2.2.3. Cementogenesis

Cementum, a thin calcified avascular tissue between dentin and periodontal ligament, is produced by cementoblasts. Cementum contains collagen type I, bone sialoprotein, osteopontin, glycoproteins and proteoglycans arranged in a fibrous network with hydroxyapatite deposits. Various types of cementum are present in distinct regions of mature tooth roots: thin acellular cementum is deposited around the cervical tooth area and below, while thick cementum with entrapped cementocytes and their processes penetrating cementum locates at the root apexes (Figure 1C). Histological studies also indicate that a thin layer of dense acellular cementum lies beneath the cellular cementum at the root apex and plays an important role in cementum mineral metabolism. The cementum volume is enlarging over the lifespan and is not subjected to remodeling such as bones. Cementoblast precursors are present in the periodontal ligament and can be mobilized for cementum regeneration if needed [13,41,112]. Ex vivo, cementoblasts can be generated from periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFSCs), and iPSCs [75,113,114].

By analogy with dentinogenesis, TGF-β, and BMPs, Wnt and ameloblast-derived factors regulate cementum structural matrix protein expression. The central transcription factor of cementogenesis is Osx, which is activated by Wnt and TGF-β/BMP signaling. Osx is abundantly expressed in cementoblasts and cementocytes during cementum deposition, where it regulates DMP1, BSP, OCN, and ALP expression. It is proposed that Osx regulates cementogenic differentiation, while it inhibits cementoblast proliferation [35,115]. Stabilization of β-catenin leads to increased cementum formation via the upregulation of Osx, which is achieved by β-catenin binding to the Osx promoter, thus pointing to the direct regulation of Osx by β-catenin [36]. Additionally, Osx regulates the expression of dickkopf-related protein 1 (DKK1), an antagonist of β-catenin, and the transcription factors T-cell factor 1 (Tcf1) and lymphoid enhancer-binding factor 1 (Lef1), which together with β-catenin form a transcription initiation complex with β-catenin in the cell nucleus. It is therefore evident that cross-regulation of β-catenin and Osx plays a central role in cementogenesis [36,115].

In addition to Wnt/β-catenin regulation, Osx is regulated via the TGF-β/Smad axis, as Smad3 plays an important role in *Osx* gene expression during cementogenesis [37]. BMP2 and -4 likewise regulate Osx expression via a BMP-Smad-Runx2 cascade, but also Runx2-independently [34]. Despite the suggested beneficial role of Wnt/β-catenin in cementogenesis, another point of view has been expressed, according to which excess Wnt may inhibit cementogenesis under normoxic conditions, while hypoxia reverses this effect [24]. BMP2/4 signaling, which promotes cementogenesis in several ways, is negatively regulated by FGF2 in a concentration-dependent manner. This has been shown in periodontal ligament cells undergoing cementogenesis, thus implying that FGF2 is not beneficial for differentiation, but is important for cellular stemness [75]. This is in line with similar results in amelogenesis or very early stages of odontogenic differentiation [27,75,87]. Contrarily, in vivo, local FGF2 infusion was shown to promote cementum formation during periodontal injury regeneration by recruiting, enhancing and accelerating the proliferation of endogenous cemento/ostogenic cells [116].


**Table 1.** Cell Sources and signaling modulators useful for amelogenesis, dentinogenesis, and cementogenesis.

a studies of epithelial invagination/development; b studies in vivo; the rest are cell culture-based reports.

The enamel-derived signaling components, amelogenin and its alternatively-spliced isoforms, regulate cementogenesis by modulating the expression of various matrix proteins. Full-length amelogenin application induced the expression of osteopontin (OPN), cementum attachment protein (CAP), OCN, Cola1, BSP, DMP1, and ALP mRNA; upregulated OPN and Col1a1 proteins; and improved the mineralization of an immortalized mouse cementoblast cell line (OCCM-30). Moreover, amelogenin positively regulated its putative receptor lysosome-associated membrane glycoprotein 1 (LAMP1) in murine dental follicle cells and OCCM-30 cells, thus confirming its role as an important ligand regulating cementogenesis [38,39]. Amelogenin derivates, such as leucine-rich amelogenin peptide (LRAP), modulate gene expression in a slightly different manner: LRAP inhibited OCN expression, while promoted OPN and osteoprotegerin (OPG) expression in a dose-dependent manner and had a negative effect on cementoblast mineralization. The effects are probably mediated through the ERK1/2 pathway since ERK inhibition annuls the LRAP effects [79].

Similar to dentin and dentinogenesis, cementogenesis has a central transcription factor: Osx, which regulates cementogenesis-specific gene expression. Besides Osx, Runx2, and ERK1/2 are involved in cementogenic differentiation. In addition, ameloblast-derived proteins are important ligands positively regulating cementum matrix-associated gene expression (Figure 2C). Modulators of herein discussed cementogenic pathways are listed in Table 1.

Gained knowledge about molecular cues shaping dental tissue genesis may help to establish novel stem cell selection, culture, and differentiation methods and develop functionalized scaffolds and biomaterials, which will support and promote amelogenic, dentinogenic, and cementogenic differentiation in vitro. Thus, it will approximate the era of dental tissues regeneration using most suitable odontogenic cells with adequately functionalized biomaterials.

#### **3. Sca** ff**olds and Drug Release Materials for Tooth Regeneration**

#### *3.1. Sca*ff*olds for Enamel, Dentin, and Cementum Regeneration*

Sca ffolds and biomaterials are essential components in dental tissue regeneration since they can be used as a template for tissue regeneration by serving as a site of attachment for the regenerative cells from the surrounding tissues or act as a delivery platform for implantable odontogenic cells with the ability to di fferentiate towards the desired cell type [122,123]. Additionally, the sca ffold material may be used as a delivery platform for bioactive molecules such as drugs or proteins (especially growth factors) that further enhance the regenerative potential [60,61,63,124].

In general, sca ffold materials used in tissue regeneration need to be readily available and meet criteria such as biocompatibility and biodegradability without any toxic metabolites. In the case of sca ffolds for tooth regeneration, biomaterials are subjected to the challenging environment of the oral cavity—including mechanical forces due to mastication, the presence of microorganisms, and varying conditions regarding temperature and pH. The intended biomaterial has to face these challenges without limitations in its biocompatibility [125]. Since it is generally intended to mimic the native extracellular matrix by using biomaterials, properties besides biocompatibility are imposed by the tissue which should be regenerated. Thus, in the case of sca ffold materials for dental tissue engineering, the used material systems di ffer greatly depending on whether enamel, endodontic, or periodontic tissue is intended to be regenerated. Categories for biomaterials used in tooth regeneration are natural organic, synthetic organic materials, or inorganic materials [126]. Natural organic materials involve peptides such as collagen or gelatin and polysaccharides such as chitosan, alginate, or agarose. Frequently used synthetic organic materials are poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and poly(caprolactone) (PCL), while commonly used inorganic materials are bioactive glasses or calcium phosphates such as hydroxyapatite (HA), β-tricalcium phosphate (TCP), and cementitious systems of calcium phosphate (CPC) or calcium silicate (e.g., mineral trioxide aggregate, MTA). Polymeric materials often lack mechanical and biological properties but are able to establish three-dimensional porous structures, thereby providing a highly hydrated matrix in vivo that facilitates the transport of nutrients, anabolites, and catabolites. In turn, inorganic biomaterials used in tissue engineering often comprise preferable biological properties but have disadvantages such as brittleness and lacking in the supply of nutrients. Thus, composite materials comprising both organic and inorganic constituents gain increasing interest in recent years due to their inherent combination of the desirable properties of the single components [127]. In the following subsection, the challenges, approaches, and recent studies for the targeted and sca ffold-assisted regeneration of enamel, dentin, and cementum are presented. Injectable biomaterials are a central and highly desirable class in the context of dental regeneration, but are not extensively reviewed here due to the very recent and detailed publication of a distinct review on this topic by Haugen and coauthors [128].
