flow cytometry. Differentiation intoosteogenic, chondrogenic and adipogenic lineageswas induced and evaluated by histologic staining andimmune-regulation assay. Gingival tissue cells ful-filled the criteria proposed by the International Soci-ety for Cellular Therapy for mesenchymal stem cells(29). In culture, stem cells from human gingival tissuehad the ability to adhere to plastic and showed afibroblast-like spindle shape (Fig. 9A,B). Cell charac-terization was consistently positive for the mesenchy-mal stem cell markers CD90, CD105, CD73, CD44 andCD13 and negative for the hematopoietic markersCD34, CD38, CD45 and CD54 (29; Fig. 10). Weobserved differentiation with positive staining foradipogenic, chondrogenic and osteogenic lineages;

furthermore, gingival cells showed immune-modula-tory capacity (68; Fig. 11).

As mesenchymal stem cells are nonimmunogenicand have immune-modulatory capability, transplan-tation of allogeneic mesenchymal stem cells to a hostmay not require immunosuppression. Humanmesenchymal stem cells impair the maturation andfunction of dendritic cells and inhibit, in vitro, theproliferation, differentiation and mobility of humanT- and B-cells (19). We evaluated the inhibitory effectof stem cells from human gingival tissues on mito-gen-stimulated peripheral blood mononuclear cells(Fig. 12). Gingival tissue mesenchymal stem cellswere co-cultivated, at different ratios, with peripheralblood mononuclear cells, and a dose-dependentimmunosuppressive capacity of gingival tissue mes-enchymal stem cells was observed. The peripheralblood mononuclear cells showed a proliferation rateof 55.3% in the absence of gingival stem cells, but aproliferation rate of only 7.3% when cultured with thesame number of gingival stem cells. Our results are inaccordance with those of Wada et al. (107), who dem-onstrated an immunosuppressive effect of periodon-tal ligament and dental pulp mesenchymal stem cellson peripheral blood mononuclear cell proliferation.The cellular and molecular mechanisms by whichmesenchymal stem cells exert their immune-modula-tory effect on lymphocytes is a matter of intenseresearch (25) as it represents a potentially promisingtool in cell therapy.

Very limited information is available on the mor-phological and structural characteristics of dentaladult mesenchymal cells, and even less information isavailable on their differentiated cell lineages, such asosteogenic cells. Subsequently to describing the pres-ence of mesenchymal stem cells in gingival connec-tive tissue (68), we studied the morphological andultrastructural characteristics of these cells under

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Fig. 9. Morphology of stem cells from human gingival tis-sue. Phase-contrast image demonstrating the morphologyof gingival cells (A) at passage 0 on day 3 of culture (103magnification; scale bar = 50 lm) and (B) on day 15 of cul-

ture (103 magnification; scale bar = 50 lm). The presenceof a homogeneous fibroblast-like population was evidentduring the in-vitro expansion. One representative experi-ment is shown.


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transmition electron microscopy. We also comparedthe morphologycal characteristics of the diferentiatedMesenchymal Stem Cells to Osteoblastic linages tomature rabbit Osteoblasts. A fibroblast-like morphol-ogy was observed with cellular interconnections andsurface structures compatible with phyllopodous(Fig. 13A,B). Figure 14A,B shows the fibroblast-likeshape of the cells and the presence of nodule-likestructures displaying the characteristics of calciumdeposits. This finding agrees with the results obtainedof cultures of mesenchymal stem cells stained withAlizarin Red, a stain used to identify differentiation ofcells into osteoblasts. Transmission electron micros-copy evaluation of gingival stem cells showed exten-sion of nucleoli with dense chromatin andintracellular structures, similar to phagolysosomes(Fig. 15A,B). Structures in the periphery of the stemcells resembled blebs. Figure 16A–C shows collagenfibers, arranged parallel to the cell body, in the extra-cellular matrix produced by the osteblasts. Fig-ure 17A,B depicts a mature osteoblast obtained froma rabbit, which exhibits similarity with the stem cell-derived osteoblasts. A fibroblast-like cell resembling

an osteoblast can be distinguished and, behind it, anendotheliocyte. Multiple black dots representing col-lagen fibers, in cross section, are also evident in theextracellular matrix of bone as incremental lines. Thatstem cells from human gingival tissues may differen-tiate into osteoblastic lineages is indicated by thepresence of two or three extended nucleoli, mito-chondria with extended morphology, expression ofvacuoles in the process of exocytosis, extracellulargranular and nongranular matrix, collagen fibers andareas of early mineralization between the collagenfibers. Similar features are present during the metab-olism of a mature osteoblast (39; Fig. 16A–C). Thepotential of stem cells from human gingival tissues todifferentiate into osteoblast linages and to serve asbone producers, open up new clinical approaches intissue engineering (41, 42, 61).

Conclusions

A biological solution to a biological problem is thecurrent mantra in medical science. Regeneration of

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Fig. 10. Analysis of stem cells fromhuman gingival tissue. (A) Flowcytometry. Cells were stained withantibodies against the cell-surfaceproteins indicated (green histograms).FITC, fluorescein isothiocyanate; PE,phycoerythrin; Tri Color, TC. Control:isotype IgG (black histograms). (B)Expression of commonly usedmesenchymal stem cell surface mark-ers (>90% positive) in contrast tohematopoietic markers (<1% positive).The results are representative of fiveindependent experiments. Data areexpressed as mean � standard devia-tion.

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damaged organs or tissues is the optimal outcome oftreatment, and both tissue engineering and regenera-tive medicine are emerging as promising treatmentoptions. Stem cells, specifically mesenchymal stem

cells, are promising candidates for tissue regenera-tion. Numerous body sites have been described fromwhich somatic or adult stem cells can be isolated.Adult mesenchymal stem cells were originallythought to be developmentally restricted to specificcell lineages related to the tissue from which theywere isolated, but it was recently shown that adultmesenchymal stem cells have a broad differentiationpotential covering virtually all mesenchymal-derivedtissues. Since the discovery of mesenchymal stemcells, basic and animal research has suggested thatstem cell therapy can be successfully used in humans.Bone marrow has been the major source of mesen-chymal stem cells for clinical applications. However,isolation of cells from bone marrow is an invasiveprocedure, which can be painful and may occasion-ally be associated with infectious complications.Moreover, the number of mesenchymal stem cellsharvested from bone marrow can be low, which maynecessitate an ex vivo expansion step of cells.

Autologous adult stem cells should ideally be easyto obtain, result in minimal patient discomfort andbe available in substantial numbers to avoid exten-sive expansion culturing. The mouth, and specifically

Fig. 12. Immunosuppressive effects of mesenchymal stemcells from gingival Human gingival tissue. Proliferation ofperipheral blood mononuclear cells mitogenically stimu-lated with phytohemagglutinin in co-culture with gingivalconnective tissue stem cells. Proliferation of mononuclearcells reached maximal suppression at the at a PBMC:MSCs1:1 ratio. Data from five independent experiments areshown as mean � standard deviation. MSC;Mesenchymalstem cells, PBMC; peripheral blood mononuclear cells.

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Fig. 11. Differentiation of stem cellsfrom human gingival tissue. (A, B)Osteogenic differentiation. (A) Con-trol group: negative staining withAlizarin Red (203 magnification;scale bar = 50 lm). (B) Experimentalcell group after osteogenic differenti-ation: positive staining with AlizarinRed (203 magnification; scalebar = 50 lm). (C, D) Chondrogenicdifferentiation. (C) Control group:negative staining with Safranin O(203 magnification; scalebar = 50 lm). (D) Experimental cul-ture after differentiation toward thechondrogenic lineage: positive stain-ing with Safranin O (203 magnifica-tion; scale bar = 50 lm). (E, F)Adipogenic differentiation. (E) Con-trol group: negative staining with OilRed O (203 magnification; scalebar = 50 lm). (F) After adipogenicdifferentiation: positive staining withOil Red O. Drops of fat (stained red)are visible inside the cells (203 mag-nification; scale bar = 50 lm). Theresults are representative of at leastfive independent experiments.


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the gingiva, constitutes one of the most accessibleharvesting sites for stem cells and gives rise to littleor no morbidity as a result of the rapid healingcapacity of gingiva. However, although tissue engi-neering comprises a highly attractive treatment pros-

pect, cell-based therapy is still limited to clinicaltrials and is not performed in a routine clinical set-ting. The next steps in the study of stem cell therapyinvolve the development of protocols to standardizethe preparation of the biological products and to

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Fig. 13. Scanning electron micros-copy of undifferentiated stem cellsfrom human gingival tissue. (A)2003 magnification. (B) 1,0003magnification.

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Fig. 14. Scanning electron micros-copy of stem cells from human gin-gival tissue differentiated intoosteoblasts, showing nodules ofgranular material. (A) 5003 magnifi-cation. (B) 2,0003 magnification.

A B

Fig. 15. Transmission electronmicroscopy of undifferentiated stemcells from human gingival tissue,with blebs. (A) 4,4003magnification.(B) 12,0003 magnification.

A B C

Fig. 16. Transmission electron microscopy of stem cells from human gingival tissue differentiated into osteoblasts. (A)Fibroblast-like shape and extended nucleus (3,0003 magnification). (B) Mitochondria with extended morphology andexpression of vacuoles (3,0003 magnification). (C) Collagen fibers forming the extracellular matrix in vitro (3,0003 magni-fication).

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develop appropriate vehicles for cell transportationthat are safe both for the cells themselves and forthe patient receiving the cells. Only when theseand other issues have been satisfactorily resolvedcan stem cell therapy become a true therapeuticalternative.

Acknowledgments

The authors express their appreciation to Carolina I-nostroza, Macarena Hernandez, Constanza Jim�enez,Claudia Brizuela and Antonio Quintero for their con-tribution in the laboratory procedures, data analysisand administrative assistance. Special thanks aregiven to the Immunology laboratory of the Faculty ofMedicine at the University of the Andes. The Univers-idad de los Andes funded the present research underthe grant FIC- ODO (2010–2012) and provided finan-cial support.

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