The Open Dentistry Journal




ISSN: 1874-2106 ― Volume 13, 2019

Dental Stem Cells and their Applications in Dental Tissue Engineering



S Lymperi1, 2, C Ligoudistianou2, #, V Taraslia2, E Kontakiotis1, E Anastasiadou2, *
1 Department of Endodontics, Dental School, University of Athens, Greece
2 Department of Genetics and Gene Therapy, Biomedical Research Foundation of Academy of Athens, Greece

Abstract

Tooth loss or absence is a common condition that can be caused by various pathological circumstances. The replacement of the missing tooth is important for medical and aesthetic reasons. Recently, scientists focus on tooth tissue engineering, as a potential treatment, beyond the existing prosthetic methods. Tooth engineering is a promising new therapeutic approach that seeks to replace the missing tooth with a bioengineered one or to restore the damaged dental tissue. Its main tool is the stem cells that are seeded on the surface of biomaterials (scaffolds), in order to create a biocomplex. Several populations of mesenchymal stem cells are found in the tooth. These different cell types are categorized according to their location in the tooth and they demonstrate slightly different features. It appears that the dental stem cells isolated from the dental pulp and the periodontal ligament are the most powerful cells for tooth engineering. Additional research needs to be performed in order to address the problem of finding a suitable source of epithelial stem cells, which are important for the regeneration of the enamel. Nevertheless, the results of the existing studies are encouraging and strongly support the belief that tooth engineering can offer hope to people suffering from dental problems or tooth loss.

Keywords: Dental stem cells, mesenchymal stem cells, tissue engineering, biomaterials, bone regeneration, bioengineered tooth.


Article Information


Identifiers and Pagination:

Year: 2013
Volume: 7
First Page: 76
Last Page: 81
Publisher Id: TODENTJ-7-76
DOI: 10.2174/1874210601307010076

Article History:

Received Date: 2/8/2012
Revision Received Date: 29/1/2013
Acceptance Date: 31/1/2013
Electronic publication date: 26 /7/2013
Collection year: 2013

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© Lymperi et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the Biomedical Research Foundation of Academy of Athens 4 Soranou Ephessiou St., 115 27 Athens, Greece; Tel: +30 210 6597000; Fax: +30 210 6597545; E-mail: anastasiadou@bioacademy.gr# Both authors contributed equally




INTRODUCTION

Stem cells are undifferentiated cells, with the ability to divide and give rise to identical, undifferentiated cells. Under specific condition, they can differentiate to various cell types that comprise the human body. Therefore, the stem cells are capable of developing to mature cells, with distinctive figures and specific functions, such as neural and muscle cells. The stem cells are divided in two groups: 1) the embryonic stem cells and 2) the adult stem cells. The latter are located in human tissues such as bone marrow, skin, adipose tissue and dental pulp [1Hall PA, Watt FM. Stem cells the generation and maintenance of cellular diversity Development 1989; 106(4): 619-33.-4Ulmer FL, Winkel A, Kohorst P, Stiesch M. Stem cells--prospects in dentistry Schweiz Monatsschr Zahnmed 2010; 20(10): 860-3.].

Due to their properties, stem cells have the potency to become an important tool of tissue engineering and regenerative medicine. Tissue engineering is merging the principles of engineering and bioscience aiming to develop biological substances for the restoration, conservation and/or improvement of the tissue function [5Langer R. Vacanti JP , Burdick J. Cellular encapsulation in 3D hydrogels for tissue engineering J Vis Exp 2009; (32): ]. Tissue engineering is aiming to provide the stimulus to the organism to regenerate the tissue from the inside or to develop the tissue externally which could be transplanted as natural tissue. The applications are based on the ability of the cells to proliferate and differentiate as well as on the construction of structures through cell and bioscaffold interactions [6Khetan S, Burdick J. Cellular encapsulation in 3D hydrogels for tissue engineering J Vis Exp 2009; (32): pii. 1590-., 7Schneider RK, Puellen A, Kramann R, et al. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds Biomaterials 2010; 31(3): 467-80.].

The loss or the absence of a tooth is a situation that could occur due to various conditions such as dental caries, periodontitis, traumatic injury or various pathological syndromes. The replacement of the lost tooth is very important not only for aesthetic reasons but also for functional purposes. Recent efforts in the field of biomaterials have led to the creation of osseointegrated implants from biocompatible materials which invade in the jaw bone to replace the missing tooth or to provide support. However, the successful outcome of this application depends on many parameters. Although the success rate of the technique is relatively high it is not always adequate as the implant is of different consistency to the recipient substrate. To overcome this shortcoming novel approaches are suggested, deriving from the field of stem cells and tissue engineering. Recently, tooth tissue engineering is providing the scientific ground for novel therapeutic applications regarding the tooth loss, compensating the existing prosthetic methodology. Dental tissue engineering is focusing on three main parameters: the type of cells that should be used, the scaffolds where the cells should be seeded on and the growth factor/molecular signals that should be applied. The present review is focusing on the properties of dental mesechymal stem cells and the current studies in the field of dental engineering and the applications of these cells.

DENTAL EPITHELIAL CELLS

Two main cell types are involved in dental tissue formation: the ameloblasts, of epithelial origin, that form enamel and the odontoblasts of mesenchymal origin, that are responsible for dentin production.

Enamel is formed from ameloblasts that derive from epithelial stem cells. They are the only cells of ectodermal origin that play a role in tooth development. These cells and their ancestor are lost just after the eruption of the tooth and as a result they do not exist in permanent teeth and cannot therefore be stimulated in vivo, to produce enamel.

In animal models, epithelial stem cells were isolated from the 3rd molar of newborn or young (still growing) animals. Initially, the epithelium was isolated and the cells were enzymatically separated and propagated, in vitro. They were combined with mesenchymal stem cells that were isolated from the same tooth and exposed to biomaterials [8Young CS, Terada S, Vacanti JP, Honda M, Bartlett JD, Yelick PC. Tissue engineering of complex tooth structures on biodegradable polymer scaffolds J Dent Res 2002; 81(10): 695-700.-10Honda MJ, Tsuchiya S, Sumita Y, Sagara H, Ueda M. The sequential seeding of epithelial and mesenchymal cells for tissue-engineered tooth regeneration Biomaterials 2007; 28(4): 680-9.].

The above approaches are promising regarding the tooth formation/regeneration. However, the clinical applications are challenging because a tooth germ from children is required. The use of autologous stem cells is most wanted and a good reliable source is needed.

DENTAL MESENCHYMAL STEM CELLS

The mesenchymal stem cells are non hematopoietic, multipotent cells that can proliferate and differentiate into a range of cell types comprising various tissues. They were characterized for the first time, 48 years ago, by Dr Friedenstein and his scientific group, when they identified a specific cell population in the bone marrow [11Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs Exp Hematol 1976; 4(5): 267-74.]. These cells demonstrated specific properties such as:

  1. fibroblast-like morphology,
  2. ability to adhere on plastic tissue-culture surfaces, and
  3. osteogenic potency.

They express explicit protein markers (proteins of the cell membrane existing in these cells) such as CD90, CD73, CD105, CD44, and they are negative for CD34, CD31, CD45 [12Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement Cytotherapy 2006; 8(4): 315-7.]. They are multipotent and they can differentiate to osteoblasts, neuroblasts, cartilage, endothelial, muscle and adipose cells [13Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues Science 1997; 276(5309): 71-4.]. Mesenchymal stem cells are also found in: adipose tissue, skeletal muscle, join fluid, amniotic fluid, cord blood, umbilical cord and teeth. Due to their presence in the teeth, their properties and the relatively easy isolation technique, the dental mesechymal stem cells are currently studied extensively, becoming a potential, novel, important tool of tissue engineering and regenerative medicine.

Various mesenchymal stem cell populations exist in the tooth. According to their position in the tooth they can be grouped as:

  • Dental Pulp Stem Cells, DPSCs [14Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo Proc Natl Acad Sci USA 2000; 97(25): 13625-30.]
  • Stem cells from Human Exfoliated Deciduous teeth, SHEDs [15Miura M, Gronthos S, Zhao M, et al. SHED stem cells from human exfoliated deciduous teeth Proc Natl Acad Sci USA 2003; 100(10): 5807-12.]
  • PerioDontal Ligament Stem Cells, PDLSCs [16Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament Lancet 2004; 364(9429): 149-55.]
  • Dental Follicle Stem Cells, DFSCs [17Morsczeck C, Gotz W, Schierholz J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth Matrix Biol 2005; 24(2): 155-65.]
  • Stem Cells from the dental Apical Papilla, SCAPs [18Sonoyama W, Liu Y, Fang D, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine PLoS ONE 2006; 1: e 79-0.].

Although the above groups are similar they also demonstrate specific characteristics relevant to each population that will be analyzed in the following paragraphs.

DENTAL PULP STEM CELLS (DPSCS)

The regenerative properties of the dentin/dental pulp complex, has lead the scientists to the hypothesis that dental pulp could contain stem cells responsible for the restoration/repair of the dentin (replacing and reparative tertiary dentin). In 2000, Gronthos et al., reported for the first time, the presence of stem cells in the dental pulp of adults [14Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo Proc Natl Acad Sci USA 2000; 97(25): 13625-30.]. This study reported the isolation of DPSCs from adult 3rd molars (19-29 years old) and demonstrated that they have a typical fibroblast shape, expression pattern of protein markers similar to mesenchymal stem cells of the bone marrow (BM MSC) and higher proliferation rate in vitro, compare to the latter. This particular feature could be attributed to the fact that 3rd molars are the last permanent teeth regarding growth, formation and eruption. Thus, they exhibit a more primitive state compared to the mesenchymal stem cells of the bone marrow.

DPSCs were then transplanted along with HA/TCP powder into immunocompromised mice. After 6 weeks, they observed a dentin-pulp-like structure, similar to the one observed in human teeth. Collagenous matrix was deposited perpendicular to the odontoblast-like layer and the odontoblast-like cells extended cytoplasmic processes into the dentinal matrix, which interfaced with a pulp-like interstitial tissue infiltrated with blood vessels.

The same group, in 2002, demonstrated that DPSCs can proliferate giving rise to identical cells and differentiate to various cell types such as neuro- and adipose cells (stem cell properties). Moreover, they can generate dentin with attached pulp, in vivo [19Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stem cells J Dent Res 2002; 81(8): 531-.].

According to a recent study, it was observed that DPSCs can generate bone, following subcutaneous transplantation in mice, using ceramic ΗΑ/TCP, as carrier [20Otaki S, Ueshima S, Shiraishi K, et al. Mesenchymal progenitor cells in adult human dental pulp and their ability to form bone when transplanted into immunocompromised mice Cell Biol Int 2007; 31(10): 1191-7.]. Additionally, DPSCs stored and defrosted after 2 years remain capable of differentiating to pro-osteoblasts; the presence of specific surface antigens remained unchanged and the cells produced dental tissue [21Papaccio G, Graziano A, d'Aquino R, et al. Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair J Cell Physiol 2006; 208(2): 319-25.].

The ability of these cells to differentiate into odontoblasts, osteoblasts, muscle, adipose, neurons and chondrocytes, has been verified in vitro, while their capability to develop hepatocyte morphological features and functions has been recently demonstrated [4Ulmer FL, Winkel A, Kohorst P, Stiesch M. Stem cells--prospects in dentistry Schweiz Monatsschr Zahnmed 2010; 20(10): 860-3., 22Ishkitiev N, Yaegaki K, Calenic B, et al. Deciduous and permanent dental pulp mesenchymal cells acquire hepatic morphologic and functional features in vitro J Endod 2010; 36(3): 469-74.].

A study in 2006, reports that when DPSCs were placed in three different 3D scaffolds (a spongeous collagen, a porous ceramic, and a fibrous titanium mesh) and transplanted into mice for 6 or 12 weeks, they formed a tissue that was more like a connective tissue rather than dental-pulp-like tissue [23Zhang W, Walboomers XF, van Kuppevelt TH, Daamen WF, Bian Z, Jansen JA. The performance of human dental pulp stem cells on different three-dimensional scaffold materials Biomaterials 2006; 27(33): 5658-68.]. All these studies demonstrate that DPSCs can be used in dental tissue engineering. The major issue is to provide the scaffold, which in endodontics could be the sterile root canal inside of which the new pulp could be recreated. The stem cells could be found in the residuals parts of the live pulp if the pulp or other regions of the tooth like the dental apical papilla or the bone of periapical region has not undergone necrosis.

Researchers all over the world are still trying to find a competent scaffold to place DPSCs as well as the ideal microenvironment that will promote their differentiation. Research on the microenviroment and its role on self-renewal, differentiation and proliferation of the cells and tissue are of crucial importance for the future of regenerative medicine.

MESENCHYMAL STEM CELLS FROM DENTAL PULP OF EXFOLIATED DECIDUOUS TEETH (SHED)

In 2003, Miura M et al., presented the existence of multipotent mesenchymal-type stem cells in the dental pulp of the deciduous teeth [15Miura M, Gronthos S, Zhao M, et al. SHED stem cells from human exfoliated deciduous teeth Proc Natl Acad Sci USA 2003; 100(10): 5807-12.]. For their isolation incisors from 7-8 years old children were used and they demonstrated that SHEDs have the ability to differentiate in vitro to neuro-like cells, odontoblasts, osteoblasts, and adipocytes. Based on transplantation experiments in mice, it was shown that they have the capacity to produce dentin, as well as bone forming ability. In contrast to the mesenchymal cells from the adult dental pulp (DPSCs), they were not able to form dentin-pulp complex.

Deciduous teeth are different compared to the permanent teeth regarding developmental processes, tissue structure and function. Thus it is plausible that SHED have a higher proliferation rate, better osteoinductive ability in vivo and inability to form a dental-pulp complex [15Miura M, Gronthos S, Zhao M, et al. SHED stem cells from human exfoliated deciduous teeth Proc Natl Acad Sci USA 2003; 100(10): 5807-12.].

Based on these observations, several studies suggest the use of SHEDs in dental tissue engineering [24Murray PE, Garcia-Godoy F. Stem cell responses in tooth regeneration Stem Cells Dev 2004; 13(3): 255-62., 25Sloan AJ, Smith AJ. Stem cells and the dental pulp: potential roles in dentine regeneration and repair Oral Dis 2007; 13(2): 151-7.]. Cordeiro et al., also suggested that stem cells from deciduous teeth could also be the ideal source of stem cells for repairing damaged teeth or for the induction of bone formation [26Cordeiro MM, Dong Z, Kaneko T, et al. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth J Endod 2008; 34(8): 962-.]. This research group seeded SHEDs on a biodegradable scaffold composed of poly-L-lactic acid (PLLA) prepared inside human tooth slices and transplanted these scaffolds/slices subcutaneously in mice. SHEDs differentiated into odontoblasts and when endothelial cells were co-transplanted the scaffold was vascularised. Thus, SHEDs could be an important source of stem cells for repairing damaged dental tissues and for the induction of bone regeneration. This means that in the future therapeutic approaches the restoration of damaged dentin and pulp could be successful with the use of autologous stem cells of the deciduous dentition which could have been previously extracted and preserved. However, the emerging question is whether we could successfully use heterologous stem cells for such therapeutic approaches.

MESENCHYMAL STEM CELLS FROM THE PERIODONTAL LIGAMENT (PDLSCS)

Periodontal ligament is found between the tooth and the alveolar bone and it consists of fibers that keep the tooth attached to the jaw. It can be isolated from the root of extracted teeth and it contains stem cells that self-renew and differentiate to form other tissues, like cementum and the alveolar bone [16Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament Lancet 2004; 364(9429): 149-55., 27Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. Recovery of stem cells from cryopreserved periodontal ligament J Dent Res 2005; 84(10): 907-12.]. PDLSCs can be differentiated in vitro into adipocytes, osteoblasts and chondrocytes [28Gay IC, Chen S, MacDougall M. Isolation and characterization of multipotent human periodontal ligament stem cells Orthod Craniofac Res 2007; 10(3): 149-60.].

Upon transplantation in mice, these cells formed structures similar to bone, cementum, cartilage and periodontal ligament, whereas in a study in pigs, PDLSCs were used to cure periodontal lesions [29Liu Y, Zheng Y, Ding G, et al. Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine Stem Cells 2008; 26(4): 1065-73.]. In another study, when PDLSCs combined with stem cells from the dental apical papilla, (SCAPs), isolated from the 3rd molar, were seeded in a scaffold and transplanted in the alveolar bone of young pigs, the formation of root and periodontal complex were observed and were able to support an artificial ceramic crown. This restored the normal function of the tooth [18Sonoyama W, Liu Y, Fang D, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine PLoS ONE 2006; 1: e 79-0.].

A more recent study of Orciani et al., showed that human PDLSCs were differentiated into osteoblasts which exhibited a high production of Ca2+ and nitric oxide. Based on these observations they suggest that the local transplantation of proliferating PDLSCs, together with nitric oxide could be a new promising method for the treatment of periodontal lesions [30Orciani M, Trubiani O, Vignini A, Mattioli-Belmonte M, Di Primio R, Salvolini E. Nitric oxide production during the osteogenic differentiation of human periodontal ligament mesenchymal stem cells Acta Histochem 2009; 111(1): 15-24.]. Moreover, PDLSCs that had been previously cultured with APTG-CM (apical tooth germ cells) produced structures of cementum / periodontal ligament, upon transplantation in mice [31Yang ZH, Zhang XJ, Dang NN, et al. Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues J Periodontal Res 2009; 44(2): 199-210.]. It seems that the periodontal ligament is another alternative source of stem cells, which could be used in cellular therapies to restore lesions.

MESENCHYMAL STEM CELLS FROM DENTAL FOLLICLE (DFSCS)

The dental follicle surrounds the developing tooth and plays an important role in the formation of cementum, periodontal ligament and alveolar bone. DFSCs were isolated from the follicle of 3rd molars and it was documented that they can remain in cell cultures until passage 15 [17Morsczeck C, Gotz W, Schierholz J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth Matrix Biol 2005; 24(2): 155-65.]. DFSCs were differentiated into osteoblasts, adipocytes and nerve-like cells in vitro [32Coura GS, Garcez RC, de Aguiar CB, Alvarez-Silva M, Magini RS, Trentin AG. Human periodontal ligament: a niche of neural crest stem cells J Periodontal Res 2008; 43(5): 531-6.-34Yao S, Pan F, Prpic V, Wise GE. Differentiation of stem cells in the dental follicle J Dent Res 2008; 87(8): 767-1.], whilst they were able to form cementum in vivo [35Handa K, Saito M, Tsunoda A, et al. Progenitor cells from dental follicle are able to form cementum matrix in vivo Connect Tissue Res 2002; 43(2-3): 406-8.]. Another study revealed that DFSCs transplantation into mice resulted in the formation of a new periodontal ligament, 4 weeks post-transplantation [36Yokoi T, Saito M, Kiyono T, et al. Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo Cell Tissue Res 2007; 327(2): 301-11.]. These cells could be a new tool for the study of the development of the periodontal ligament and for the development of regenerative therapies and reconstructive treatments.

MESENCHYMAL STEM CELLS FROM APICAL PAPILLA (SCAPS)

SCAPs are the stem cells isolated from the upper dental papilla, which is the precursor tissue of the dental pulp. An important source of SCAPs is the 3rd molars and teeth with open apices.

It has been found that SCAPs can be differentiated into osteoblasts, odondoblasts and antipocytes, in vitro whereas in vivo they can be differentiated into osteoblasts and odondoblasts [37Kikuchi H, Suzuki K, Sakai N, Yamada S. Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture Cell Tissue Res 2004; 317(2): 173-85., 38Ikeda E, Hirose M, Kotobuki N, et al. Osteogenic differentiation of human dental papilla mesenchymal cells Biochem Biophys Res Commun 2006; 342(4): 1257-62.]. It has been demonstrated that human SCAPs possess a higher differentiation rate and are more efficient, compared to DPSCs, for the tooth formation. Furthermore, transplantation of SCAPs and PDLSCs along with ΗΑ/TGT (hydroxyapatite/tricalcium phosphate) as carrier on mice resulted in the formation of dentin and cementum/Sharpey’s fibers, respectively. Based on these results, it is suggested that the combination of dental mesenchymal stem cells could regenerate the root/periodontal ligament complex [18Sonoyama W, Liu Y, Fang D, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine PLoS ONE 2006; 1: e 79-0.]. Thus, SCAPs are a category of cells that take part in the regenerative endodontic therapy (revascularization).

EPITHELIAL AND MESENCHYMAL STEM CELLS IN DENTAL TISSUE ENGINEERING

Given that the tooth is formed from two different tissues, its construction requires the cooperation of dental mesenchymal and epithelial cells. Several trials have been attempted for the reconstruction of teeth in vitro and in vivo using the above combination of stem cells, with encouraging results [39Amar S, Luo W, Snead ML, Ruch JV. Amelogenin gene expression in mouse incisor heterotopic recombinations Differentiation 1989; 41(1): 56-61.,40Yoshiba K, Yoshiba N, Aberdam D, et al. Expression and localization of laminin-5 subunits during mouse tooth development Dev Dyn 1998; 211(2): 164-76.].

Dental mesenchymal and epithelial stem cells originating from rats, pigs and mice were cultured in the lab and seeded on the surface of different biomaterials, prior to transplantation in mice. All the present published reports indicated the formation of dentin and enamel concluding that the combination of dental stem cells could lead to their reorganization, the formation of separate independent layers and their differentiation in odondoblasts and ameloblasts [9Honda MJ, Shimodaira T, Ogaeri T, Shinohara Y, Hata K, Ueda M. A novel culture system for porcine odontogenic epithelial cells using a feeder layer Arch Oral Biol 2006; 51(4): 282-90., 10Honda MJ, Tsuchiya S, Sumita Y, Sagara H, Ueda M. The sequential seeding of epithelial and mesenchymal cells for tissue-engineered tooth regeneration Biomaterials 2007; 28(4): 680-9., 41Honda MJ, Shinohara Y, Hata KI, Ueda M. Subcultured odontogenic epithelial cells in combination with dental mesenchymal cells produce enamel-dentin-like complex structures Cell Transplant 2007; 16(8): 833-47., 42Hu B, Nadiri A, Kuchler-Bopp S, Perrin-Schmitt F, Peters H, Lesot H. Tissue engineering of tooth crown.oot.and periodontium Tissue Eng 2006; 12(8): 2069-75.].

These bioengineered teeth were formed in ectopic site and were characterized by the lack of important elements such as the complete root and the periodontal tissues that allow the correct implantation on the alveolar bone. However, in 2007 Nakao et al., published a study for tooth formation on mouse mandible. In particular, dental epithelial and mesenchymal stem cells were seeded on a drop of collagen gel and implanted in a mouse tooth cavity [43Nakao K, Morita R, Saji Y, et al. The development of a bioengineered organ germ method Nat Methods 2007; 4(3): 227-30.]. The bioengineered tooth germ, lead to the formation of a structurally correct tooth with odondoblasts, ameloblasts, dental pulp, blood vessels, crown, periodontal ligament, root and alveolar bone. It was concluded that the implantation into the mandible allowed the development, the maturation and the emergence of the tooth [43Nakao K, Morita R, Saji Y, et al. The development of a bioengineered organ germ method Nat Methods 2007; 4(3): 227-30.]. These results are an important indication that the dental stem cells could be used for the replacement of a missing tooth, in human.

Another study of 2009, demonstrated the successful functional tooth replacement in mouse by the transplantation of a bioengineered tooth on the alveolar bone, in the area of the missing tooth [44Ikeda E, Morita R, Nakao K, et al. Fully functional bioengineered tooth replacement as an organ replacement therapy Proc Natl Acad Sci USA 2009; 106(32): 13475-80.]. Dental epithelial and mesenchymal molar tooth germ-derived stem cells were cultured and combined with a biomaterial, prior to transplantation into the alveolar bone of the mouse in the area of the missing teeth. In particular, the upper first molar was extracted and the transplantation took place three weeks later, to allow the physical rehabilitation of the dental cavity and oral cavity epithelium. Finally, it was demonstrated that the bioengineered tooth that emerged in the oral area had the correct construction and the hardness of the tissues necessary for chewing (measured by the Knoop hardness test). Furthermore, it responded to experimental orthodontic treatment and to noxious stimuli, such as mechanical stress and pain challenge with various thermal and electrical stimuli. Its size was smaller than normal since the configuration of parameters forming the tooth, such as setting the width of the crown, is not possible with the current techniques.

FIRST AUTOLOGOUS DENTAL TISSUES ENGINEERING CLINICAL TRIAL IN HUMAN

In 2009, d’Aquiono et al. presented the results of their study regarding the restoration of bone loss in the lower jaw, in human, with the implantation of a bio-complex of adult dental pulp mesenchymal stem cells (DPSCs) and a collagen sponge [45d'Aquino R, De Rosa A, Lanza V, et al. Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge biocomplexes Eur Cell Mater 2009; 18: 75-83.]. Initially 17 patients participated in this clinical trial that underwent panoramic radiograph to establish the presence of impacted third molars of the mandible. It was estimated that upon extraction of the 3rd moral of the mandible bilaterally, the patients would present a bone defect, of at least 1.5 cm in height, distal to the 2nd molar secondary to impaction of the third molar on the cortical alveolar lamina. This clinical condition permits slow bone repair after extraction of the third molar, and may eventually lead to loss of the adjacent second molar. An important parameter for the enrolment of the patients in this study was to have two similar lower molars, so that we could use one as a test (T) site and the other one as a control (C) site for the implantation of the biocomplex. Furthermore, the patients should have untouched 3rd molars, the extraction of which was necessary for the isolation of DPSCs.

Upon the extraction of the maxillary 3rd molars DPSCs were isolated and cultured for 21 days. Flow cytometry was performed to detect the protein markers usually found on the cellular surface of DPSCs and were finally placed on collagen sponge. The day of the application, the patients underwent extraction of the mandibulary 3rd molars. In the control site (C), a collagen sponge without DPSCs was placed whereas in the other site, the Test site (T), the biocomplex of collagen sponge filled with DPSCs was positioned. The patients were examined one week post-surgery and they were monitored monthly for the first trimester.

Three months after the surgery the radiographs of the two sites were significantly different. The T site presented a higher rate of mineralization and the cortical level was much higher when compared to the control site (C). The T sites were completely regenerated. No infections or inflammations were observed and the functionality (eg mastication) was normal. Furthermore, a sample from T and C sites was collected for histological and immunofluorescence analyses. In the T sites a well organized and vascularized bone with a lamellar architecture surrounding the Heversian channels was observed, whereas bone from C sites was immature with incomplete and large Haversian channels and signs of bone reabsorption. Immunofluorescent analysis demonstrated the expression of osteonectin, osteocalcin, bone alkaline phosphatase in both sites but with a different allocation, whereas bone morphogenic protein 2 and vascular endothelial growth factor had a higher expression in the T site confirming different levels of bone maturation. In general, this first report of autologous bone regeneration in humans with the use of DPSCs in collagen sponge demonstrates optimal repair of bone defects of the mandible and the restoration of periodontal tissue, below the 2nd molar.

The bones of the maxilla and mandible usually undergo reabsorption following degenerative diseases like periodontal diseases, necrosis of the mandible etc. The authors suggest that DPSCs could be an easy and natural alternative for the restoration/regeneration of damaged tissues and they call them the new “tool” for bone engineering. Of course more clinical trials are necessary before this technique of using autologous stem cells in dental treatments is applied. However, the technique of using stem cell originating from the periapical bone tissue, the dental papillae or the remaining living cells in the pulp cavity to revascularize a sterile pulpal cavity is already applied in endodontics.

DISCUSSION

Tooth loss can cause many problems, like difficulty in chewing and there it can be a problem of aesthetics. The current practises for the replacement of the missing tooth, like implants and classic prosthetic procedures, are based on external materials which may lead to the final loss of teeth. Dental tissue engineering is a new promising therapeutic approach that aims to replace the missing tooth with a bioengineered tooth or to restore the damaged dental tissue. The main principle is the use of dental stem cells, seeded on the surface of biomaterials that provide the proper stimuli to create a biocomplex.

Numerous studies have shown the formation of dental tissues and/or tooth in animal models. A recent clinical trial in humans demonstrated the potential of adult dental mesenchymal stem cells attached on collagen to regenerate bone of the mandible. These results are promising and prove that dental tissue engineering can offer a new opportunity to the individuals that suffer from dental diseases or have lost teeth due to pathological syndromes, traumatic injuries or congenital absence. However, more research is necessary since the application of these techniques could be time-consuming, difficult and expensive and thus not easily accessible to the general public.

The most direct source of dental stem cells seems to be the adult dental pulp and the periodontal ligament. Even though deciduous teeth are the most easily accessible source of dental stem cells very few patients have saved SHEDs, since their isolation and storage is a very recent service. For DFSCs and SCAPs isolation the presence of the third molar is necessary. DPSCs can be isolated both from the third molar and the teeth predestined to have an endodontic treatment and still have a live pulp (50), whereas PDLSCs are isolated from the roots of extracted teeth. Even though DPSCs and PDLSCs exhibit a lower proliferation potential compared to SHEDs, they are an easy and direct source of dental stem cells for the purposed of dental tissue engineering but also for the restoration of other tissues and organs.

The major obstacle that needs to be overcome is the finding of available dental epithelial stem cells, since their combination with the dental mesenchymal stem cells seems to be the technique with the best results when trying to create a biotooth. Even though the isolation of dental epithelial stem cells for newborn or young animal is feasible, their use in humans is impossible and potentially hazardous, given that it can cause immunoreactions and rejection. Dental epithelial stem cells could be isolated from the tooth germ of childrens’ third molar and be used or saved for future use. Since this particular practice refers the child to surgery it is not ethical and thus not an easily applicable technique. Furthermore, in the case of adults, it still remains a problem, since dental epithelial stem cells are already lost after the eruption of the teeth.. According to current options, the solution of the problem of enamel regeneration could be the use of an artificial crown, which will be supported for a teeth originating for the mesenchymal stem cells.

Many in vitro and in vivo studies demonstrate encouraging and promising results for the potential future use of dental tissue engineering techniques. However, more research is needed to better understand odontogenesis and resolve the existing obstacles. The future establishment of these techniques will change the therapeutic approach of many of the dental syndromes and diseases.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENT

Declared none.

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