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 Table of Contents  
Year : 2013  |  Volume : 3  |  Issue : 1  |  Page : 32-36

A review of tooth regeneration

1 Department of Prosthodontics, People's College of Dental Sciences and Research Centre, Bhanpur, Bhopal, Madhya Pradesh, India
2 Department of Pedodontics, People's College of Dental Sciences and Research Centre, Bhanpur, Bhopal, Madhya Pradesh, India

Date of Web Publication26-Nov-2013

Correspondence Address:
Rucha Kashyap
Department of Prosthodontics, People's College of Dental Sciences and Research Centre, Bhanpur, Bhopal - 462 010, Madhya Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2231-6027.122101

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Regeneration of dental tissue is one of the highly demanding areas, in which lot of research is going on. Trauma, genetic disorders and malformations have created the need for functional, mechanical and esthetic replacement of missing teeth. Current methods of tooth replacement including dental implants, tooth transplantation and Prosthodontics are not considered permanent and significant complications exist with these methods. Ideally, the altered signaling cascade that interrupts tooth development could be treated, or an autogenous tooth replacement developed. This review discusses, so as to better understand multiple approaches and procedures of tooth regeneration.

Keywords: Missing teeth, stem cells, tooth regeneration

How to cite this article:
Kashyap R, Shashikiran N D. A review of tooth regeneration. Int J Oral Health Sci 2013;3:32-6

How to cite this URL:
Kashyap R, Shashikiran N D. A review of tooth regeneration. Int J Oral Health Sci [serial online] 2013 [cited 2023 Feb 2];3:32-6. Available from: https://www.ijohsjournal.org/text.asp?2013/3/1/32/122101

  Introduction Top

Trauma, genetic disorders and malformations have created the need for functional, mechanical and esthetic replacement of missing teeth. Tooth agenesis is the most prevalent craniofacial congenital malformation in humans. [1] At least 25% of the population may lose one-third molar, [1] agenesis of other permanent teeth ranges from 1.6% to 9.6% depending upon the population studied. [2] Traditionally, tooth agenesis is classified depending upon the number of missing teeth as either hypodontia [3] (up to six missing teeth) or oligodontia [4],[5] (more than six missing teeth). Current methods of tooth replacement include dental implants, tooth transplantation and prosthodontics; however, these options are not considered permanent and significant complications exist with these methods. [6],[7] Ideally, the altered signaling cascade that interrupts tooth development could be treated, or an autogenous tooth replacement developed. This paper discusses and reviews, so as to better understand multiple approaches and procedures of tooth regeneration.

Regeneration of dental tissue is one of the highly demanding areas, in which lot of research is going on. During regeneration of skin, bone, or cartilage, the size and shape of these tissues can be controlled by using scaffolds; on the other side it is difficult to control the size and shape of regenerated dental tissues. Tooth development includes reciprocal interactions between the epithelium and mesenchyme, which is governed by the expression of sonic hedgehog, fibroblast growth factors, bone morphogenetic proteins and wingless (Wnt) signaling families. [8],[9],[10] By controlling the interactions between the dental epithelial and mesenchymal cell and thereby tooth morphogenesis, the issues of controlling the size and shape of regenerated dental tissues can be resolved. Functional tooth was developed from transplanted tooth buds from re-aggregated E14.5 mouse tooth bud cells. [11],[12],[13],[14],[15] During the procedure of tooth regeneration cells are obtained by harvesting early stage embryonic tooth buds. For practical purposes of tooth regeneration, it is difficult to obtain autologous human embryonic dental cells, so the current challenge remains to invent reliable methods to regenerate teeth of predetermined anatomic shape and size, using cells derived from post-natal tissues. Human ameloblasts transform into human dental enamel after sequential steps. Hence, the human dental epithelial cells are difficult to obtain for regeneration purposes due to the fact that dental epithelial cells or ameloblasts have largely undergone apoptosis prior to tooth eruption by transforming into dental enamel.

  Molecular Approach for Regeneration of Missing Teeth Top

During tooth development, altered gene expression has widespread effects on other signaling pathways and may result in the cessation of tooth development. Many different approaches are being undertaken to treat different etiologies of tooth agenesis. In cases, where growth factors are absent, post-natal supplements have been proposed, while in cases of tooth avulsion or the lack of development, the growth of complete bioengineered teeth is being attempted. In one of the first studies of its kind, fetal mice with ectodermal dysplasia (ED) were treated with a recombinant Eda protein. [16] Eda was attached to the Fc portion of human immunoglobulin G (IgG). The Fc-Eda recombinant complex was stable, aggregated and capable of transplacental delivery. It was injected intravenously into the pregnant mother at set intervals. Although the mother appeared unaffected during pregnancy, all of her offspring reverted to the wild-type-like phenotype. Though, the Fc-Eda restored dental anatomy, third molars were still frequently absent. In a similar study, canine dogs with ED were treated intravenously with Fc-Eda after birth. [17] Most of the dogs that received an extended treatment of Fc-Eda responded well, developed a normal dentition and eliminated or reduced most other ED symptoms.

Currently, there are two main approaches to complete tooth generation. The first method is to develop a bio-degradable scaffold that cells will be seeded onto. In one study, a tooth bud from a rat was removed, the cells were isolated, single-cell isolates were seeded onto a polymer scaffold which was grown in a rat omentum and the teeth were removed after 12 weeks. [18] The resulting teeth exhibited mineralization and proper color, shape and size. Despite the promise of this scaffold study, the growth patterning was not fully understood and application of this technology would require tooth bud isolation during early development. Another significant complication is the development of a biocompatible, resorbable and drug delivering scaffold.

The second method for tooth regeneration is to isolate and transplant stem cells to establish epithelial-mesenchymal interactions and reconstruct the tooth germ. Five major sources of stem cells have been proposed: Dental pulp stem cells (DPSCs); stem cells from human exfoliated deciduous (SHEDs) teeth; periodontal ligament stem cells (PDLSCs); stem cells from the apical root papilla (SCAPs); dental follicle stem cells (DFSCs) [Table 1], [Figure 1]. DPSCs from extracted third molars have been shown to contain pluri-potent DPSCs that have the ability to expand into odontoblasts, osteoblasts, chondrocytes and adipocytes. [19],[20],[21],[22] In vitro, studies have demonstrated the ability for DPSCs to form calicific nodules with functional dental tissue with enamel, dentine and pulp-like complexes. [21],[22] SHEDs have increased population doublings, osteo-inductive capacity and higher population rates than DPSCs. [19],[20],[21],[22] PDLSCs may differentiate along mesenchymal cell lineages to form cementoblast-like cells, adipocytes and collagen. [19],[21] SCAPs are isolated prior to tooth eruption (such as the 3 rd molar); these cells can differentiate into odontoblasts and adipocytes and show higher rates of proliferation than DPSCs. When SCAPs and PDLSCs were implanted into an alveolar socket of a pig, dentine and a periodontal ligament (PDL) were found, suggesting the possible development of a natural tooth root implant with a PDL rather than a titanium screw implant. [21] DFSCs are isolated from the epithelium and mesenchyme prior to eruption and may form cementoblast-like cells, PDL and osteoblasts. One study isolated the epithelially activated mesenchyme, reorganized the cells, recombined the cells with epithelium and placed the epithelial and mesenchymal cells in a mouse renal capsule. [23] After allowing time to develop, the teeth were removed and shown to contain enamel, dentine and pulp tissue (not cementum). The clinical application to transform these stem cells into teeth appears promising; however, more research is needed to study the effects of combining multiple lineages of stem cells to facilitate the accelerated development of an entire tooth.
Figure 1: Stem cell harvest sites. This figure depicts a developing tooth and stem cell harvest sites: dental follicle stem cells, dental pulp stem cells, periodontal ligament stem cells and stem cells from the apical root papilla

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Table 1: Proposed stem cells for tooth regeneration

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  Most Successful Models for Tooth Regeneration Top

Dissociated cell-scaffold model, combined enamel organ derived epithelial cell layer and pulp organ derived mesenchymal cell model and collagen gel drop model. [24]

Dissociated cell-scaffold model

The dissociate cell scaffold method seeds mixed population of dental epithelial and mesenchymal cells, either pre-mixed or sequentially added to the scaffold or onto the 3D scaffolds. Using this approach, multiple teeth were formed in one construct and were smaller than naturally formed teeth.

Combined enamel organ-derived epithelial cell and pulp organ derived mesenchymal cell model

Combines an intact tooth bud enamel organ with dissociated dental mesenchymal (DM) cells. Although the teeth that formed using this model appear quite similar to naturally formed teeth, it is not a practical approach for human tooth regeneration, due to the fact that it is difficult, if not impossible, to obtain autologous human enamel organ tissues.

Collagen gel drop model

Dissociated dental epithelial (DE) and DM cells harvested from E14.5 tooth buds are injected into adjacent regions within a collagen gel drop. Although this model was successful using embryonic stage tooth bud cells, no positive results have been reported for similar approach using the post-natal dental cells.

Sequence of Procedures in Teeth Regeneration

  • Cell collection
  • Preparation of bioengineered germs
  • Transplantation of tooth germs.
Cell collection

To regenerate tooth structure, cells are required, the genetic material is modified in these cells and directed to regenerate specific tissues and later transplanted into an animal tissues to study the regeneration process. Cells can be collected from, homologous (e.g., rat epithelial + rat mesenchymal), heterogeneous (e.g., rat epithelial + human adult mesenchymal), entire dental tissue or dissociate cells from embryonic tooth buds, dental cells or non-dental cells, DPSC - DPSCs from permanent or deciduous teeth or bone marrow stem cells. [25],[26]

Preparation of bioengineered tooth germs

The cells collected to use for regeneration will not have a code to regenerate to produce specific target tissues, so various biochemical modifications are done in the genetic structure of source cells and transplanted to produce specific tissues. [25],[26] The steps include:

  • Cloning and cell culture
  • Immunocytological and histochemical treatments
  • Ribonucleic acid (RNA) isolation and polymerase chain reaction (PCR) analysis.
Cloning and cell culture

Once cells are collected they should be detached from their basement membranes, using various enzymes, if the source cells are collected from tissues attached to bone, demineralization should be done to remove the bone structure. Examples of materials used are enzymes e.g., Trypsin, Collagenase etc., Demineralization agents like ethylene diamine tetra acetic acid (EDTA).

Nutrition is supplied to cells by growing these cells on various media like Dulbecco's modified Eagle's medium, Ham's nutrient mixture, fetal calf/Bovine sera, Anterior chamber of eye works as an excellent culture system. To prevent infections cells are treated with various Antibiotics like penicillin, streptomycin and other useful agents are insulin, transferrin, cholera toxin and sodium selenite etc. [26]

Immunocytological and histochemical treatment

To prevent host cell response, after cell collection all the cells are treated so that the cells do not express any surface proteins. The cells are treated with primary antibiotics such as Anti-cytokeratin 14 (K14), Anti-K18, Anti-vimentin, mouse monoclonal antibodies, Anti-amelogenin, rabbit polyclonal antibody and secondary antibodies like fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG serum, FITC-conjugated anti-rabbit IgG serum. [27]

  RNA Isolation And RT-PCR Analysis Top

Include RNA extraction from cell line using systems like SV total RNA isolation system. Reverse transcription is performed using first strand complementary deoxyribonucleic acid (cDNA) synthesis kit. First strand of cDNA is then used as a template for PCR with following primer sets; amelogenin, antisense, ameloblastin, antisense, Msx 2, antisense, NGFRp75, antisense and b-actin, antisense. Then PCR amplification is performed with Taq polymerase, finally amplified products are analyzed by electrophoresis. [25],[26]

  Transplantation Top

It is of two types, orthotopic transplantation and ectopic transplantation. Orthotopic transplantation is transplanting bio-engineered tooth germ or scaffold in to a freshly extracted teeth site. Ectopic transplantation is transplanting into areas like renal capsule of nude mice, omentum of mice, dorsum of mice, jawbone, or any area other than usual alveolar socket. [12]

  Slide Preparation Top

As the regeneration of the whole tooth is not yet complete, evaluation of regeneration method is essential. For this purpose, slide preparation is essential. Few weeks later, in both types of trans plantations, tissues are dissected out and fixed in, solutions like 4% of paraformaldehyde, de-mineralized with 4.5% EDTA in ultra-pure water, dehydrated in graded ethanol, embedded in paraffin and sectioned to 6-8 μ. Once slides are prepared, they can be stained with stains like Hematoxylin and Eosin, Goldner's trichrome and 1% of alizarin red; Von Kossa staining. [12],[25],[26],[28]

  Conclusion Top

Multiple approaches are being studied for replacement of missing teeth. Preliminary trials of signaling factor supplementation with animals that have ED appear promising, while treatment options for the other genetic disorders still must be elucidated. Remarkable knowledge has been gained from previous studies in tooth morphogenesis and many laboratories are currently investigating various aspects of tooth morphogenesis. Other studies are developing methods to grow new teeth with different dental stem cell populations. Future studies will need to examine the effect of growth factor supplementation on tooth development and experiment with stem cell population combinations to grow functional teeth. The study of tooth development is now stepping into a new dimension in which all the applicable techniques could be used for producing a human tooth.

  References Top

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3.Arte S, Nieminen P, Pirinen S, Thesleff I, Peltonen L. Gene defect in hypodontia: Exclusion of EGF, EGFR, and FGF-3 as candidate genes. J Dent Res 1996;75:1346-52.  Back to cited text no. 3
4.Schalk-van der Weide Y, Steen WH, Bosman F. Distribution of missing teeth and tooth morphology in patients with oligodontia. ASDC J Dent Child 1992;59:133-40.  Back to cited text no. 4
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8.Thesleff I, Jernvall J. The enamel knot: A putative signaling center regulating tooth development. Cold Spring Harb Symp Quant Biol 1997;62:257-67.  Back to cited text no. 8
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10.Yen AH, Sharpe PT. Stem cells and tooth tissue engineering. Cell Tissue Res 2008;331:359-72.  Back to cited text no. 10
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12.Kim K, Lee CH, Kim BK, Mao JJ. Anatomically shaped tooth and periodontal regeneration by cell homing. J Dent Res 2010;89:842-7.  Back to cited text no. 12
13.Ikeda E, Morita R, Nakao K, Ishida K, Nakamura T, Takano-Yamamoto T, et al. Fully functional bioengineered tooth replacement as an organ replacement therapy. Proc Natl Acad Sci U S A 2009;106:13475-80.  Back to cited text no. 13
14.Mantesso A, Sharpe P. Dental stem cells for tooth regeneration and repair. Expert Opin Biol Ther 2009;9:1143-54.  Back to cited text no. 14
15.Nakao K, Morita R, Saji Y, Ishida K, Tomita Y, Ogawa M, et al. The development of a bioengineered organ germ method. Nat Methods 2007;4:227-30.  Back to cited text no. 15
16.Gaide O, Schneider P. Permanent correction of an inherited ectodermal dysplasia with recombinant EDA. Nat Med 2003;9:614-8.  Back to cited text no. 16
17.Casal ML, Lewis JR, Mauldin EA, Tardivel A, Ingold K, Favre M, et al. Significant correction of disease after postnatal administration of recombinant ectodysplasin A in canine X-linked ectodermal dysplasia. Am J Hum Genet 2007;81:1050-6.  Back to cited text no. 17
18.Duailibi MT, Duailibi SE, Young CS, Bartlett JD, Vacanti JP, Yelick PC. Bioengineered teeth from cultured rat tooth bud cells. J Dent Res 2004;83:523-8.  Back to cited text no. 18
19.Peng L, Ye L, Zhou XD. Mesenchymal stem cells and tooth engineering. Int J Oral Sci 2009;1:6-12.  Back to cited text no. 19
20.Sloan AJ, Waddington RJ. Dental pulp stem cells: What, where, how? Int J Paediatr Dent 2009;19:61-70.  Back to cited text no. 20
21.Volponi AA, Pang Y, Sharpe PT. Stem cell-based biological tooth repair and regeneration. Trends Cell Biol 2010;20:715-22.  Back to cited text no. 21
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23.Yamamoto H, Kim EJ, Cho SW, Jung HS. Analysis of tooth formation by reaggregated dental mesenchyme from mouse embryo. J Electron Microsc (Tokyo) 2003;52:559-66.  Back to cited text no. 23
24.Zhang W, Ahluwalia IP, Yelick PC. Three dimensional dental epithelial-mesenchymal constructs of predetermined size and shape for tooth regeneration. Biomaterials 2010;31:7995-8003.  Back to cited text no. 24
25.Honda MJ, Tsuchiya S, Sumita Y, Sagara H, Ueda M. The sequential seeding of epithelial and mesenchymal cells for tissue-engineered tooth regeneration. Biomaterials 2007;28:680-9.  Back to cited text no. 25
26.Komine A, Suenaga M, Nakao K, Tsuji T, Tomooka Y. Tooth regeneration from newly established cell lines from a molar tooth germ epithelium. Biochem Biophys Res Commun 2007;355:758-63.  Back to cited text no. 26
27.Yu J, Shi J, Jin Y. Current approaches and challenges in making a bio-tooth. Tissue Eng Part B Rev 2008;14:307-19.  Back to cited text no. 27
28.Arany S, Kawagoe M, Sugiyama T. Application of spontaneously immortalized odontoblast cells in tooth regeneration. Biochem Biophys Res Commun 2009;381:84-9.  Back to cited text no. 28


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  [Table 1]


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