Enhanced understanding of the biological processes involved in both tooth development and repair are creating conditions to enhance reparative responses and tissue regeneration. The dentin matrix contains innumerous proteins and growth factors capable of stimulating tissue repair. These growth factors are secreted by pulp cells and deposited within the dentin matrix during mineralization [24, 25] where they remain protected in active forms. Demineralization of the dentin after application of cavity-etching agents or dressing for pulp capping [26], or even during cavity preparation [27, 28], releases these growth factors and makes them available to the surrounding cells. They seem to play key roles in controlling many of the events of reparative dentin formation [10, 29]. Growth factors, especially those of the transforming growth factor (TGF)-beta family, are important players in cellular signaling; they stimulate odontoblast differentiation and dentin matrix secretion, leading to increased reparative dentin formation [29].

Another important family of growth factors in tooth development and regeneration are the bone morphogenetic proteins (BMP). Recombinant human BMP-2 stimulates the differentiation of adult pulp stem cells into an odontoblast-like cell type in culture [30, 31]. Similar inductive effects of TGF-beta 1–3 and BMP-7 have been demonstrated in cultured tooth slices [32, 33]. Recombinant BMP-2, BMP-4, and BMP-7 induce formation of reparative dentin in vivo [30, 34]. The application of recombinant human insulin growth factor (IGF)-1 has been found to induce dentin bridging, comparable to a calcium hydroxide-based capping agent [35]. In addition to growth factors, other molecules have been shown to stimulate pulp cell differentiation. Dentin matrix protein (DMP)-1, a noncollagenous protein involved in mineralization processes, induces cytodifferentiation, collagen synthesis, and calcified deposits after direct pulp capping in animal models [36]. Dexamethasone, a synthetic glucocorticoid, reduces cell proliferation and induces expression of alkaline phosphatase and dentin sialophosphoprotein in primary human dental pulp cells [37]. The addition of beta-glycerophosphate to the cell culture medium in explants from human teeth induced a change in cell morphology, collagen synthesis, and mineral formation [38]. Combinations of dexamethasone with inorganic phosphate have been used for dental stem cell differentiation and the induction of mineralization in several studies [8, 9]. The key message here is that there are known biological regulators of dental pulp cell behavior that have been considered as potentially viable therapeutic strategies for regenerative endodontic procedures.

Scaffolds are carriers for cells, which provide a 3D environment for cell adhesion, migration, and differentiation. Ideally, they should biodegrade at the same rate as the new tissues form. Scaffold materials should be biocompatible and nontoxic. For dental pulp tissue engineering, the injectable materials are advantageous. Natural polymers, such as collagen, elastin, glycosaminoglycans, fibrin, alginate, silk, and chitosan, have long been used as carriers [39]. Although these materials provide structural strength and are biocompatible and biodegradable, they offer few possibilities for controlling the structure or making compositional modifications to improve their performance. Synthetic polymers, on the other hand, provide high control of the mechanical and chemical properties. Polylactic acid (PLA), polyglycolic acid (PGA), and their copolymer poly(lactic-co-glycolic acid) (PLGA) have been widely used for tissue engineering applications including regenerative dentistry [40, 41]. Hydrogel is particularly interesting because of their tissue-like viscoelastic properties, efficient transport of nutrients and metabolic products, uniform cell encapsulation, and the possibility of injection and gelation in situ. Gels have been made from polyethylene glycol fibrin, glycosaminoglycans, or peptide-based building blocks. Based on their chemistry, they can be chemically or physically cross-linked, and modifications such as the incorporation of biofunctional molecules or growth and differentiation factors are possible [42].

The concept of tissue engineering was conceived by Langer and Vacanti in the early 1990s to describe the technique for biological tissue regeneration [43]. Tissue engineering refers to the science of generating new living tissues to replace, repair, or augment the diseased/damaged tissue and restore tissue/organ function [44].

The objectives of tissue engineering procedures in endodontics are to regenerate a pulp-like tissue and ideally the pulp–dentin complex. An immature tooth with a necrotic pulp does not have residual progenitor pulpal cells to continue root development [45–47]. The main goals of conventional root canal therapy, including complete cleaning and shaping and appropriate obturation, cannot be achieved in an immature root. Moreover, the short, weak, and fracture-prone root of an immature tooth [48] becomes even weaker after mechanical instrumentation of the root canal. Apexification with Ca(OH)₂ [49] or an apical plug [50] may solve many of the problems associated with conventional root canal therapy; however, there is still the risk of horizontal root fracture especially at the cervical area and tooth mobility due to compromised crown-to-root ratio (Chap. 8).

Apexification with Ca(OH)₂ was proposed by Frank (1966), in which Ca(OH)₂ is used in multiple visits during a long period of treatment to induce an apical barrier [49]. In addition to the extended treatment period, long-term contact of dentin with Ca(OH)₂ decreases the mechanical strength of dentin, making the tooth susceptible to fracture [51, 52]. Cvek (1992) reported a higher incidence of cervical root fracture in root-filled immature teeth compared to mature teeth following dental trauma [48]. Therefore, preserving (or restoring) the pulp vitality of immature teeth with deep caries or dental trauma is of great importance.

To keep the tooth vital, several research groups are pursuing the idea of engineering dental pulp, which was initially proposed by David Mooney’s group at the University of Michigan in the nineties [53, 54]. Dental pulp fibroblasts were cultured on PGA scaffolds for 60 days, and the cells were proliferated and formed tissue similar to native dental pulp [53]. Bohl et al. tested whether different scaffold materials influenced the proliferation and collagen synthesis of dental pulp fibroblasts 2 years later and found PGA to be the most conducive compared with alginate and collagen type I hydrogel [54]. A dental pulp-like tissue was generated using DPSCs in combination with collagen scaffolds and DMP-1 as a morphogenic factor on dentin slices after transplantation in vivo [55]. Our group used SHED cells seeded onto polymer scaffolds within tooth slices and transplanted these constructs into immunocompromised mice. A pulp-like tissue formed within the tooth slices after 2–4 weeks, the cells lining the dentin showed expression of odontoblast/osteoblast markers, and microvessel formation was observed [16]. Notably, SHED differentiated into odontoblasts that synthesized new tubular dentin, confirming their differentiation into fully functional odontoblastic cells [17].

Galler’s group demonstrated the growth and differentiation of stem cells derived from different dental tissues in a PEGylated fibrin hydrogel. The cells degraded the fibrin, replaced it with a collagenous matrix, and deposited mineral after osteogenic induction. Furthermore, SHED and DPSCs were tested for compatibility with a self-assembling peptide hydrogel, which offers several possibilities to modify the matrix. The optimization of a peptide sequence through the incorporation of the cell adhesion motif RGD (arginine–glycine–aspartic acid) and a matrix metalloproteinase (MMP)-2-specific enzyme cleavable site led to increased proliferation rates and enhanced migration of SHED and DPSCs into these matrices [56]. Furthermore, studies with peptide-based hydrogels showed that the differentiation of dental pulp stem cells can be stimulated in this type of matrix [57]. Further steps toward a tailor-made system include the incorporation and slow release of growth and differentiation factors, such as TGF-beta1, FGF2, and VEGF.

The following are possible approaches (Fig. 9.2) that have been explored for dental pulp tissue regeneration: (A) root canal revascularization via blood clotting, (B) postnatal stem cell transplantation using injectable scaffolds, and (C) pulp transplantation using stem cells grown in sheets. These techniques are based on the basic tissue engineering principles and include specific consideration of stem cells, growth factors, and scaffolds. Of these approaches, the only one that is currently approved for general clinical use in the USA and in several other countries throughout the world is the “root canal revascularization via blood clotting” procedure, as called simply “revascularization” (see Chap. 8). Nevertheless, the approaches employing stem cell transplantation are areas of intense basic and translational research that should lead to clinical transplantation in the not so distant future.