Whole-Tooth Regeneration: It Takes a Village of Scientists, Clinicians, and Patients

Vision of a Solution: A Clinical Procedure for Tooth Regeneration

We looked to developmental biology, coupled with engineering, for the strategies needed to regenerate human teeth. We hypothesized that, by recapitulating the developmental signals from canonical teeth, naïve stem cells could be induced to odontogenic potential (Figure 3⇓). Epithelial-mesenchymal interactions are the hallmark of tooth development. These interactions are characterized by the reciprocal exchange of signals between these two naïve germ layer tissues and result in the emergence of unique terminal phenotypes with their supporting cells (Figure 3A⇓).^1,2 The simplicity of back and forth signals was the leverage point for our regeneration strategy.

Figure 3.

Rationale for regenerating a human tooth

Panel A defines the reciprocal signals occurring between all epithelial-mesenchymal interacting tissues. Fundamental to organo-genesis is the ability of one tissue to induce in another tissue the expression of previously quiescent genes that determines the induced tissue to acquire a new identity. Panel B describes the use of growth factors, transcription factors, and matrix factors to provide the signals inducing a naïve stem cell population to adopt odontogenic potential. Panel C is a strategy for screening a variety of stem cell populations, such as mesenchymal stem cells (MSC), embryonic stem cells (ESC), and epithelial stem cells (EpSC) for their ability to adopt odontogenic capacity under the influence of an odontogenic tissue, such as porcine dental epithelia (pDE) or porcine dental mesenchyme (pDM). Panel D is the time line required to bring the laboratory steps together for the in vitro manipulation of stem cell populations to regenerate a cap stage tooth bud and to initiate clinical training with release to the dental community within a decade.

Efforts to use such strategies have been previously identified.^3–5 In the continuously developing rodent incisor, for example, the determination and differentiation of stem cells to terminal phenotypes and their unique signals have been well defined and provided some of the necessary molecular details needed for regeneration.^6–9 A systems biology and bioinformatics approach was launched to define in high resolution all of the known gene products that define the canonical pathways for tooth development (Figure 2⇑).

We also sought ways to estimate our ignorance by predicting the extent of the factors that were not yet known and then devising the means to fill in these gaps in knowledge.^10-12 The team paid particular attention to the fate of the supporting tissues of the tooth, the periodontal ligament, cementoblasts, and supporting alveolar bone. The emphasis on regenerating the supporting tissue of the tooth was held as a central tenet to our design plans, because the team felt strongly that the key to successfully integrating a regenerated tooth would lie in regenerating the periodontium.^13,14

Our strategy was to exploit stem cells, adult or embryonic (Figure 3B⇑), in a milieu of growth factors that replicated those signals normally exchanged at each of the appropriate stages of odontogenesis. In addition, we planned the use of a nanofabricated bio-informative matrix for both cell support and informational content.^15,16 It had already been experimentally shown that cell differentiation could be enhanced by using a supporting matrix that closely matched the material properties of the authentic matrix.¹⁷ Moreover, tailoring the stiffness of the supporting matrix could also be used to control proliferation of embedded cells in the matrix.^18,19

Another function of the nanodesigned matrix was information content.²⁰ For example, peptide amphiphiles containing laminin epitopes have been used for the induction of neurons from stem cell precursors. By mating soluble signaling factors with the proper physical clues, we planned to engineer an optimal environment for cell differentiation.²¹

Finally, we envisioned the need to engraft the regenerated cap stage to the host. For this, a series of peptide amphiphiles was designed that could provide a transiently protected environment that would suppress inflammatory responses predicted to be detrimental to engraftment, while providing a pro-angiogenic and pro-reinnervation environment that would support the continued development of the host-engrafted cap stage primordia.²²

In our efforts to identify the type of stem cells that would exhibit ideal growth characteristics, as well as being competent for odontogenic induction, a number of stem cell precursors were to be screened (Figure 3C⇑). A series of inductive signals was envisioned to be exchanged between members of the naïve stem cell population when they were challenged by their reciprocal odontogenic tissue (Figure 3A⇑). The success of the interactions would be assessed by using gene expression parameters achieved by the stem cells after induction. This would be done by comparing the gene expression profiles obtained from these samples to those obtained from authentic developing teeth. These profiles were to be stored as a searchable computer database created as part of the molecular blueprint (Figure 1⇑). Our goal was to ascertain if the gene expression profile from the induced stem cell population faithfully matched the gene expression profile from canonical teeth, thus ensuring that development was progressing unhindered. In this manner, we would replicate the signal cascade that normally occurs during tooth development, and these signals would yield an authentic copy of the dental tissue created from the naïve stem cell population.

Human embryonic stem cell lines (hESC) approved for use by the National Institutes of Health, adult mesenchymal stem cells (MSC), and human epithelial stem cells (hEpSC) would be surveyed with the goal of identifying the optimal stem cell population for production of the regenerated tooth.^23-26 Sonoyama et al. have recently shown that the fabrication of a dental root stock engineered from a stem cell population could be engrafted into the jaw of a pig, where it functioned with a traditionally fabricated dental crown.²³

With this carefully outlined strategy, the team forged a vision around the long-term scientific objective of a clinical procedure for tooth regeneration. We proposed to create a cap stage tooth organ fabricated in vitro that would be transplanted into a patient and subsequently grown autonomously into a fully formed adult tooth in vivo. The cap stage would be created by inducing odontogenic fate in stem cells using a marriage of engineering sciences with cell and developmental biology that would enable near-natural ideal-control of the biochemical and biomechanical environment of a cell culture. The underlying hypothesis was that recapitulating the quantitative and qualitative parameters of gene expression identified in the canonical developing tooth would be transferred to the stem cells and would result in a regenerated tooth that was a copy of the original. Toward this end, quality assurance points were introduced at selected stages of regeneration to ensure that the gene expression parameters being sought were achieved. A strategy for virtual, real-time, continuous quality assurance was transplanted from the engineering discipline, where it had been used for evaluating complex systems and had enabled significant improvement in compliance with design specifications.

The cap stage was chosen as the ideal point in tooth development for transplantation because it contains all the cell types and associated signals required to form the adult tooth, as well as the ability to induce the host to provide vasculature and innervation. We hypothesized that the odontogenic potential in the regenerated cap stage tooth would contribute to odontogenic induction to the host’s stem cells resident at the engraftment site. This would further promote the formation of a new periodontal support, including cementoblasts, periodontal ligaments, and alveolar bone. Some of these tissues would likely be provided by stem cell populations resident in the host. Under the influence of the regenerated tooth, these host stem cells would participate in the regenerative process, with the cap stage acting as an organizing center. In favor of our design, the cap stage tooth organ was also the simplest vehicle for transferring to the patient the outcome of all the in vitro assembly processes described herein.

This regenerative procedure would offer an alternative to the mechanical solution of synthetic dental implants. Also, by providing a biological solution to the problem of tooth loss, the procedure would transform the prognosis for edentulous patients as well as for patients with severely diseased teeth or diminished jawbone. We hypothesized that it is a realistic ultimate goal to carry out the majority, if not all, of the tooth regeneration by cell biology routes.

A tooth regenerated as a living organ would have important clinical advantages over a synthetic tooth, including a biological hermetic boundary between the gingivae and the root, participation in the immune system, innervation, sensory perception, and vascularization. The presence of a regenerated root, cementum, and periodontal ligament would confer to the regenerated tooth the ability to repair itself in response to injury and for the periodontium to remodel and adapt to the mechanical environment, whether arising from orthodontic procedures or mastication. The regenerated tooth would be a familiar organ to dentists, who could apply to it all of the advantages of modern dental therapeutics.

We therefore proposed a strategy of addressing all the challenges peculiar to biologic methods. We would use synthetic or biomimetic materials primarily to create an ideally controlled environment for cell development, and secondarily as an alternative if the biologic solutions met an obstacle that could not be overcome by this creative team of scientists and clinicians.

Vision of the Scientific Objectives for the First Five Years of the Program

We believed that an approved clinical procedure of tooth regeneration, via an implanted biomanu-factured cap stage, could be realized by 2017, in a ten-year plan. In the first five years, we proposed short-term scientific objectives that could bring the procedure to the point of animal trials.

We projected that these objectives would likely also provide tangible scientific and clinical benefits as we progressed through the regeneration strategy in its entirety. Among these benefits would be the following: 1) establish a goal-driven management team leading a network of outstanding senior scientists and a generation of newly trained young investigators, who would unite the disciplines of cell biology, materials science, informatics, mechanics, dentistry, and system engineering into a single research community focused on tooth regeneration; 2) identify an uninterrupted signaling cascade predicted to induce odon-togenesis in optimally chosen stem cell populations forming the cap stage tooth organ; 3) create a map of gene expression and cell signals associated with the progression of naïve stem cells to their odontogenic fate; 4) create a new science for controlling the biochemical and biomechanical environment of a cell culture to induce organogenesis (this science would include a proteome-based pharmacologic repertoire to manipulate cell signal pathways); nano-textured, information-rich matrices that would guide odontogenic fate; and fast new experimental techniques for non-destructive in situ monitoring of cell development; and 5) establish an understanding of the cell and molecular biology required for the regeneration of tooth roots and their adnexa necessary to ensure successful biological integration of the cap stage with the jawbone following transplantation.

These outcomes, when attained, would enrich the ongoing efforts to regenerate the periodontium, and would be readily transferred to an already well-developed technology base existing in the dental profession. To contribute to regenerative medicine in general, we hoped to recreate a generalized source of stem cells for future applications. In addition, we would create an assembly sequence for the manipulation of stem cells that could be adapted for other organs based on their use of common early development patterns, capabilities, and progression. Finally, just as the space program has yielded many inventions and technologies adapted to improving health, we would create deliverable intermediary biological devices adapted from the organ regeneration program that could address clinical patient needs. Such intermediary products would include the fabrication of the dentine enamel junction as a means of improving bonding between dental tissues and restorative ceramics and the creation of a biomimetic periodontal ligament based on the hydroxyapatite binding peptides, identified by Oren et al. and Ma et al.^27,28

Where We Stand Today on Tooth Regeneration

This is a story that does not yet have an ending. What remains remarkable about this story is that there is no single author: all efforts to date have been written by teams of investigators cross-fertilizing each other with newly won techniques and technologies that will push the process forward. Some chapters in this story have been written and evaluated by scientific, peer-review panels who have weighed in on the merits of our ideas. The story has had mixed outcomes up to now. The NIDCR has decided not to accept any of the applications submitted to it for further consideration of support. The “Request for Proposals” for tooth regeneration by the NIDCR is closed, having been a one-time solicitation.

Some aspects of tooth regeneration have evolved in the form of several intertwined research proposals recently submitted as part of the NIH Roadmap Initiative. A team of investigators from Harvard University plans to regenerate human teeth, as well as the pancreas and heart. The team I have described in this report wishes them every measure of success, as our dreams and their future accomplishments can measurably improve the health of the American people and guide worldwide regenerative medicine strategies. In the words of Kurt Vonnegut (1922–2007), “and so it goes.”