Direct coupled electrical stimulation towards improved osteogenic differentiation of human mesenchymal stem/stromal cells: a comparative study of different protocols

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols.


Mobini et al. 2016, Considerations on FEM modeling in COMSOL.
The geometric model described in the previous step was imported into COMSOL.Since the culture dish, electrode materials, and culture medium electrical properties were not measured, estimated, or reported in the Mobini et al. (2016) manuscript, approximate values for those properties were obtained from the literature.Electrical properties for the culture medium were obtained from Visione et al. (2018), considering an electric conductivity of 1.5 S/m and a relative permittivity of 80.1.Polystyrene material electrical properties were retrieved from professionalplastics.com with an electrical conductivity of 10 -18 S/m (reported >10 18 Ohm/m) and an electrical relative permittivity of 2.6 (interval 2.4 to 3.1).Platinum material electrical conductivity was considered with a value of 9.52x10 6 S/m (reported 1.05x10 -9 Ohm/m), and relative permittivity for all metals at lower frequencies is considered 1.The electric stimulus applied in Mobini et al. (2016) setup was a 2.2 V direct current, generating a reported electric field of 100 mV/mm for that input electrical potential.To translate this to COMSOL, two boundary conditions were applied, one for ground in one electrode tip and another considering the 0.07 mA current condition DC floating potential on the opposite electrode tip (as reported later by Srirussamee et al. (2019) directly measuring it on Mobini's setup).A physics-controlled mesh with normal size was generated that consists of 19320 domain elements, 8080 boundary elements, and 721 edge elements.The solution converged in 2 seconds and calculated a volumetric average electric field in the cell culture medium of 0.59 V/m.

DEVELOPED DCOUPLED SETUP CAD and COMSOL files availability.
All developed CAD geometries and COMSOL models are available in Figshare for download at the following link: https://doi.org/10.6084/m9.figshare.23629926.v1 Considerations on modeling geometrical variations in the developed setup.
The impact of varying electrode size, tilt position, and culture medium volume was evaluated with COMSOL for the developed one-well model.The results are summarized in the following tables A, B, and C. All values consist of the volumetric average estimates at the region of interest (a disk shape region with a radius of 7.5 mm and a height of 0.5 mm, placed in the center and bottom of the cell culture well).All studies were modeled in basal medium at 37ºC.Here we made available in high resolution the print screens from the oscilloscope measurements that are part of the main manuscript Figure 4 and Figure 5. Figure S3 -Oscilloscope measurement for the electric potential drop for a single well.This is the response of the developed DCoupled system for the application of a potential step waveform.
Figure S4 -Oscilloscope measurement for the electric potential drop for a single well.This is the response of the developed DCoupled system for the application of a current step waveform.

Stability of reference genes expression between the di erent experimental groups
The normalization of the RT-qPCR results using two reference genes (GAPDH and RPL13A) was done by determining the geometric mean of these genes and then normalizing the Besides, the normalization of the target genes expression against the two reference genes (GAPDH and RPL13A), additional RT-qPCR experiments were performed to test the expression stability under the di erent experimental conditions studies.As it is possible to observe in Table D, the CT values obtained for the two reference genes (GADPH and RPL13A) are quite similar for all the experimental conditions described (variations in CT values between the two reference genes are shallow (all < 3.1%)), which suggests that the reference genes are stably expressed between the di erent treatment groups, confirming the reliability of the RT-qPCR methods and results presented by the authors.
This suggests the e ects of normalizing our target gene expression also against the RPL13A reference gene on our obtained results were minimal.

Figure S1 -
Figure S1 -The considered geometry of a single well according to the data reported by Mobini et al. (2016), including the described assumptions where information was lacking.Measurement values are in millimeters.

Figure S2 -
Figure S2-Oscilloscope measurement for the electric potential drop between three wells in series.This is the response of the developed DCoupled system for the application of a potential step waveform.

Table A -
Impact of di erent distances between electrodes and input current in the electric field, for a fixed culture medium height of 2 mm.

Table B -
Impact of di erent well liquid volumes in the electric field for a constant electric current of 0.05 mA.The green values indicate the variation in the EF due to liquid volume change, while the red values indicate the variation imposed by the longer versus shorter electrodes (+-5 mm size).

Table C -
Worst case scenario for a twist angle 5º up/down for a fixed culture medium height of 5 mm.The percentage di erence from the no-twist scenario under the same conditions is represented in red.

Table D -
Average CT values (from n=3 independent samples) and respective percentage of variation of CT values calculated from the RT-qPCR data to compare the two di erent reference genes (GADPH and RPL13A) and confirm the stability of their expressions between the di erent treatment groups.