RETRACTED ARTICLE: Stem Cell Mediated Tooth Regeneration: New Vistas in Dentistry

Abstract

The generation of dental structures and/or entire teeth in the laboratory depends upon the manipulation of stem cells and requires a synergy of all cellular and molecular events that finally lead to the formation of tooth-specific hard tissues, dentin and enamel. This review focuses on the different sources of stem cells that have been used for making teeth in vitro and their relative efficiency. Embryonic, post-natal and adult stem cells were assessed and proved to possess an enormous regenerative potential, but their application in dental practice is still limited due to various parameters that are not yet under control such as the high risk of rejection, cell behaviour, long tooth eruption period, appropriate crown morphology and suitable colour. Nevertheless, the development of biological approaches for dental reconstruction using stem cells is promising and remains one of the greatest challenges in the dental field.

Introduction

Tooth loss or absence is a common and frequent situation that can result from numerous pathologies such as periodontal and carious diseases, fractures, injuries or even genetic alterations. In most cases this loss is not critical, but for aesthetic, psychological and medical reasons (e.g. genetic aberrations) replacement of missing teeth is important. Recent efforts made in the field of biomaterials have led to the development of dental implants composed of biocompatible materials such as titanium that can be inserted in the maxillary and/or mandibular bone to replace missing teeth. However, implants are still not completely satisfactory and their successful use greatly depends on their osteointegration. The quantity and quality of the bone, as well as its interaction with the surface of the implant are some crucial parameters that can influence the success of implant placement. Although innovative materials and techniques (e.g. surface treatment) have been used for the improvement of implant osteointegration [1], the metal/bone interface does not ensure complete integration of the implant, thus reducing its performance and long-term stability. Furthermore, dental implant technology is dependent on bone volume, as devices can be implanted only in patients possessing a sufficient amount of bone. Quite often, there is a need for alveolar bone volume increase before any implant fixing. To overcome these difficulties, new ideas and approaches have emerged recently from the quickly developing fields of stem cell technology and tissue engineering.

Tooth Regeneration and Tissue Engineering

Tooth regeneration has been a major goal of tissue engineering in the dental field. Efforts to regenerate teeth have been conducted for decades. Studies have suggested that adult stem cell populations can form bone and teeth by tissue-engineering techniques and imply that a pure population of stem cells is not necessary. This may have important implications for the further development of these procedures in humans.

Discovery that human mature pulp tissue contains a population of multi-potent mesenchymal dental pulp stem cells with high proliferative potential for self renewal and the ability to differentiate into functional odontoblast has revolutionized dental research and opened new avenues in particular for reparative and reconstructive dentistry and tissue engineering in general.

Tissue engineering and regenerative medicine needs three major ingredients [2] which are:

1. Morphogenic signals such as growth factors and differentiation factors. These factors play an important role in the multiplication and differentiation of stem cells into the specifically needed type of cells. BMPs (bone morphogenic proteins) and cytokines play a major role in organogenesis, and in the dental aspect, specifically GDf-11 (growth/differentiation factor 11) which is a novel member of BMP/TGF family and is expressed in differentiating odontoblasts. It plays a major role in differentiation of dental pulp stem cells into odontoblasts which is the corner stone in teeth tissue engineering.

2. Responding stem cells which are originally harvested from the patient and preserved under good conditions to maintain their special ability to differentiate into a wide range of cells.

3. Scaffold of extra cellular matrix, which provide these cells with the environment and mold to grow into what we want them to become and function.

Three approaches have been investigated by different labs to implant stem cells from teeth in humans [3] and these are:

1. Placing the stem cell into a mold of tooth crown which is made of enamel-like substance with a scaffold material and this scaffold is will be implanted elsewhere in the body and once they are mature, these teeth are extracted and implanted in the oral cavity.

2. Harvesting wisdom teeth of a person and releasing stem cells from their pulp tissue. The stem cells are then implanted in a severely injured tooth, and these stem cells will help to regenerate the pulp of the injured teeth sparing them root canal treatments.

3. If there are no teeth present in the oral cavity from which stem cells can be harvested, stem cells can be harvested from unerupted wisdom tooth.

Stem Cells

The term stem cell was proposed for scientific use by Russian histologist Alexander Maksimov in 1908. In general there are two broad types of stem cells which are: embryonic stem cells (ESCs), and adult stem cells. The main principles in the definition of stem cell include the following: (1) self-renewal, or the ability to generate at least one daughter cell with characteristics similar to the initiating cell, (2) multilineage differentiation of a single cell, and (3) in vivo functional reconstitution of a given tissue or cell type.

Stem Cell Types and Sources

Stem cells are immature, undifferentiated cells that can divide and multiply for an extended period of time, differentiating into specific types of cells and tissues. Autogenous stem cells are derived from the patient being treated, while allogenous stem cells are derived from other individuals. Stem cells available commercially are currently mainly allogenous (donor-derived). While it is believed that allogenous stem cells will not produce an immune response, this is not known with certainty. Autogenous stem cells, on the other hand, reduce the risk of rejection and, provided they are handled correctly, remove the risk of cross-infection from allogenous transplanted tissue. In addition, autologous stem cell transplant recipients will not require immunosuppressive drugs to combat rejection [4]. Stem cells may be totipotent, multipotent or unipotent; i.e. be able to differentiate into any tissue, several types of tissue or one type of tissue, respectively. The process by which stem cells are derived from one type of tissue and differentiate into other types of tissue is referred to as plasticity or transdifferentiation. Multipotent stem cells consist of three major types—ectodermal, mesodermal or mesenchymal and endodermal. The two main categories of stem cells are ESCs and adult stem cells, defined by their source.

Embryonic Stem Cells

ESCs are derived from the cells of the inner cell mass of the blastocyst. ESCs are characterized as being pluripotent, that is, they have the potential to develop into many tissues in the body. As the embryo develops, ESCs begin down a path of differentiation and maturity, at which time they lose this potential [5]. A potential disadvantage of ESCs is their ability to differentiate into any cell lineage and to proliferate endlessly unless controlled.

Adult Stem Cells

Adult stem cells are defined as the undifferentiated cells that are found in a differentiated adult tissue, residing in a specific area of each tissue where they remain quiescent in the body until they are activated by epigenetic and/or environmental factors, such as mechanical forces, disease, or trauma [6, 7]. These cells have been identified in the craniofacial complex including stem cells from craniofacial bone, dental pulp, periodontal ligament, and developing tooth bud. Adult stem cell populations serve to regenerate multiple tissues that are damaged or are in need of repair or regeneration.

Induced Pluripotent Stem Cells (iPS)

iPS cells are adult or somatic stem cells that have been coaxed to behave like ESCs. iPS cells have the capacity to generate a large quantity of stem cells as an autologous source that can be used to regenerate patient-specific tissues [8, 9]. Recent reports have cautioned that any carcinogenic potential of iPS cells should be fully investigated before any commercialization can be realized.

Amniotic Fluid-Derived Stem Cells (AFSCs)

AFSCs can be isolated from aspirates of amniocentesis during genetic screening. An increasing number of studies have demonstrated that AFSCs have the capacity for remarkable proliferation and differentiation into multiple lineages such as chondrocytes, adipocytes, osteoblasts, myocytes, endothelial cells, neuron like cells and live cells [10–15]. The potential therapeutic value of AFSCs remains to be discovered.

Umbilical Cord Blood Stem Cells (UCBSCs)

UCBSCs are derived from the blood of the umbilical cord. There is a growing interest in their capacity for self-replication and multilineage differentiation, and UCBSCs have been differentiated into several cell types that resemble cells of the liver, skeletal muscle, neural tissue, pancreatic cells, immune cells and mesenchymal stem cells (MSC) [16–24]. The greatest disadvantage of UCBSCs is that there is only one opportunity to harvest them from the umbilical cord at the time of birth. Similarly, amniotic stem cells can be sourced only from amniotic fluid and are therefore subject to time constraints.

Bone Marrow-Derived Stem Cells (BMSCs)

BMSCs consist of both hematopoietic stem cells that generate all types of blood cells and stromal cells (MSC) that generate bone, cartilage and other connective tissues and fat [25]. BMSCs are currently the most common commercially available stem cell. They can be isolated from bone marrow aspiration or from the collection of peripheral blood-derived stem cells following chemical stimulation of the bone marrow, by means of subcutaneous injection, to release stem cells.

Adipose-Derived Stem Cells (ASCs)

ASCs are typically isolated from lipectomy or liposuction aspirates. They have been differentiated into adipocytes, chondrocytes, myocytes, and neuronal and osteoblast lineages, and may provide hematopoietic support [26–31]. While ASCs have an advantage in that adipose tissue is plentiful in many individuals, accessible and replenishable, the ability to reconstitute tissues and organs using ASCs versus other adult stem cells has yet to be comprehensively compared and documented.

Dental Stem Cells (DSCs)

DSCs can be obtained from the pulp of the primary and permanent teeth, from the periodontal ligament, and from other tooth structures. Periodontal ligament derived stem cells are able to generate periodontal ligament and cementum. Extracted third molars, exfoliating/extracted deciduous teeth and teeth extracted for orthodontic treatment, trauma or periodontal disease are all sources of dental stem cells from the dental pulp. The dental pulp offers a source of stem cells postnatally that is readily available, with minimally invasive process that results in minimal trauma [32–37]. Exfoliating or extracted deciduous teeth offer extra advantages over other teeth as a source of stem cells. Stem cells from deciduous teeth have been found to grow more rapidly than those from other sources, and it is believed that this is because they may be less mature than other stem cells found in the body [35, 37–39]. Additional advantages of sourcing stem cells from exfoliating deciduous teeth are that the cells are readily available, provided they are stored until they may be needed later in life; the process does not require a patient to sacrifice a tooth to source the stem cells; and there is little or no trauma.

Various Sources for Dental Stem Cells

Stem Cells from Human Exfoliated Deciduous Teeth (SHED)

Miura et al. [39] stated that post-natal stem cells can be isolated from the pulp of human deciduous incisors. These cells were named SHED (stem cells from human exfoliated deciduous teeth) and exhibited a high plasticity since they could differentiate into neurons, adipocytes, osteoblasts and odontoblasts. In vivo SHED cells can induce bone or dentin formation but, in contrast to dental pulp, DPSC failed to produce a dentin–pulp complex.

Adult Dental Pulp Stem Cells (DPSC)

It has been shown that adult dental pulp contains precursors capable of forming odontoblasts under appropriate signals [6, 39–44]. Dental pulp progenitors have not been clearly identified but some data suggest that pericytes, which are able to differentiate into osteoblasts, could also differentiate into odontoblasts [42, 45, 46]. Tooth repair is a lifetime process thus suggesting that MSC might exist in adult dental pulp. The in vivo therapeutic targeting of these adult stem cells remains to be explored.

Stem Cells from the Apical Part of the Papilla (SCAP)

Sonoyama et al. [47] stated that stem cells from the apical part of the human dental papilla (SCAP) have been isolated and their potential to differentiate into odontoblasts was compared to that of the periodontal ligament stem cells (PDLSC). SCAP exhibit a higher proliferative rate and appears more effective than PDLSC for tooth formation. Importantly, SCAP are easily accessible since they can be isolated from human third molars.

Stem Cells from the Dental Follicle (DFSC)

DFSC have been isolated from follicle of human third molars. These cells can be maintained in culture for at least 15 passages. DFSC’s can differentiate into cementoblasts in vitro [48] and are able to form cementum in vivo [49]. Immortalized dental follicle cells are able to re-create a new periodontal ligament (PDL) after in vivo implantation [50].

Periodontal Ligament Stem Cells (PDLSC)

Continuous regeneration of the PDL is thought to involve mesenchymal progenitors arising from the dental follicle. PDL contains cells that maintain certain plasticity since they can adopt adipogenic, osteogenic and chondrogenic phenotypes in vitro [51]. It is thus obvious that PDL itself contains progenitors, which can be activated to self-renew and regenerate other tissues such as cementum and alveolar bone [52].

Bone Marrow Derived Mesenchymal Stem Cells (BMSC)

BMSC have been tested for their ability to recreate periodontal tissue. These cells are able to form in vivo cementum, PDL and alveolar bone after implantation into defective periodontal tissues. Thus, bone marrow provides an alternative source of MSC for the treatment of periodontal diseases [53]. BMSC share numerous characteristics with DPSC and are both able to form bone-like or tooth-like structures. However, BMSC display a lower odontogenic potential than DPSC [54].

Epithelium-Originated Dental Stem Cells

No information is currently available for dental epithelial stem cells (EpSC) in humans. The major problem is that dental epithelial cells such as ameloblasts and ameloblasts precursors are eliminated soon after tooth eruption. Therefore, epithelial cells that could be stimulated in vivo to form enamel are not present in human adult teeth. Stem cell technology appears to be the only possibility to re-create an enamel surface [55].

EpSC from Developing Molars

Several studies describe the use of EpSC isolated from newborn or juvenile animals, usually from third molar teeth. In these studies, epithelia were removed and cells dissociated enzymatically. Precursors were then amplified and associated with MSC (originated from the same tooth). These approaches are promising for tooth formation and/or regeneration [55]. However, the clinical application is difficult, if not unrealistic, since it would require the donation of a tooth germ from children. The use of autologous stem cells is desirable but raises the question of a good and reliable source.

Tooth Organogenesis—Current Scenario

Teeth are highly mineralized organs resulting from sequential and reciprocal interactions between the oral epithelium and the underlying cranial neural crest-derived mesenchyme [56]. Tissue recombination experiments have shown that the oral epithelium contains the inductive capability for odontogenesis. This potential allows conditioning of the underlying mesenchyme, which in turn regulates the differentiation of epithelial cells. Numerous growth factors have been shown to be involved in different stages of the embryonic tooth development (i.e. initiation, morphogenesis, cytodifferentiation). Members of the transforming growth factor beta (TGF) superfamily such as bone morphogenic protein 2 (BMP-2) and BMP-4 are key signalling molecules in regulating epithelial mesenchymal interactions during odontogenesis.

Two major cell types are involved in dental hard tissue formation: the mesenchyme-originated odontoblasts that are responsible for the production of dentin and the epithelium-derived ameloblasts that form the enamel [55]. The aim of regenerative medicine is to stepwise re-create in vitro all the mechanisms and processes that nature uses during initiation and morphogenesis of a given organ. The possibility of manipulating stem cells in situ using specific signalling molecules or by expanding them ex vivo is an exciting outcome of basic research. As tooth formation results from epithelial–mesenchymal interactions, two different populations of stem cells have to be considered: EpSC, which will give rise to ameloblasts, and MSC that will form the odontoblasts, cementoblasts, osteoblasts and fibroblasts of the periodontal ligament. Thus, tooth engineering using stem cells is based on their isolation, association and culture as recombinants in vitro or ex vivo conditions to assess firstly tooth morphogenesis and secondly cell differentiation into tooth specific cells that will form dentin, enamel, cementum and alveolar bone. Understanding the microenvironment of adult stem cells and their regulation is the key for the successful reproduction and for the ex vivo engineering of an organ with ensured functional homeostasis.

Interaction Between EpSC and MSC

Building a tooth logically requires the association/cooperation of odontogenic mesenchymal and epithelial cells. The recombination of dissociated dental epithelial and mesenchymal tissues leads to tooth formation both in vitro and in vivo [57]. Numerous attempts have been made in order to form teeth in vivo with very promising results. Single cell suspensions obtained from rat, pig or mice tooth germs have been seeded onto the surface of selected biomaterials (e.g. collagen-coated polyglycolic acid, calcium phosphate material, collagen sponges) and successfully re-implanted into the omentum of immunocompromised animals [58, 59]. All these reports describe the presence of both dentin and enamel. This indicates that the recombined cells could re-organize themselves and form individual layers and, furthermore, that they can differentiate properly into odontoblasts and ameloblasts.

Making entire teeth with enamel and dentin structures in vivo is a reality. However, these bioengineered teeth have been produced in ectopic sites and are still missing some essential elements such as the complete root and periodontal tissues that allow correct anchoring into the alveolar bone. Recently, a new approach has been proposed for growing teeth in the mouse mandible [60]. In this study, epithelial and mesenchymal cells were sequentially seeded into a collagen gel drop and then implanted into the tooth cavity of adult mice. With this technique the presence of all dental structures such as odontoblasts, ameloblasts, dental pulp, blood vessels, crown, periodontal ligament, root and alveolar bone could be observed [60]. Thus, the implantation of these tooth germs in the mandible allowed their development, maturation and eruption indicating that stem cells could be used in the future for the replacement of missing teeth in humans.

Conclusions

Taken together, these findings clearly indicate that the control of morphogenesis and cytodifferentiation is a challenge that necessitates a thorough understanding of the cellular and molecular events involved in development, repair and regeneration of teeth. The identification of several types of epithelial and mesenchymal stem cells in the tooth and the knowledge of molecules involved in stem cell fate is a significant achievement. In vitro and in vivo experiments using these cells have provided promising results illustrated by the generation of a complete tooth with all dental structures including cells and extracellular matrix deposition. Clinical usage of this technology still remains a question. The use of animal cells for human diseases is restricted by immune rejection risks. Additionally, isolating autologous stem cells requires a source of easily accessible cells without the need for a surgery. It may be possible to replace dental mesenchymal stem cells with stem cells of another origin. At present, it does not appear that this is the case for EpSC. A reliable source of EpSC for that purpose remains to be determined. The engineering of tridimensional matrices (either polylactic acid polymers or collagen sponges) which are a composition more or less similar to that of the organs to reconstruct, and the addition of growth factors such as FGF, BMP or PDGF might facilitate the transplantation and the differentiation of stem cells. However, the engineering of tooth substitutes is hard to scale up, costly, time-consuming and incompatible with the treatment of extensive tooth loss. Scientific knowledge is not enough and the main challenge in stem-cell therapy is to find a compromise between the benefits to the patients, regulatory agencies, increased stem cell requirements and the cost of the procedure.