Introduction

Regenerative endodontics is concerned with the development of biologically based treatment modalities that are used to replace diseased portions of the dental pulp or to allow complete formation of a dental pulp–like tissue that will act as the original dental pulp. Today, the major effort in regenerative endodontics appears to use several types of stem cells placed on a scaffold inside the diseased root canal system of a tooth. With the addition of growth factors, externally or from dentin and/or remaining dental pulp, a pulplike tissue forms. (Please see a review of tooth formation from embryonic tissues to a fully formed tooth by Tziatas and Kodonas. ) A form of regenerative endodontics began many years ago with the development of direct and indirect pulp-capping procedures. The need for a scaffold, vascular supply, growth factors, signaling mechanisms, migration of cells, and differentiation were not well known, nor were the actual events that occurred during the formation and regenerative processes known. Today’s placement of a direct calcium hydroxide pulp cap leads to growth factor activation from surrounding hard tissues, inclusion of native stem cells from the remaining pulp tissue, and hard tissue formation (dentinlike hard tissue) that may also act as a scaffold and as a source for growth factors. The body of work in regenerative endodontics has grown exponentially, therefore this article is concerned with the future possibilities of an understanding of the processes and mechanisms to restore a vital, healthy tissue within a tooth in situ and include a review of the progress of laboratory studies that may lead to greater knowledge of the interactions at the cellular and molecular levels of tissue engineering.

In a healthy tooth, the pulp/dentin complex undergoes dentin matrix formation with eventual mineralization. The dentin formed is in a physiologic process. The dentin formed is very similar to primary dentin, with a tubular structure that covers primary dentin with the dentin tubules being continuous between both primary and secondary dentin formations. In teeth that have been injured in some manner (caries, restorations, trauma), another type of dentin forms called tertiary dentin. This is a unique type of dentin that is not tubular in its formation but rather occurs as an atubular structure. There are 2 types of tertiary dentin: reactive and reparative. Reactive dentin is formed by the remaining, original odontoblasts forming a matrix that becomes almost completely solid with no tubules; however, tunneling has been seen to occur in this form of dentin. Reparative dentin is a matrix formed by new odontoblastlike cells that form precursor and stem cells found in the remaining vital pulp tissue. It also is formed without tubules.

Regenerative endodontic procedures use biologically based treatment modalities and pulpal cells. The information available in regenerative studies to date, however, indicate that more must be learned about the interactions that occur between all cells, growth factors, proliferation and differentiation of cells, and the ability to use materials that will result in a well-formed, functioning tooth.

Pulp repair and regeneration

An excellent review by Goldberg suggests that there are more questions than answers in the ability to effect pulp repair and pulp regeneration. The following is a summary of the questions he poses as to the future of pulp repair, pulp regeneration, and tissue engineering.

A distinction between endodontic repair and regeneration must be understood before one can understand the processes that occur in repair and regeneration. Repair indicates that healing occurs because the remaining damaged tissue is vital, original odontoblasts survive, and the pulp tissue can be restored to a normal-like form and function. In the dental pulp, odontoblasts are reactivated, a dentin matrix is formed that becomes mineralized (reactionary dentin), and the pulp retains, for the most part, its biologic functions.

Regeneration indicates that the pulp is completely necrotic (complete degradation) and a tissue (pulplike) must be formed that may function as the original tissue. Questions arise as to how the resultant cells and tissue react in relation to the original tissue. Studies have demonstrated that a completely necrotic pulp, combined with a periapical lesion and incomplete root formation, is the usual clinical finding. Initial treatment is undertaken to remove the infection and heal the lesion. This is followed by formation of the tooth root, creation of new odontoblastlike cells functioning as a pulplike tissue. In these cases, the new pulplike tissue continues to form a hard dentinlike tissue, generally without tubules closing the root canal space in what appears to be an event that occurs rapidly. The events occurring in these teeth may cause the need for further therapeutic interventions, including root canal or surgical root end therapy. The mechanisms for these events are not fully understood, hence the term “-like tissue.” Can the processes be controlled to produce a more natural tissue reaction that occurs over many years without closing down the root canal space? Ideally, the process should mimic the development of secondary dentin formation, which is physiologic in nature and occurs in an uninvolved pulp as a natural part of the aging process.

The future of repair and regeneration depends on answers to the questions posed in the Goldberg article. What is the nature of the stem cells that should be used to regenerate pulp tissue? This is of great importance, as researchers appear to have isolated several different stem cell lines, which, in itself generates several other questions. Greater attention to the analysis of the biologic properties of dental tissue–derived mesenchymal stem cells using both in vitro and in vivo systems is necessary. A recent study concluded that both dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) could differentiate into odontoblastlike cells with the potential to migrate and mineralize leading to 3-dimensional dentinlike structures; however, SCAPs had a higher population capacity and proliferation rate compared with DPSCs. This may be an advantage for dental tissue repair and regeneration from the standpoint of cryopreservation of the cells in large quantities and a high mineralization rate may shorten the process.

Pulp repair and regeneration

An excellent review by Goldberg suggests that there are more questions than answers in the ability to effect pulp repair and pulp regeneration. The following is a summary of the questions he poses as to the future of pulp repair, pulp regeneration, and tissue engineering.

A distinction between endodontic repair and regeneration must be understood before one can understand the processes that occur in repair and regeneration. Repair indicates that healing occurs because the remaining damaged tissue is vital, original odontoblasts survive, and the pulp tissue can be restored to a normal-like form and function. In the dental pulp, odontoblasts are reactivated, a dentin matrix is formed that becomes mineralized (reactionary dentin), and the pulp retains, for the most part, its biologic functions.

Regeneration indicates that the pulp is completely necrotic (complete degradation) and a tissue (pulplike) must be formed that may function as the original tissue. Questions arise as to how the resultant cells and tissue react in relation to the original tissue. Studies have demonstrated that a completely necrotic pulp, combined with a periapical lesion and incomplete root formation, is the usual clinical finding. Initial treatment is undertaken to remove the infection and heal the lesion. This is followed by formation of the tooth root, creation of new odontoblastlike cells functioning as a pulplike tissue. In these cases, the new pulplike tissue continues to form a hard dentinlike tissue, generally without tubules closing the root canal space in what appears to be an event that occurs rapidly. The events occurring in these teeth may cause the need for further therapeutic interventions, including root canal or surgical root end therapy. The mechanisms for these events are not fully understood, hence the term “-like tissue.” Can the processes be controlled to produce a more natural tissue reaction that occurs over many years without closing down the root canal space? Ideally, the process should mimic the development of secondary dentin formation, which is physiologic in nature and occurs in an uninvolved pulp as a natural part of the aging process.

The future of repair and regeneration depends on answers to the questions posed in the Goldberg article. What is the nature of the stem cells that should be used to regenerate pulp tissue? This is of great importance, as researchers appear to have isolated several different stem cell lines, which, in itself generates several other questions. Greater attention to the analysis of the biologic properties of dental tissue–derived mesenchymal stem cells using both in vitro and in vivo systems is necessary. A recent study concluded that both dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) could differentiate into odontoblastlike cells with the potential to migrate and mineralize leading to 3-dimensional dentinlike structures; however, SCAPs had a higher population capacity and proliferation rate compared with DPSCs. This may be an advantage for dental tissue repair and regeneration from the standpoint of cryopreservation of the cells in large quantities and a high mineralization rate may shorten the process.

Stem cells

All stem cells in odontogenesis, with the exception of ameloblast progenitor cells, originate in the mesenchyme and are said to be of ectomesenchymal origin. DPSCs are isolated from the dental pulp and can regenerate into new stem cell lines that can differentiate into other cell lines. As the developmental ability of these cells in vitro is limited, they are more useful in in vivo studies, as more complex tissues arise. For example, dentin/pulplike tissues arise from DPSCs, such as dentinlike and pulplike tissues.

SHEDS are stem cells from exfoliated, human, deciduous teeth, which are a readily accessible source of adult stem cells from impacted third molars. In vivo, removal of these teeth led to collection of multipotent stem cells having the potential to differentiate into odontoblastlike cells, neurons, and osteoinductive cells. Periodontal ligament stem cells (PDLSCs) can form cementum and periodontal ligament and, when transplanted into mice, bone and cementum structures were seen. Dental follicle stem cells (DFSCs) are collected from the follicles that surround developing third molars. These cells have a major role in the genesis of cementum and cementoblastlike cells.

SCAPs are harvested from the apex of a developing tooth. The papilla is a precursor of the dental pulp. As in other stem cells, SCAPs express early mesenchymal surface markers. A reading of the quoted articles will show that the previously listed references demonstrate that, in many instances, the studies compare one type of stem cell to another or several others. These stem cells are proliferative with characteristic markers such as Stro-1, CD146/MUC18, and CD44 (see articles by Sedgley and colleagues and Law and colleagues elsewhere in this issue). This leads to questions as to which markers should be recognized that will allow collection and development of a cell line that can be maintained and colonized and introduced into a tooth as an in vivo treatment option. A primary question must be asked as to what type of pulplike tissue should be the result of implantation?

Is it possible to obtain a functional, nonmineralized pulp that is vascularized and innervated as the original tissue would be? Or is the aim to develop a pulp tissue that would induce an increased amount of mineralization that could serve as a substitute for root canal therapy? Cell differentiation can lead to either adult progenitor or an odontoblastlike/osteoblastlike cell, which is divergent from other results obtained.

The question of using multipotent stem cells remains unsettled, especially when attempting to regenerate pulpal tissue. The cells necessary are present in the pulp and can be associated with odontoblast and osteoblast cells, endothelial cells, and, later, formation of neurons. Therefore, is the use of multipotent progenitors or nonpotent cells, the cells of choice?

In the future, it may be possible to minimally invade and isolate suitable stem cells, have them undergo differentiation in vitro, and combine and develop them into tooth structures. Pulp cells differentiate in vitro into odontoblastlike stem cells. The dentin formed, as previously mentioned, is atubular. Is there a possibility of dental pulp cells producing tubular dentin? A recent study mixed pulp cells with a hydroxyapatite (tricalcium phosphate powder) and generated a dentin-pulplike tissue. Bartouli and coworkers transplanted tubular dentin on the surface of dentin-pulp slices and generated increased amounts of tubular dentin; however, the origin of the progenitor cells giving rise to new odontoblasts (tubular dentin) and the signaling pathways in cell differentiation have not been clearly identified and remain a matter of debate.

Greater knowledge related to the location and identity of odontogenic precursor cells that participate in reparative dentin formation is required. Implant experiments have begun to identify genuine progenitor cell markers and molecular signal pathways that allow stem cell recruitment. Implantation experiments using pulp-derived precursor cell lines have started to provide evidence that, in the absence of carriers or biomolecules, exogenous stem cells have the capacity to promote efficient tooth repair.

Because repair and regeneration have different targets, the expectations of a particular therapy must be clear. Is regeneration of a nonmineralizing pulp the proper goal or is generation of a tissue that may become a completely mineralized root canal system the proper treatment option? Each aim uses specific tools that are valid for bioengineering treatment modalities.

Caries may be the most common and dangerous of all types of injury, provoking adverse stimuli to the dental pulp. Understanding caries management (see article by Chogle and colleagues elsewhere in this issue for a review of the pulpal response to caries) has led to improved understanding of mineralization of teeth, further leading to therapies necessary to restore the biologic behavior of the entire pulp-dentin complex.

The pulp-dentin complex is protective in nature, as its main roles are to manufacture dentin matrices and to restart dentinogenesis to protect the new pulplike tissue from injury or insult. Many of the processes involved are thought to be the same as the initial pulp developmental processes occurring embryonically. Because the onset of injury in the dental pulp may be a result of caries, markers of inflammation are different, depending on the depth of the inflammatory process of the lesion.

Still somewhat unclear is how inflammation may overwhelm and cause degeneration in the pulp, as opposed to its role in the regeneration of that tissue. To understand the treatment prognosis, understanding the balance between infection and inflammation is necessary, together with an understanding of proinflammatory and anti-inflammatory mediators and how they relate to the innate and adaptive immune systems.

Many studies have reported that several populations of stem cells in and around the tooth pulp are able to be used to repair or regenerate the pulp/dentin complex. These populations of cells include DPSCs, SCAPs, PDLSCs, and mesenchymal stem cells. SHEDs are human pulp cells of the dental follicle collected from impacted third molars. These cells may have the dual ability to repair (heal), regenerate a particular tissue, or differentiate in a manner that causes a change in the ability of these cells to form original tissue. As previously mentioned, odontoblasts normally secrete a tubular dentin (both primary and secondary) as a normal physiologic function throughout life that maintains the tubular structure in both dentins. However, insults to the pulp may cause newly formed odontoblastlike cells to form an atubular (tertiary) dentin that is not tubular and not physiologic. Rather it is formed as a result of the pulplike tissue reacting as a defender of that tissue. To be able to use these cell lines clinically, translational research in the future will require both researchers and skilled clinicians who can develop new and novel therapies that can eventually be tested and used in clinical environments to answer these questions.

Scaffolds

A scaffold is thought of as a 3-dimensional construct or support substance used for several tissue engineering applications. When stem cells are seeded on scaffolds, they are expected to attach, proliferate, and differentiate into new tissues that will eventually replace the scaffold. Scaffolds should be biocompatible, not elicit an inflammatory response or be cytotoxic, support cell organization and vascularization, allow new regenerated tissue to form, be sterilizable, and be stable while maintaining mechanical form and strength. They should have an inductive ability with added growth factors and morphogens for a more rapid cell attachment, proliferation, migration, and differentiation into a specific tissue.

The choice of a scaffold is critical in tissue regeneration. Most scaffolds are organic in nature and used to provide surfaces on which cells may adhere, grow, and organize. Scaffolds chosen for laboratory studies are diverse, including natural or synthetic polymers, extracellular matrices (EMCs), self-assembling systems, hydrogels, and bioactive ceramics. Recently, a synthetic polymer polycaprolactone was successful in growing increasing numbers of SCAP stem cells with apparent identification of NOTCH signaling expression.

Although the number of scaffolds has increased (see the excellent review by Sakai and colleagues ), questions remain that must be addressed. For example, are scaffolds able to support various kinds of stem cells or are they stem cell–specific? Are stem cells able to be seeded with like results on more than one scaffold? What are the limitations of the use of one or another scaffold that may be natural or synthetic scaffolds? The use of a self-assembling peptide system that allows a “bottom-up” approach of generating EMC materials, offering high control at the molecular level, will be a major step forward in constructing future scaffolds. The peptide system is referred to as a tunable matrix with several features that possibly allow scaffolds to be designed, as different requirements are needed to regenerate a tissue.