Experimental setup
The in vitro experimental setup included a 3D-printed chamber made of PLA, a plate and two discs made of Ti6Al4V, a bipolar constant current stimulator (DS5, Digitimer) and an Arbitrary Function Generator (AFG-2112–12MHz, Gwinstek).
The PLA chamber was a rectangular box with integrated design features (Fig. 5a-b). The chamber contained two different positioners, one for the implant and two for the electrodes. The implant positioner had two separated components that allowed the implant to stand up by sliding into two slots. The slot nearest the chamber wall was designed with an output that allowed the wire to exit the chamber. The electrode positioner contained a cylindrical extrusion with an opening closest to the wall in order to let the wire exit. The chamber had three rectangular slots in the upper part of the wall, two located at the long side and one positioned at the short side. Those slots were designed to prevent rotation and restrict movements of the implant and the electrodes during the experiment.
The Ti6Al4V plate was chosen to imitate the implant fixture with a size of 40x4 mm and thickness of 1 mm. The discs were chosen to act as electrodes with a diameter of 4 mm and height of 3 mm. The wires that connected the plate and the discs to the current generator were made of titanium grade 1 (Sargenta AB) with a length of 10 mm where the part of the wire that was in the cell culture medium was isolated within a silicone tube. To prevent corrosion between the wire and the implant, and leakage due to the capillary effect in the silicone tube, a small droplet of silicone glue (Med-1037, Nusil) was used to cover the welding and prevent the media from being sucked out through the tube. The function generator was used to control the bipolar constant current generator that sent out the pulse with desired settings. The implant was connected to the negative output, thus functioning as a cathode, and the electrodes were connected to the positive output, thereby serving as anodes (Fig. 5c). In order to produce replicates with the same amount of applied current, the chambers were coupled in series where the implant in one chamber was connected to the electrodes in another chamber.
Expansion of MC3T3-E1 cells and preculture on Ti6Al4V
The same vial of passage 10 osteoblastic cell line MC3T3-E1, established from C57BL/6 mouse calvaria, was used for every experimental cycle. Cells were precultured on the implant surface in a 2 mL Eppendorf tube at 37°C in 95% humidity and 5% CO2 for 16 h with Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco™, USA) containing 4.5 g/L D-glucose, L-glutamine, and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and supplemented with 10% foetal bovine serum, 1% penicillin-streptomycin, and 0.25 mg/mL amphotericin-B (sDMEM). The implant surface facing the electrodes in the experimental setup was placed upwards and a seeding density of 105 cells per implant was applied. Six randomly-selected implants were counted at 16 h to determine number of cells attached to the surface prior to start of stimulation and two randomly-selected implants were qualitatively investigated by SEM imaging after 16 h of preculturing before stimulation.
The implant with precultured cells was removed from the Eppendorf tube and carefully placed into position in the PLA chamber. The electrodes were placed in their positioners and connected to the generators. 12 mL osteogenic differentiation media (sDMEM supplemented with 1% L-ascorbic acid 4.5 mM, 1% dexamethasone 1 mM and 2% β-glycerophosphate 1 M) were added to the chamber before placement in a non-CO2 incubator (Heratherm IMC 18, Thermo Scientific). The experiment started when electrical stimulation was applied.
Pulsed electrical stimulation
The electrical stimulation consisted of charge-balanced, cathodic, rectangular, biphasic asymmetric (10:1), current-controlled pulses (Fig. 5d). The cathodic phase (negative pulse) was followed by an inter-pulse break (zero amplitude) and a recovery phase (positive pulse) that was 10x smaller in amplitude and 10x longer in duration than the cathodic phase. Each stimulation pulse was followed by a charge recovery phase where any residual charge was recovered back to zero to ensure that charge accumulation cannot occur.
Stimulation treatment
Pulsed electrical stimulation was applied for a continuous duration of 72 h at 3 combinations of negative pulse amplitude (denoted “A”, 10 and 20 µA) and frequency (denoted “F”, 50 and 100 Hz), e.g., A10F50, A20F50, A20F100. Fixed pulse parameters included negative pulse width (500 µs), inter-pulse break (50 µs) and sample frequency (100 kSPS). To adjust for evaporation, 2 mL fresh medium was added per chamber every 24 h. The three first replicates in each stimulated group were evaluated for cell count and the two last replicates was prepared for SEM imaging. Every replicate was evaluated for collagen production.
EVALUATION ASSAYS
Cell distribution, morphology, and attachment
Distribution, morphology, and attachment of cells on the titanium implant were qualitatively evaluated using SEM imaging (n = 2 per group). Samples were fixed in 4% paraformaldehyde for 2 h at room temperature and stained with 1% OsO4 for 2 h. After rinsing with buffer, the samples were briefly dehydrated in a graded ethanol series for 5 min per cycle (50, 70, 80, 90, 95 and 100% ethanol) and allowed to air dry. The samples were sputter-coated with gold before examination in an Ultra 55 FEG SEM (Leo Electron Microscopy Ltd, UK) with settings of 5 kV accelerating voltage, 5 mm working distance and 30 µm aperture size.
Cell proliferation
Number of cells attached to the implant were counted using a NuceloCounter at 72 h of stimulation. Briefly, each implant was removed from the PLA chamber and placed into a 2 mL Eppendorf tube. Lysis buffer (200 µL; Reagent A100, Chemometec) was added and the tube was vortexed for 30 s to detach cells. Next, stabilisation buffer (200 µL; Reagent B, Chemometec) was added and the tube was vortexed again for 30 s. The solution (detached cells and both buffer solutions) was taken up in a NucleoCounter cassette (NucleoCassette™, 941-0002) for counting.
Collagen production
The amount of soluble collagen present in the cell culture medium at 72 h was measured using a collagen detection kit (Sircol Soluble Collagen Assay, Biocolor). The medium for every replicate in each experimental group was collected and diluted to 11.5 mL in consideration of uneven evaporation. Samples were prepared according to the manufacturer’s protocol and absorbance measurements were performed at 555 nm by a microplate reader (FLUOstar Omega, BMG LABTECH). OD555nm values were transformed to µg collagen by the standard curve function, y = 5.1528*x – 0.7766, R2 = 0.9665. Three technical replicates per sample were measured and each sample is presented as the mean value of the technical replicates.
pH measurement
Cell culture media pH was measured using a pH meter (Beckman, USA). Two technical replicates per sample were measured, and each replicate is presented as the mean of the technical replicates.
Statistical analyses
For all statistical analyses, the unpaired, two-tailed, homoscedastic Student’s t-test was used and p values < 0.05 were considered statistically significant.