The tooth on-a-chip: a microphysiologic model system mimicking the biologic interface of the tooth with biomaterials.

Microdevice fabrication The device design was created using a Computer Aided Design (CAD) software (Autodesk Fusion 360, Autodesk Inc, San Rafael, CA, USA) and a positive template was laser-cut (Boss LS1416, Boss laser, Sandorf, FL, US) in a polymethylmethacrylate (PMMA) board. Next, the templates were attached to the base of an impression container, molded with PDMS (poly dimethyl siloxane, Dow Corning) pre-polymer ( Fig. 1A ), and cured at 80 °C overnight ( Fig. 1B ). The cured PDMS mold was removed from the template ( Fig. 1C ) and four reservoirs were prepared with an 8 mm punch ( Fig. 1D ). The device is comprised of two parallel channels, two perfusable chambers (300 μm W × 1 mm L × 1 mm H ), and a central groove that holds a dentin fragment ( Fig. 1E ). Human dentin from third molars extracted for orthodontic reasons according to the institutional ethics committee guidelines was used. Teeth were sectioned into fragments of 500 μm W × 1 mm H × 4.5 mm L cut perpendicular to the dentin tubules using an low speed saw (Accutom 5) (ESI † Fig. S1 ). The PDMS piece and a coverslip were then plasma treated (Plasma Cleaner, PDC-32G, Harrick Plasma, Ithaca, NY, US), as to increase the silanol groups (−OH) at the surface of the PDMS to form covalent Si–O–Si bonds with the glass. Of note, we did not subject the dentin fragments to plasma treatment to prevent any chemical or structural change to the dentin matrix. Immediately after plasma treating the PDMS mold and coverslips, a dentin fragment was carefully inserted into each PDMS mold using tweezers, and the system was assembled onto the glass coverslip using slight pressure, forming a sealed and leak-proof microdevice ( Fig. 1E and F ) with two chambers separated by a semi-permeable membrane (dentin) creating distinct microenvironments for each chamber ( Fig. 1G and H ). Although the dentin fragments had4.5 mm in length, the borders were placed within the PDMS holders ( Fig. 1G and H ), thus the dentin area exposed to dental treatments and cell attachment in the device were about 2–2.5 mm L × 1 mm H and 0.5 mm W . Each tooth-on-a-chip was then filled with sterile water until use to prevent dentin dehydration. The fully assembled microdevice replicates the interface of dentin with the dental pulp on one side and the dental material with dentin on the other side, thus forming two accessible chambers representing the “pulp side” and the “cavity side”, respectively ( Fig. 1G and H ). The system was tested for leakage around the dentin fragments with a liquid dye, and our experiments suggest that the coloured medium would typically penetrate through the dentin tubules and intertubular matrix in the presence or absence of active flow (ESI † Fig. S2 and S3 ). The microfluidic devices were then sterilized with ultraviolet light (EXFO Acticure 4000, 365 nm, at 8.5 cm distance, light density: 45 mW cm −2 ) for 40 min prior to use.

Cell culture Stem cells from apical papilla

(SCAPs) were cultured for 10 days in odontogenic medium (OM) (ESI † ) to pre-differentiate cells into an odontoblast-like lineage. This is to more closely mimic the natural tooth anatomy where odontoblasts form a monolayer interfacing with the dentin. Dentin was treated with 17% EDTA for 45 seconds to remove the smear layer, thoroughly rinsed with water, and seeded with a suspension of 20 μL with 10 5 SCAP per ml on the ‘pulp side’ chamber. The device was incubated for 1 h to promote cell contact and attachment onto the dentin wall. Next, ( Fig. 1H ) the reservoirs were filled with 100 μL of cell medium and cells were cultured for 7 days with daily cell medium changes.

Live-cell imaging

To demonstrate that the tooth-on-a-chip enables live imaging of the cells in close contact with the dentin, a monolayer of odontogenically differentiated SCAP was imaged overnight, every 30 minutes using a spinning disk confocal microscope (ESI † ). For analyses of cell morphology, on days 1 and 7, chips ( n = 4) were fixed with 4% paraformaldehyde, stained for actin filaments and nuclei, and imaged using a confocal microscope (LSM 880 Zeiss) (ESI † ). The whole monolayer in contact with dentin was photographed in 3 consecutive images and analyzed using ImageJ (Fiji, NIH, Maryland, USA). Cytotoxicity After the monolayer formation, three dental materials were tested: (a) HEMA dissolved in cell culture medium at a known cytotoxic concentration of 10 mM, (b) 37% phosphoric acid gel (PA) (Ultradent Products, South Jordan, UT, USA) used to etch the dentin for 15 s, and (c) 35% PA plus Adper Single Bond 2 (SB) (3M/ESPE, St Paul, MN, USA) applied per manufacturer recommendations (ESI † ). The materials were introduced to the ‘cavity side’ of the device, thus forming an interface similar to the dentin–pulp complex in a restored tooth ( n = 4). Live-cell images of SCAPs assembling as a monolayer on the dentin surface were obtained using a Yokogawa (Japan) CSU-X1 spinning disk field scanning confocal system. Cells were imaged using Nikon 10× objective lens (numerical aperture = 0.5). The temperature was maintained at 37 °C using an all-in-one stage incubator. To identify dead cells, SCAPs were incubated with 50 nM of Helix NP NIR (Biolegend, San Diego, CA) diluted in cell culture medium 10 minutes before imaging. The Helix NP dye does not require rinsing, so cells continued to be imaged to provide a baseline. After three minutes of imaging, 20 mM of HEMA was added to the opposite side of the dentin, and live-cell images were taken every 10 minutes for one hour. We then compared on-chip experiments against experiments performed using the ISO-10993–1 (ref. 25 ) standard, which we refer to as the off-chip group. To that end, 10 4 SCAPs/well were seeded in 96-well plates, and after 24 h, cells were supplemented with the ISO recommended concentrations of HEMA, PA, and extracts of SB obtained by immersing the photopolymerized SB disks in culture media ( n = 6) (ESI † ). Next, cells were incubated for 24 h with the conditioned medium of each dental material, and the medium was replaced with the untreated medium for 7 days. Controls were samples cultured in standard culture medium not exposed to the dental materials. Cell metabolic activity was measured using Alamar Blue (ThermoFisher) on days 0, 1, 3, 5, and 7. Gelatinolytic activity of hybrid layer on-a-chip After determining the cytotoxicity of HEMA, PA, and SB, we evaluated the contribution of MMPs released by dental pulp cells in the degradation of the hybrid layer formed after SB treatment. For proper bonding of resin restorations to the tooth, dentin needs to be conditioned with an acid to partially expose the collagen network present in the dentin organic matrix, and then an adhesive agent is applied to fabricate the hybrid layer, which consists of a hybrid system of collagen fibrils embedded with an adhesive resin. The final quality and longevity of the dental restoration depend significantly on the integrity of the hybrid layer, and it has been shown that matrix metalloproteinases (MMPs) contribute to the its degradation. 26 We hypothesized that the immediate response of pulp cells to the acid attack and monomer exposure might stimulate cells to secrete substantial amounts of proteases, which may further promote the hybrid layer degradation in vivo – a phenomenon that cannot be measured using traditional off-chip gelatinolytic assays. To that end, we applied SB as previously described to the ‘cavity side’ of the chips fabricated with or without SCAPs ( n = 4). The gelatinolytic activity at the hybrid layer was performed as detailed in ESI † using fluorescein-conjugated gelatin (DQ™ gelatin, EnzChek Gelatinase Assay Kit, ThermoFisher) incubated for at least 48 h, after which the proteolytic activity was imaged via confocal microscopy.

Statistics

Results were analyzed using either a Student’s t -test, one-way ANOVA or two-way ANOVA followed by Tukey post-hoc tests ( α = 0.05) on GraphPad Prism 8.

Effect of dental materials on SCAP morphology and proliferation Each tested material elicited apparent cellular injury with as early as 24 h after treatment for both on-chip ( Fig. 4A – D ) and off-chip ( Fig. 4E – H ) groups. The monolayer in the HEMA group consisted of poorly connected round cells with pyknotic nuclei ( Fig. 4B ), while off-chip, HEMA ( Fig. 4F ) resulted in a visible reduction in cell number relative to the untreated controls. PA etching of the dentin on-chip caused more discrete monolayer disorganization and cytoplasmic injuries ( Fig. 4C ). Similar cytoplasmic changes were seen off-chip, but, overall, samples had a more visible effect ( Fig. 4G ). SB treatment on-chip also caused cytoplasmic changes characterized by a dim actin stain and increased intercellular spaces ( Fig. 4D ), while off-chip samples presented a visible decrease in cell numbers ( Fig. 4H ). After 7 days, there were striking differences between on-chip ( Fig. 4I – L ) and off-chip ( Fig. 4M – P ) samples regarding cell number and morphology, except for the untreated control samples ( Fig. 4I and M ). Untreated cells on-chip showed little change from day 1 ( Fig. 4A and I ), while samples treated with HEMA ( Fig. 4J ), PA ( Fig. 4K ) and SB ( Fig. 4L ) had visible morphological changes. Samples cultured off-chip ( Fig. 4M ) had confluent monolayers when left untreated for 7 days, but had very few cells with faint cytoplasms when treated with HEMA ( Fig. 4N ), PA ( Fig. 4O ), and SB ( Fig. 4P ). Quantitatively, cell numbers were significantly higher on-chip compared to off-chip controls after 7 days for all treatments, with both HEMA and SB leading to a significant reduction in cell number relative to untreated controls on day 1 ( Fig. 5A ), and for all treatment groups on day 7 ( Fig. 5B ) ( p < 0.05).

Supplementary Material 1 2 3 4 Supplement