Dental care for children adheres to the principle of preserving the deciduous teeth until physiologic exfoliation, thereby maintaining the dental arch integrity, and guiding the eruption of the permanent successors. In deciduous teeth with deep carious lesions and reversible pulpal injuries, vital pulp therapy (VPT) is indicated, aiming to preserve the tooth structure and pulp vitality, thus allowing phonation, esthetics, and masticatory function during the deciduous and mixed dentition. This includes two treatment modalities, namely indirect pulp capping (IPC) in deep dentinal cavities and direct pulp capping (DPC) or pulpotomy in cases of pulp exposure [1]. The latter techniques require the use of a biocompatible material, placed in contact with the amputated pulp tissue, to promote the wound healing process [2].

Recent research on materials used for VPT of deciduous teeth is directed towards the use of bioactive endodontic cements, such as mineral trioxide aggregate (MTA) or other bioactive calcium silicate-based cements, presenting favorable outcomes regarding the long-term treatment success [3]. Material bioactivity refers to its ability to develop a mineralized interfacial layer between the cement and dentinal wall, as well as formation of apatite crystals on the material surface [4], [5]. Bioactive materials exert their action on progenitor pulp cells, promoting their activity and subsequently pulp tissue repair. More specifically, pulp stem cells are stimulated to proliferate, migrate, and differentiate into odontoblast-like cells secreting reparative dentin matrix, thereby sealing the remaining pulp tissue from external noxious stimuli. Additionally, angiogenesis is crucial in this process, since blood supply is established, bringing nutrients, oxygen, and progenitor cells to the injured site [6], [7].

MTA was the first calcium silicate-based cement introduced in the 1990 s [8], and up to this date extensive research supports its use as a gold standard material in VPT [9]. Its composition is based on Portland cement, containing tricalcium silicate, tricalcium aluminate, dicalcium silicate, and tetracalcium aluminoferrite as main constituents, in addition to bismuth oxide and calcium sulfate [10]. Despite its superior biological properties, drawbacks regarding the long setting time, difficulty in handling and discoloration, led to the development of improved MTA-based calcium-silicate cements, which are widely used in current clinical practice [11]. In recent years, a new bioactive calcium silicate-based cement, namely Biodentine™ (Septodont, Saint Maur des Fossés), has been also widely applied. Biodentine™ is a tricalcium silicate-based cement composed of a powder and liquid. The solid form comprises of 80% tricalcium silicate, 15% calcium carbonate and 5% zirconium oxide as radiopacifier, while the liquid is made up of water with some additions of calcium chloride and a water-soluble polymer [12]. Both MTA and Biodentine™ are indicated as pulp capping materials, possessing antibacterial properties and the ability to induce a hard tissue barrier (dentin bridge) formation [13]. Furthermore, Biodentine™ can be applied as a dentine substitute, creating a firm anchorage to dentinal tubules, and presenting improved mechanical properties [14].

Clinical trials and systematic reviews comparing the clinical effectiveness of the aforementioned bioactive cements used in pulpotomy of deciduous teeth conclude that there is no superiority of one material towards the other for up to 18 months after placement [3]. However, evidence on using these cements in DPC of deciduous teeth is scarce. To deepen our understanding of the way these materials interact with the deciduous pulp stem cells, and perhaps propose clinical protocols regarding the use of DPC, the idea for the current study was born.

Numerous in vitro studies aimed to decipher the biological mechanisms behind the favorable action of these cements [7]. Traditionally, monolayer cultures have been utilized to evaluate the cytotoxicity and biological interactions of dental biomaterials in vitro. However, tissues are three-dimensional (3D) and organized by complex cell-cell and cell-extracellular matrix (ECM) interactions, which two-dimensional (2D) experimental systems cannot recapitulate. In this way, conventional models lack accuracy regarding the in vitro to in vivo extrapolation (IVIVE), rendering the novel 3D cultures more suitable to mimic the clinical situation. Within the latest experimental systems, interaction with neighboring cells and ECM is enabled, which stimulates cell proliferation and promotes differentiation [15], [16]. Hadjichristou et al. (2020) established a three-dimensional tissue engineering-based dentin/pulp analogue as an advanced cytotoxicity assessment tool [17]. The DentCytoTool incorporates a dentin-component, comprising a human treated dentin matrix underlined by a layer of human odontoblast-like cells, and a vascularized pulp analogue, consisting of co-cultured endothelial and dental mesenchymal stem cells, encapsulated in a collagen/fibrin hydrogel. Collagen is a natural constituent of the dental pulp and fibrin is biodegradable with the ability to sustain a microvascular network [17], [18].

The biological effects of MTA and Biodentine™ have been well studied on 2D cultures of human dental pulp stem cells of permanent teeth (hDPSCs), though fewer studies are investigating these effects on stem cells from human exfoliated deciduous teeth (SHED) [19]. According to the literature, both cell populations have the ability to differentiate into multiple cell lineages, including odontogenic, angiogenic and neurogenic [20], [21]. In comparison to hDPSCs, SHED exhibit higher proliferative capacity and migration ability [22], [23]. To the knowledge of the authors, there are no studies so far that evaluate the effect of these VPT materials on a tissue-engineered 3D organotypic model involving SHEDs. In an effort to deepen our understanding regarding the biological interactions of MTA and Biodentine™ with the deciduous pulp, the pulp analogue of the DentCytoTool was utilized in the current study. Originally the tool’s pulp analogue comprises of a co-culture of stem cells from the apical papilla (SCAP) and human umbilical vein endothelial cells (HUVEC) encapsulated in a collagen/fibrin hydrogel, but for the purposes of the current study, it was modified to include SHED instead of SCAP cells, to resemble the cell population dwelling in a deciduous tooth pulp. The described pulp analogue not only provides the advantages of 3D culture systems mentioned above, but also promotes regeneration through secretion of angiogenic and odontogenic factors, better resembling the vascularized pulp tissue [24]. Based on the above, the aim of the current study was to evaluate the effects of MTA and Biodentine™ on this organotypic 3D deciduous pulp analogue in terms of cell viability/ proliferation, and morphology, as well as regarding the expression of a wide panel of angiogenic and odontogenic markers.