Isolation and culture of dental pulp cells
Human DPC were harvested in a sterile environment from extracted third molars without pulpitis, with a prior informed consent . Pulp tissue underwent explant cultures to isolate DPC through outgrowth. Cells were cultured in alpha-minimal essential medium (α-MEM, Sigma-Aldrich, St. Louis,MO, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo FischerScientific, MA, USA), penicillin G, streptomycin and amphotericin B (Gibco) at 37 °C, 5% CO2, and 95% atmospheric moisture. The experiments were performed with three donors to take into consideration the donor-dependent variability in response and to rule out bias. The protocol was accepted by the ethics committee of the Medical University of Vienna (631/2007).
Fabrication of rod microtissues
3D rod-shaped microtissue patterns of DPC were created using 3D Petri dishes® embracing the manufacturer’s instructions (Microtissues, Inc., Providence, RI, USA). Nominal dimensions of each 3D culture trough are 2200 μm in length × 800 μm in depth × 400 μm in width. Pouring liquid agarose into flexible 3D Petri dishes® embodying a trough-shaped arrangement formed agarose molds with 24 troughs for constructing DPC rods. The agarose molds were soaked in cell culture medium and positioned into 24-well plates, with one mold per well. Cell suspensions of 480,000 cells in drops of 70 μL were pipetted into each mold as described by the manufacturer instructions. Each well then received 1 mL of cell culture medium and plates were incubated for 24 h.
Rod microtissues of DPC were incubated with 1 mg/mL MTT at 37 °C for 30 min in a 24-well plate after the 24-h incubation period. Formazan formation was observed under light microscope and images were taken .
To assess the viability of the self-assembly of the DPC, samples were stained with the Live/Dead Cell Staining Kit (Enzo Life Sciences AG, Lausen, TX, USA) embracing the manufacturer’s guidelines in a 24-well plate following the 24 h incubation period . The vitality of the rod microtissues was analyzed using fluorescence microscopy for green and red with a B-2A filter (excitation filter wavelengths: 450–490 nm), respectively after 24 and 240 h. Vital cells were detected as green while the dead cells as red. Images were taken.
Resazurin-based toxicity assay
Resazurin-based toxicity assay was performed according to the manufacturer’s instructions to assess the cell viability of DPC rod microtissues. Sixty microliters of resazurin dye (Merck, Darmstadt, Germany) were added into each well of the 24-well plate, 24 h after seeding the cells into agarose molds. Following 8 h of incubation at 37 °C, fluorescence was evaluated using a Synergy HTX multi-mode reader (BioTek, Winooski, VT, USA) at a wavelength of 600 nm, using an excitation wavelength of 540 nm (Klara Janjić et al. 2018). The data was calculated relative to the control (only the cell culture medium).
Contraction dynamics of dental pulp cell rod microtissues
Geometrical and morphological alterations in the length and width of the rod microtissues of DPC were measured over a period of 10 days. Cell culture medium was changed every 3 days. Images of the self-assembly of DPC in the agarose mold troughs were taken at 0, 2, 6, 24, 48, 72, 96, 120, and 240 h after seeding at fourfold magnification. Length and width were calculated in millimeters using the Call EZ software.
Hematoxylin and eosin (H&E) staining of DPC rod microtissues was performed after a 24-h incubation period using the following protocol: samples were stained for 7 min in Mayer’s hematoxylin and rinsed in distilled water for 30 s. After differentiating with 0.1% HClOH for 20 s, they were rinsed for 10 min in tap water followed by rinsing in distilled water for 30 s. Next, they were stained for 3 min in 0.5% Eosin G (with two droplets of glacial acetic acid) and rinsed in tap water for 10 s, followed by dehydration in 70% EtOH, 96% EtOH, 100% EtOH, and xylene for 2, 2, 10, and 5 min, respectively. Finally, they were permanently embedded.
DPC were incubated with human TGF-β at 5 μg/mL and its inhibitor SB431542 at 10 mM as described above to examine the role of TGF-β on the contraction of their rod-shaped microtissue patterns. The concentration was based on previous publications . Twenty-four hours after incubation, images were taken using fourfold objective and OptoCapture software 2.2. Length and width were calculated in millimeters using Call EZ software. Data are displayed as compared with the untreated cells, referred to control.
PI3/AKT and MAPK signaling
DPC were incubated with the pharmacological inhibitors LY294002 (PI3K-inhibitor; Sigma), SB203580 (p38- inhibitor; Sigma), SP600125 (JNK-inhibitor; Calbiochem, San Diego, CA, USA), and U0126 (ERK-inhibitor; Cell Signaling Technology, Beverly, MA, USA), all at 10 μM as described above, to examine the role of PI3K and MAPK signaling. The concentration was based on previous reports . Twenty-four hours after incubation, images were taken and processed as described above.
Statistical analysis was done with IBM SPSS Statistics Version 24 (IBM Corporation, Armonk, NY, USA), using the ANOVA and the post hoc Dunnett’s test. The level of significance was set at p < 0.05.