The tooth on-a-chip: a microphysiologic model system mimicking the pulp-dentin interface and its interaction with biomaterials

Abstract

The tooth has a unique configuration with respect to biomaterials that are used for its treatment. Cells inside of the dental pulp interface indirectly with biomaterials via a calcified permeable membrane, formed by a dentin barrier which is composed of several thousands of dentinal tubules (~2 µm in diameter) connecting the dental pulp tissue to the outer surface of the tooth. Although the cytotoxic response of the dental pulp to biomaterials has been extensively studied, there is a shortage of in vitro model systems that mimic the dentin-pulp interface, enabling an improved understanding of the morphologic, metabolic and functional influence of biomaterials on live dental pulp cells. To address this shortage, here we developed an organ-ona-chip model system which integrates cells cultured directly on a dentin wall within a microdevice which replicates some of the architecture and dynamics of the dentinpulp interface. The tooth-on-a-chip is made out of molded polydimethylsiloxane (PDMS) with a design consisting of two chambers separated by a dentin fragment.To characterize pulp cell responses to dental materials on-chip, stem cell-derived odontoblasts were seeded onto the dentin surface, and observed using live-cell microscopy. Standard dental materials used clinically (2-hydroxyethylmethacrylate -HEMA, Phosphoric Acid -PA, and Adper-Scotchbond -SB) were tested for cytotoxicity, cell morphology and metabolic activity on-chip, and compared against standardized off-chip controls. All dental materials had cytotoxic effects in both onchip and off-chip systems in the following order: HEMA>SB>PA (p<0.05), and cells presented consistently higher metabolic activity on-chip than off-chip (p<0.05).Furthermore, the tooth-on-a-chip enabled real-time tracking of odontoblast monolayer formation, remodeling, and death in response to biomaterial treatments, and gelatinolytic activity in a model hybrid layer (HL) formed in the microdevice. In conclusion, the tooth-on-a-chip is a novel platform that replicates near-physiologic conditions of the pulp-dentin interface, and enables live-cell imaging to study dental pulp cell response to biomaterials.

Advanced in Vitro Experimental Models for Tissue Engineering-based Reconstruction of a 3D Dentin/pulp Complex: a Literature Review

“…In the case of the entire tooth culture and the tooth slice organ culture, the two tissues (dentin and the dental pulp) were naturally in contact beforehand. Nylon mesh 1 [10] Dentin disc 16 [10,12,[29][30][31][32][33][34][35][36][37][38][39][40][41][42] Polyamide mesh 6 [12,[39][40][41][42][43] Dentin fragments 1 [25] Polystyrene scaffold 1 [38] Tooth cavities 4 [14,15,17,44] Cell matrix 1 [18] Single cell suspension 1 [18] GelMa 2 [19,20] Cell sheet layers 1 [19] Gelatin 1 [45] GelMA 1 [20] Natural pulp 10 [14,15,46,17,[30][31][32][33][34]37,44] Gelatin 1 [45] Fibrin/fibrinogen 2…”

“…The dentin/pulp complexes of this category are comprised of: spheres within rings with different stiffness values [45], slices of dentin with the preservation of the dental pulp [37], cells encapsulated in fibrin including Growth factors enhancing angiogenesis (VEGF) accompanied with BMP-2 coated dentin discs [36], stacking fibrinogen -of various concentrations -in a bioprinting process within a polycaprolactone frame for dentin and the pulp [47], hanging culture inserts dividing two compartments -a dentin analogue and a pulp analogue -through a membrane with a dentin slice [35] and lastly, a three-dimensional tooth on a chip assembly with two perfusable compartments representing dentin and the dental pulp. In this category cellular populations were all of human origin, mostly DPSCs [35,36,45,47] and SCAP [25], while Hadjichristou et al used a combination of HUVEC and SCAP for the pulp compartment and DPSCs for the dentin compartment [35]. The observation period of these experiments varied from 3 days [37] up to 4 weeks [45] and the main outcome of these studies was to evaluate the feasibility of a dentin/pulp complex implementation via mineral deposition quantification [45,47], marker gene expression for angiogenic (vWF, VEGF, PECAM-1, VEGFR-2, ANGPT-1, TIE-2) [35][36][37] as well as odontogenic markers (BSP, DMP-1, OCN and CBFA, DSPP, RunX2, BMP-2) [35,36,47] and thyrotropinreleasing hormone degrading enzyme (TRHDE) and syndecan3 which are highly expressed in the natural pulp [45].…”

“…The observation period of these experiments varied from 3 days [37] up to 4 weeks [45] and the main outcome of these studies was to evaluate the feasibility of a dentin/pulp complex implementation via mineral deposition quantification [45,47], marker gene expression for angiogenic (vWF, VEGF, PECAM-1, VEGFR-2, ANGPT-1, TIE-2) [35][36][37] as well as odontogenic markers (BSP, DMP-1, OCN and CBFA, DSPP, RunX2, BMP-2) [35,36,47] and thyrotropinreleasing hormone degrading enzyme (TRHDE) and syndecan3 which are highly expressed in the natural pulp [45]. Some of the constructs were further utilised to deduce the cytotoxicity of dental materials such as eluates of resin monomers (HEMA, TEGDMA) [35] or of resin monomer (HEMA) and phosphoric acid [25] using techniques such as MTT, LDH and Live cell imaging. All results seemed promising in their utilisation as 3D biocompatibility investigation tools, all with potential improvements and high output capabilities.…”

“…3D printing with bio-inks [47], and by incorporating state of the art with the latest updates in the research field, researchers have managed to include dentistry within these innovative ideas. Another example of this is the "tooth on a chip" which has borrowed the idea from the medical field through the "organ-on-a-chip", finding place also in dentistry [25]. It is essential to mention that some studies sought ways to modify already existing knowledge from two-dimensional studies and bringing insight into the three-dimensional environment [35].…”

“…These small devices -microchips-that fit between the two fingers are constructed by PDMS and they replicate the microarchitecture and basic functions of human organs such as kidney [21], blood-brain barrier [22], lung [23], liver [24]. Dentistry followed this trend and devised the "tooth on a chip" [25]. This model would serve as a biocompatibility assessment tool by combining a pulp and a cavity chamber in two compartments separated by a dentin fragment.…”