Tooth Repair and Regeneration

The dental pulp is a soft connective tissue, containing different cell populations, which is encased in a thick, porous mineralized chamber. As a clinical approach, keeping the vitality of the dental pulp has always been the goal for a successful long-term restorative dental treatment. In the adult pulp, cell division and the secretory activity of odontoblasts decrease in comparison to the developing stage; however, these processes are re-activated following pulp damage.

In shallow enamel and enamel/dentinal damage, odontoblasts can survive, and its activation supports repair, protecting the dental pulp via reactionary dentine formation [19, 20]. However, in situations of deep cavitation or trauma that involves dental pulp exposure, odontoblasts may not survive and will have to be replaced, requiring a cascade of stem cell activation, proliferation, and differentiation into new odontoblast-like cells that will culminate into reparative dentine secretion [21]. Because of the capacity of teeth to repair themselves and their accessibility, Gronthos and collaborators [22] researched and identified a population of new cells isolated from the dental pulp of human third molars. They termed these cells dental pulp stem cells (DPSCs) and showed that these cells produce dentine in vitro [23]. Although these data show that dental pulp cells have the capacity to repair damaged dental tissues, when it comes to in vivo tooth repair, the dental reparative capacity is limited to the critical size of the damage [24, 25]. In large, exposed pulpal injuries, the pulp is exposed to microorganisms from the oral cavity, and if the infected dentine is not completely removed before applying a biocompatible material while directly capping the exposed pulp, the dental pulp will undergo necrosis. This highlights the clear need to generate a therapy that stimulates the full biological potential the dental pulp has to repair injuries comprising dentine and pulp.

During primary and secondary dentin deposition, odontoblasts secrete growth factors and proteins that are fossilized in the dentine matrix after mineral maturation [26]. As decay demineralizes the dentine, these growth factors and proteins are released to the dental pulp to activate odontoblasts, immune cells, and organize the reactionary dentine matrix secretion [27]. Hydraulic silicate cements, e.g., mineral trioxide aggregate (MTA), Biodentine, total fill, endosequence, calcium-enriched cement etc., claim to be bioactive. As demonstrated by Loison-Robert and colleagues [28], Biodentine promotes mineralization when in contact with dental pulp stem cells (DPSCs); however, it does not affect DPSC proliferation. Interestingly, the only biological factor that Biodentine has shown to increase is TGF-β1 [29]; however, Neves and Sharpe [30••] showed that TGF-β and BMP are not pivotal for reactionary dentine formation but for tubular organization of reactionary dentine secretion. Reactionary dentine secretion is affected, however, by changes in the Wnt pathways. Wnt activation via GSK-3 inhibitor drugs increased reactionary dentine secretion, although Wnt inhibition did not impair reactionary dentine secretion (Fig. 1a) [30••].

One clinical specialty that could benefit from the advances of a reactionary dentine promoting material is prosthetic dentistry. The creation of a material that cements a crown and stimulates reactionary dentine formation might change the prosthetic planning approach, as it increases the rate of pulp vitality post tooth preparation to receive a crown. Biomaterials with the capacity to incorporate Wnt activators, such as Bioglass, have been developed [31], but further testing is still to be done.

When a dental injury reaches the dental pulp, a more complex clinical and biological approach is taken to preserve pulp vitality and reconstruct the lost dentine. Studies have revealed that dentine matrix derivatives and breakdown products from the dental pulp influence pulp cell migration. Recruited cells exhibited increased stem cell marker expression indicating that dental ECMs and their breakdown products selectively attract progenitor cells that contribute to repair processes [25, 32]. These mobilized resident dental pulp mesenchymal stem cells differentiate into new odontoblast-like cells that secrete a form of tertiary (reparative) dentine [33•, 34•, 35‐37]. This dentine is laid in a form of a thin band of dentine (dentine bridge) that walls off the pulp from bacterial infection. Studies have focused in understanding the underlying mechanisms of this process of “natural healing,” in order to learn how to pharmacologically trigger this event for promoting reparative dentine formation [34•].

A recent preclinical study showed that small molecules delivered via a biodegradable collagen sponge were able to provide an effective repair of experimentally induced deep dental lesions by stimulating Wnt/β-catenin signaling and hence promote reparative dentine formation [38]. This study was based on the natural underlying mechanisms and pathways that are pivotal for the healing mechanisms of the dental pulp. The activation of Wnt/β-catenin signaling is an immediate early response to tissue damage; moreover, Wnt receiving cells become odontoblast-like cells, proving they are stem cells [34•, 38‐42]. After confirming that Wnt/β-catenin is upregulated following tooth damage, the study showed that an addition of small-molecule Wnt agonists, tested in human clinical trials for Alzheimer, can stimulate the dental natural response, triggering reparative dentine formation and thus restoring the lost dentine structure with naturally generated new dentine. The striking “simplicity” of this technique makes it ideal for a translatable clinical technique; also, it helps opening a new door for more “biomimetic” approaches aiming to repair the dentine-pulp complex naturally (Fig. 1a). The fact that these particular small-molecule Wnt agonists are undergoing clinical trials already contributes further to the potential of translation into future dental treatments through an adequate incorporation into a dental material as a carrier.

When microorganisms or their endotoxins reach the dental pulp and it is diagnosed with irreversible pulpitis, because this condition cannot be reversed, the common treatment for this situation has been pulpectomy, regardless of the amount of the remaining unaffected pulp tissue. With the standard protocol, the entire pulp has to be removed, followed by root canal treatment, disinfecting the pulp space and replacing it with inorganic materials [43]. However, new approaches on de novo regeneration of the dental pulp have been investigated and will be further discussed below.

An approach for repairing/regenerating the lost dental pulp tissue, following a root canal treatment is the “de novo” regeneration of dental pulp tissue. Currently, clinical procedures are postulated to successfully revascularize infected teeth with incomplete root formation to achieve full root length development and dentine thickness. A variety of promising cases have been published that indicated pulp revascularization with pulp- and dentin-like tissue formation and healing of apical inflammation [44‐46]. However, this procedure is limited to infected or non-infected immature teeth with open apices [47]. To address tissue regeneration in fully formed teeth, procedures based on cellular approaches should be further considered and developed. Among the potential sources of stem cells isolated from teeth (DPSCs, stem cells from human exfoliated deciduous teeth [SHED] and stem cells of the apical papilla [SCAP]), Cordeiro and colleagues [48] suggested that SHED was the most valuable cell source for dental pulp tissue engineering due to its capacity to generate pulp tissue in human tooth slices with similar architecture to those of a physiologic dental pulp.

In 2010, Huang et al. [49] used different dental stem cells isolated from human dental pulp of permanent teeth (DPSCs) and stem cells isolated from the apical papilla region of human third molars (SCAP cells), to demonstrate de novo regeneration of dental pulp in empty root canal spaces. These cells were seeded onto poly-D,L-lactide/glycolide scaffold and inserted into the canal space of root fragments followed by subcutaneous transplantation into immunocompromised-SCID mice. After a period of 3 to 4 months, a histological analysis revealed that the root canal space was filled with pulp-like tissue with well-established vascularization. Moreover, a continuous layer of mineralized tissue resembling dentine was deposited on the existing dentinal walls of the canal. This dentine-like structure appeared to be produced by a newly formed layer of odontoblast-like cells [49].

In a more advanced attempt to regenerate whole dental pulp in a real clinical scenario, a recent clinical pilot study in humans assessed the therapeutic potential and safety of mobilized dental pulp stem cells (MDPSCs) to regenerate the dental pulp de novo in teeth that suffered from irreversible pulpitis without any periapical lesions [50, 51]. DPSCs were isolated from a small amount of pulp tissue of autologous discarded teeth using a granulocyte colony stimulating factor (G-CSF)-induced mobilization method. MDPSCs were successfully transplanted in previously disinfected, empty root canal spaces in vivo. Magnetic resonance imaging (MRI) and cone beam computed tomography (CBCT) revealed changes in the dental pulp indicating a pulp-like regenerated tissue and new dentine apposition after 24 weeks in most cases with positive response to electrical pulp testing (Fig. 1a) [50, 51].

While these studies focused on the usage of different dental stem cells in dental pulp tissue engineering, other studies focused on exploring new mechanical systems as options for scaffolds in pulp regeneration [52]. New injectable microsphere systems, where vascular endothelial growth factor (VEGF) binds with heparin and is encapsulated in heparin-conjugated gelatine nanospheres of a biodegradable poly L-lactic acid (PLLA) microsphere, mimic natural ECM-like collagen structures and hence act as carrier for DPSC leading to pulp tissue formation by promoting their proliferation/differentiation. Additionally, these systems provide a controlled release of the VEGF resulting in newly formed blood vessels within the regenerated tissue [52, 53].

Over the last decades, conservative endodontic procedures focused on techniques to enhance root canal disinfection and irrigation in order to address infection control of complex anatomical structures like isthmuses, and lateral canals or the apical delta. Since the presence of microbes in such areas can contribute to persistent inflammation, all procedures of de novo regeneration need to follow a procedure of complete root canal disinfection. From a clinical perspective, all the above-mentioned cellular-based cases of de novo pulp regeneration have been performed in teeth with relatively simple root canal anatomy and rather large canals. Hence, clinical applicability of teeth with more complex root canal anatomy may be questioned.

The idea of supporting the seeded dental stem cells, in this case dental pulp stem cells from adult teeth (DPSCs), providing a three-dimensional, controlled environment with added growth factors, emphasizes once more the underlying concept of the “regenerative” approach, where the biological system is mimicked and recreated in its complexity.

Several decades ago, classical tissue recombination experiments demonstrated sequential signaling between the dental epithelium and the mesenchyme of different origins and stages, where the epithelium acted as an inductive tissue [61, 62]. This odontogenic induction in the epithelium is lost early, as it naturally happens during the embryonic tooth development and then switches to mesenchyme. Thus, the mesenchyme becomes the odontogenic inductive source.

In 2003, Yamamoto and collaborators [63] showed the ability of embryonic tooth germ cells to re-aggregate following dissociation and form teeth. Other studies followed where epithelium and mesenchyme tissues from E14.5- to E12.5-stage mouse tooth germs were separated and the cells dissociated and recombined to form normal teeth [64‐67].

This reciprocal tissue induction that takes place during the early stages of tooth development, whereby the epithelium first induces tooth formation in the mesenchyme followed by a reciprocal induction from mesenchyme to epithelium, has been utilized to suggest a basis for whole-tooth bioengineering that could employ adult cells [59, 68, 69].

In 2004, a study by Ohazama et al. [69] showed that when mesenchyme cells derived from adult bone marrow are combined with inductive-stage embryonic dental epithelium, tooth formation is induced, and the adult mesenchymal cells respond and fully contribute to tooth development [69]. Another study in 2013 [70] showed that human gingival epithelial cells were able to respond to the inductive signal of mouse tooth embryonic mesenchyme, resulting in formation of fully formed teeth.

However, in spite of the fact that adult stem cells can respond to an inductive odontogenic signal and participate in tooth formation, the only cells that have been shown to be capable of tooth-inductive capacity are odontogenic embryonic cells, derived from inductive embryonic tooth-germ tissue (epithelium or mesenchyme) [54‐56, 57••, 58]. Furthermore, in all experiments reported to date, the inductive cells, whether epithelial or mesenchymal, do not retain their inductive capacity following in vitro expansion [71]. This defines one of the biggest challenges in the field of biotooth engineering, which is to identify adult cell populations that retain their odontogenic potential and can be expanded in large numbers. Moreover, these cells should ideally be allogeneic, where one population, either mesenchyme or epithelium, has tooth-inducing capacity, avoiding any issues that use of non-allogeneic cells may have for generation of nonessential organs such as teeth, in a clinical treatment.

The arrangement of cells within a tissue plays an essential role in organogenesis, including tooth development [72]. In the condensed mesenchyme, during the tooth development, cells change their shape and size dynamically.

The size and shape of the condensed cell mass also dictate the final three-dimensional form of the organ, and abnormal condensation can result in developmental defects [73]. Mechanical stimuli can modulate cell lineage commitment and control development of various tissues during embryogenesis, and studies with cultured cells suggest that cell fate can be controlled mechanically by altering cell shape [74, 75]. These observations raise the possibility that physical alterations in cells that result from cell compaction in the condensed mesenchyme also could play an active role in the differentiation process [73].

In order to preserve the odontogenic signal in cells that have been expanded in vitro, Kuchler-Bopp et al. [72], have proposed a three-dimensional micro-culture system, where they tested the hanging drop method to study mixed epithelial-mesenchymal cell reorganization in a liquid medium, showing that the system offers the microenvironmental conditions for tooth histogenesis and organogenesis. It was shown that this method can provide control of the proportion and number of cells to be used, and the forming micro tissues showed homogeneous size.

In 2016, Yang and colleagues [57••] proposed another approach to preserve the odontogenic signals in embryonic tooth germ cells that have rapidly lost their tooth-inducing capacity, once expanded in vitro. The study suggested that uncultured embryonic tooth germ mesenchymal cells were able to rescue cultured cells and enable them to fully participate into bioengineered tooth development, giving rise to dental pulp cells and odontoblasts. Although this rescue effect was not observed with postnatal dental pulp mesenchyme cells, this finding indicates that the presence of fresh (non-cultured) embryonic tooth germ cells can have a “community effect,” identified during embryonic development as a process that enables mixtures of different cells to differentiate along the same pathway.