**1. Introduction**

Dental injuries and diseases such as caries and periodontitis are a ffecting significant fractions of populations worldwide and are the main reason for dental tissue regeneration e fforts [1,2]. Caries lesions cause local enamel resorption and dentin damage due to oral microbiota activities in the morbid tooth. Although relatively easily manageable at early stages, if left untreated caries causes excessive dentin damage and poses a need for reparative treatment [3]. Periodontitis is a complex inflammatory disease, where pathogenic oral microbiota and host immune response dysregulation lead to the gingiva, periodontal ligament, cementum, and alveolar bone damage [4]. Excessive periodontitis damage cannot be regenerated naturally, thus requires specialized soft and hard calcified tissues regeneration approaches. Next to infectious/inflammatory oral diseases, several heritable disorders of dental tissue formation exist (e.g., amelogenesis imperfecta, dentinogenesis imperfecta, and tooth agenesis), which affect tooth formation, eruption, calcification, or maturation [5–8]. In addition to disrupted teeth

integrity, dental diseases often create an unaesthetically looking oral cavity, thus affecting patients emotionally, which makes dental tissues regeneration critical in both aspects: health and aesthetics.

Dental tissues have no or very limited capacity for self-regeneration [2,3,9,10]. Specifically, enamel becomes acellular after it is formed; dentin regeneration is limited and dependent on the dental pulp stem cell pool, which deteriorates in the case of an infection and inflammation; and cementum has no remodeling capacity and limited regrowth in the case of disease-induced resorption [10–13]. Each dental tissue contains a defined amount of inorganic matter (hydroxyapatite crystals), matrix proteins arranged in a scaffolding network, and microstructures such as lacunae in cellular cementum and microchannels, which accommodate cellular processes in dentin and cementum. The complex microarchitecture of the tooth poses a need for appropriate replacement materials, which have to be biocompatible and wear-resistant [14]. Additionally, the development of enamel and dentin relies heavily on mesenchymal–epithelial interactions, thus making it challenging to recapitulate the process in vitro even using existing odontogenic cell lines and adult stem cell culture methods [10,12,15–17]. Although a lot is already known about tooth formation and molecular cues shaping this process [5,6,18], signaling patterns involved in dental tissue differentiation in vitro, postnatal calcified tissue metabolism and regeneration are being actively studied and more research is expected in the future [18–47].

Efforts in whole tooth regeneration have been made for decades [48] and include biological, bioengineering, and genetic approaches. Revitalizing the odontogenic potency of the successional dental lamina (SDL) rudiment for lost tooth regeneration might be one possibility to induce tooth formation in vivo in the adult [49]. Whole-tooth restoration using autologous tooth germ cells and bioengineered tooth germ transplantation is another promising opportunity [50,51]. However, due to limited sources of tooth germ cells, the risk for immune rejection of allogeneic or xenogeneic cells, as well as ethical and legal constraints, adult stem cells of various sources or induced pluripotent stem cells (iPSCs) may be used instead [52–54]. Recently, combining cells of mesenchymal and epithelial origin of various plasticity is being actively explored for tooth regeneration using novel culture methods [55–57].

Although the implantation of recombined embryonic or adult cells may give rise to tooth-like organs in vivo, the combination with scaffold material may improve tooth formation. Scaffolds can influence the biological behavior of cells and can give mechanical support to tissue constructs. Their consecutive degradation should parallel the formation of the native extracellular matrix and promote the assimilation of constructs after implantation [58]. In contrast to periodontal bone and other bone grafts, where numerous scaffold compounds have been developed and tested within the last decade [59–64], studies on artificial scaffolds for tooth regeneration are still rare due to the rather complex nature of teeth [14,65]. Recent studies in biomaterial development involve hybrids and composites of inorganic/organic components to be used as scaffolds to mimic the complex composition of the natural tooth [55,58,66]. New investigations have shown also that the functionalization of scaffolds using cell-free methods is possible. Vesicles, small RNAs, or exosomes from cultured stem cells or embryonic cells can be used onto or within scaffold material to address regenerative functions [65]. Besides, scaffolds can be loaded with drugs, growth factors, and/or receptor ligands to guide the stem cell differentiation process during dentogenesis [19,64,67,68]. However, very few artificial materials have been tested thus far in clinical trials [9,69].

In this review, the most recent discoveries regarding cellular signals guiding dental tissue differentiation in vitro and in vivo are summarized. Current developments of biocompatible functionalized scaffolds, drug-release materials, and their applications are addressed as well. Finally, whole-tooth generation approaches using various cellular sources and dilemmas in tooth regeneration are elucidated. An interdisciplinary approach is taken to cover tooth regeneration issues from molecular, via structural to biological aspects.

#### **2. Hard Dental Tissues and Their Genesis**

#### *2.1. The Complexity of Dental Tissues*

The process of teeth formation starts within embryogenesis and proceeds in multiple phases throughout the prenatal period, childhood and adolescence resulting in an eruption of permanent teeth. Each dental tissue forms in a unique way and in a tightly regulated manner, where one tissue is guiding or supporting the formation of the other [11,30,40]. Early odontogenesis is characterized by an epithelial–mesenchymal interaction, which is also a blueprint for the formation of other organs such as hair follicles or exocrine glands [70]. The epithelium is derived from the embryonic endoderm, while the mesenchyme is derived from the cranial neural crest. Placodal thickenings of the oral epithelium along the dental lamina first induce a cellular condensation of the underlying mesenchyme. The tooth primordium then undergoes di fferent morphological stages forming a bud, cap, and later bell stage. While the epithelium gives rise to enamel, the mesenchyme is the source of the later pulp, periodontal apparatus and hard substances such as dentine and cementum. Then, epithelial components lose their inductive odontogenic competence while a reciprocal induction starts from the mesenchyme. These reciprocal crosstalks are governed by a signaling program consisting of a large number of molecules interacting in signaling pathways. Major examples of these factors are families such as the Bone Morphogenic Proteins (BMPs), Fibroblast Growth Factors (FGFs), Wingless/Int1 (Wnt), Hedgehog (Hh), or Ectodysplasin (EDA) functioning as morphogenetic inducers [18,65,71]. The morphogenesis is driven by signaling centers, which orchestrate tissue interactions and are involved in the size and shaping of the single tooth. In addition to cellular signaling, tissue forces, e.g., through an epithelial contraction, mesenchymal condensation, or bone biomechanics, participate in the formation of tooth morphology [71,72].

During tooth development, several stem cell niches have been identified. Epithelial stem cells are located, e.g., in the cervical loop, which is the apical end of the advancing epithelium consisting of an outer and inner layer and active until the onset of tooth root formation. These stem cells play a role in continuously growing teeth, e.g., mouse incisors. The elongation of the cervical loop as a double-layered structure is named Hertwig's epithelial root sheath and is the signaling center for tooth root formation. It should also be mentioned that tooth formation depends on the interaction with the developing alveolar bone, which therefore should be considered in strategies for whole tooth regeneration [71,73].

The mature tooth is a complex organ consisting of non-vascularized hard tissues: enamel, dentin, and a soft vascularized innervated dental pulp. The dental pulp is closely associated with dentin and harbors odontoblasts, dental pulp stem cells (DPSCs), pericytes, and other cellular populations. Blood vessels penetrating the pulp nourish the resident cells, while nerves participate in the sensory information exchange between the pulp and oral environment (Figure 1B). In the case of excessive dental injury (e.g., deep caries), odontoblasts, their precursors, and DPCSs can be recruited from the dental pulp and participate in dentin repair [74]. The tooth is surrounded by the periodontal ligament, which is a complex attachment tissue harboring odontogenic stem cells [69,75], linking the tooth to the alveolar jawbone (Figure 1). The mature molar tooth macrostructure and microstructures of dental tissues containing cell niches are depicted in Figure 1.

#### *2.2. Signaling Pathways Modulating Hard Dental Tissue Generation*

Many signaling cascades such as FGF, sonic hedgehog (Shh), transforming growth factors beta (TGF-β), BMPs, and Wnt/β-catenin are involved in the regulation of dentogenesis during development and adulthood [11,45,76–78]. Specific functions elicited by activation of these pathways are noted during distinct phases of dental tissue di fferentiation, some of which are beneficial for cell stemness and proliferation (FGF, Shh) while others such as Wnt, TGF-β, and BMPs act in postnatal di fferentiation phases and promote polarization, migration, and calcification [23,25–28,30,31,37,77,79,80]. Next to this, purinergic signaling function is gaining research attention in dental tissues metabolism [32,81,82]. Most ligands activate transcription factors such as runt-related transcription factor 2 (Runx2), osterix (Osx or Sp7), and extracellular signal-regulated kinase 1/2 (ERK1/2 or MEK1/2), which are central regulators of gene sets crucial for calcified tissues [33–35,83]. Epithelial–mesenchymal interactions are also involved in odontogenic and cementogenic differentiation [23,24,30,38,39].

**Figure 1.** Tooth structure and dental tissues with the respective stem cell populations. (**A**) The odontoblast niche is bordering dental pulp beneath the dentin with odontoblast processes projecting towards enamel. (**B**) Diverse cell populations are found in dental pulp, DPSCs, which can give rise to odontoblasts. (**C**) Cementocytes are residing in the lacunae of cellular cementum at the root apex with their cellular processes projecting towards the periodontal ligament.
