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. Author manuscript; available in PMC: 2025 Jan 1.
Published in final edited form as: Macromol Biosci. 2023 Apr 14;24(1):e2300044. doi: 10.1002/mabi.202300044

Conductive Hydrogel Scaffolds for the 3D Localization and Orientation of Fibroblasts

Christina M Tringides 1,2,3,4, David J Mooney 2,3,*
PMCID: PMC10551049  NIHMSID: NIHMS1892790  PMID: 37016832

Abstract

Dermal wounds and their healing are a collection of complex, multi-step processes which are poorly recapitulated by existing 2D in vitro platforms. Biomaterial scaffolds that support the 3D growth of cell cultures can better resemble the native dermal environment, while bioelectronics has been used as a tool to modulate cell proliferation, differentiation, and migration. We describe a porous conductive hydrogel scaffold which mimics the properties of dermis, while promoting the viability and growth of fibroblasts. As these scaffolds are also electrically conductive, the application of exogenous electrical stimulation directs the migration of cells across and/or through the material. The mechanical properties of the scaffold, as well as the amplitude and/or duration of the electrical pulses, are independently tunable and further influence the resulting fibroblast networks. This biomaterial platform may enable better recapitulation of wound healing and can be utilized to develop and screen therapeutic interventions.

1. INTRODUCTION

Fibroblasts are both responsible for the formation of healthy connective tissues and the repair of injured tissues[1,2]. These stromal cells are responsive to various cues including mechanical, chemical, and electrical[3–7]. The latter has been long-known, as the transepithelial electrical potential of wound edges has been measured and is thought to guide the migration of cells into the site of injury[8–11]. The potency of electrical cues to direct cells, such as fibroblasts, through electrotaxis[12], combined with the ready tuning of electrical stimulation has led to significant efforts to exploit in wound healing. Despite many advancements in the last two decades, many of those studies evaluate fibroblasts on 2D surfaces, such as tissue-culture plastic. While this allows exploration of how cells can be directed to fill a simple defect, such as a scratch, this poorly mimics the 3D environment of a wound. A more physiologically relevant, 3D culture system would likely allow for advancements in the understanding and treatment of various wounds, including trauma injuries and diabetic ulcers[13–15].

Biomaterial scaffolds can be engineered to optimize interactions with a particular cell type, and enable the growth of cellular networks in 3D. In particular, hydrogel biomaterials can mimic the mechanical properties of tissues, and support the growth of cells[16–18], and organoids[19–21] for multiple days or weeks. Further, recent work on the development of conductive hydrogel scaffolds has introduced materials which can be used to grow cells on and in the materials, while applying exogenous electrical stimulation to the cell-laden scaffolds can direct the cell location and/or differentiation[22–27]. Our approach to develop conductive hydrogels has enabled mechanically soft (<15 kPa) materials that are also viscoelastic [28], and these can be used as living electrodes to support the growth of cells for more than 12 weeks while electrical pulses can be applied to further direct the cell differentiation and resulting networks[27].

Here, we investigate how conductive hydrogels based on alginate and carbon nanomaterials can be used to support the growth of fibroblasts in both 2D and 3D. First, a conductive hydrogel with mechanical properties that match the dermis layer of skin was fabricated, and the conductivity of the scaffold was manipulated to promote the differentiation of fibroblasts. Since the scaffold is conductive, exogeneous electrical pulses were applied to the conductive scaffolds through external electrodes. Electrical pulses applied across the scaffolds altered cell migration to control the distribution and localization of cells, and application of electrical pulses in the z-direction directed the movement of fibroblasts in 3D.

2. RESULTS AND DISCUSSION

2.1. Fabrication and characterization of a conductive hydrogel scaffold

Porous conductive hydrogel scaffolds were fabricated with varying amounts of carbon nanomaterials (e.g. graphene flakes, GF; carbon nanotubes, CNT) embedded in an alginate matrix. Briefly, these two carbon nanomaterials were selected based on our previous works[27,28], where the integration of GF and CNT into an alginate hydrogel resulted in scaffolds that were highly porous with favorable mechanical and electrical properties. Because a low amount of carbon nanomaterials was needed to achieve electrical percolation, the scaffolds retained their viscoelastic behavior. More recently we reported on metal-based conductive scaffolds[29], which were overall more stiff than carbon nanomaterial formulations. Further, as an important goal was the integration of cells into the scaffold, the incorporation of metal-based particles which were undergoing electrical stimulation could have adverse effects on the cell viability.

The well-mixed dispersion was frozen and then lyophilized to create and preserve a porous network (Figure 1A, Figure S1), as previously described[27,28]. To modulate the mechanical and electrical properties, scaffolds with different relative amounts of GF and CNT were fabricated, such that the total carbon nanomaterial content was 0.13%, 0.22%, and 0.31%. The local mechanical properties of each scaffold were assessed with nanoindentation (Figure 1B), and the storage moduli (G’) of the 0% and 0.13% carbon content formulations were found to be ~10 kPa, while the G’ of the 0.22% and 0.31% carbon nanomaterial content formulations were ~6 kPa. These differences in mechanical properties could be due to differences in how the carbon nanomaterials integrate into the hydrogel matrix. While the GF are dispersed in an aqueous solution, the CNTs are hydrophobic and tend to form aggregates until they cannot be mechanically supported in the pore, at which point they break into smaller aggregates (Figure S2). This mechanical modulus range (~10 kPa) mimics that of the dermis layer of skin[30,31].

Figure 1: A porous conductive hydrogel with skin-like mechanics.

Figure 1:

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(A) Schematic showing the organization of the highly porous hydrogel (alginate, turquoise) with the embedded carbon nanomaterials (graphene flakes, gray squares; carbon nanotubes, black lines). (B) Quantification of the local mechanical properties, G’ (left) and G” (right) of the different conductive hydrogel formulations that were tested. All numerical data are presented as mean±s.d., with at least n=7 for each formulation. (C) Quantification of the scaffold conductivity of the different hydrogel formulations. All numerical data are presented as mean±s.d., with at least n=9 for each formulation. (D) Microphotographs taken with scanning electron microscopy of a porous alginate scaffold with no conductive nanomaterials added. Scale bar: 20 μm. (E) Microphotographs taken with scanning electron microscopy show a zoomed-in representation of the porous scaffolds which contain only GF. Regions dense with GF are indicated by red asterisks (*). Scale bar: 4 μm. (F) Microphotographs taken with scanning electron microscopy to show a large area of the porous scaffolds which contain only CNT. Regions dense with CNT are indicated by blue circles. Scale bar: 5 μm.

Next, the electrical properties of the different scaffolds were compared (Figure 1C). As expected, the conductivity increased as the total % carbon nanomaterial of the scaffold increased. The porous nature of the scaffolds facilitated a conductivity of a few S/m, with <0.3% of conductive material. This conductivity would be sufficient for cells seeded inside the material to directly sense the applied electrical stimulation. The porosity of the scaffolds was assessed with scanning electron microscopy (SEM). The distribution of GF could be visualized at higher magnification, with the square flakes coating the entire surface of the alginate (Figure 1D). The CNTs were better visualized at lower magnification, where they formed bundles along the nodes of the pores (Figure 1E).

2.2. Evaluation of scaffold cytotoxicity to fibroblasts

Fibroblasts were then seeded into the conductive scaffolds and their viability was evaluated after 72 hours (Figure 2A). As a comparison, cells were also seeded on tissue-culture plastic wells (Figure 2B). In both the scaffolds and the plastic, the fibroblasts had a high (~100%) viability and there were no differences in viability across any of the conductive hydrogels. As scaffolds enabled 3D cultures of fibroblasts, the cell densities were not comparable.

Figure 2: Scaffolds enable viable, 3D fibroblast cultures.

Figure 2:

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(A) Photomicrographs of fibroblasts integrated in the 3D porous scaffolds, and stained to indicate viable cells (calcein: green) and dead cells (ethidium: red). Scale bar: 400 μm. (B) Photomicrographs of fibroblasts seeded onto tissue-culture plastic, and similarly stained to indicate viable (calcein: green) and dead cells (ethidium: red). Scale bar: 400 μm.

Next, fibroblasts were again seeded into the different hydrogel scaffolds and immunohistochemistry (IHC) was used to evaluate which carbon content condition would best support cell growth after 2 days (Figure 3). Interestingly, the 0% carbon content scaffold had the fewest number of fibroblasts at this time, despite the same number of cells being seeded in each condition, and fibroblasts generally exhibited a more circular and smaller shape in these gels. As the carbon content of the scaffolds increased, the number of cells observed in each scaffold was increased as well. In the most conductive scaffold, with 0.31% carbon nanomaterial content, most of the fibroblasts had a spindle-like phenotype with just two processes, similar to fibroblast morphology in vivo[32,33]. Since the number and appearance of the cells were most promising in the scaffold with 0.31% carbon nanomaterial content, this formulation was selected for the following experiments.

Figure 3: Electrical properties of the scaffold can affect fibroblast differentiation and morphology.

Figure 3:

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Photomicrographs of fibroblasts seeded in various conductive scaffolds for 2 days, with n=3 scaffolds compared per condition. Cells are stained for phalloidin (green) and Hoechst (blue). Scale bars: 220 μm.

We next quantified the maximum amount of voltage (volts, V) that could be applied to the cell-laden scaffold without affecting the cell viability. For testing, scaffolds were placed in the center of two parallel platinum wires, and electrical stimulation was applied to the wires for a duration of 20 minutes. Afterwards, the cell viability was assessed with a live-dead stain. While cells in scaffolds that experienced 0V, 1V, 2V, and 3V had no changes in their viability (Figure S3A), most of the cells in the scaffolds that experienced 4V were dead (Figure S3B). Additionally, the live-dead staining showed that the cells were distributed throughout the entire scaffold, which was confirmed by fixing samples after 2 hours of seeding, and imaging the fibroblasts (Figure S4). This was confirmed with a Griess assay, where the amounts of nitric oxide produced by the cultures with different levels of applied stimulation were quantified (Figure S5). From these observations, the highest voltage tested on the samples for additional studies was 3V.

2.3. Application of horizontal electrical stimulation across the scaffolds

The compatibility of the conductive scaffolds with exogeneous electrical stimulation was evaluated next. A set-up to provide electrical pulses in across (x,y plane) the scaffolds was first developed (Figure 4A, 4B), and square wave monopolar pulses were applied to the cell-laden 0.31% carbon content scaffolds which were placed at the center of the platinum wires (Figure 4C). As an initial validation, pulses were applied for 15 minutes at 3V and then the scaffolds were fixed. Subsequent analysis revealed very few fibroblasts on the side of the scaffold closest to the cathode, while many more fibroblasts were concentrated and aligned on the anode-adjacent side (Figure 4D).

Figure 4: Development of a 3D platform for the application of horizontal electrical stimulation to direct fibroblast migration.

Figure 4:

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(A) Schematic of the set-up for electrical stimulation in 2D. Two platinum wires (blue) are fixed parallel to one another and spaced 20 mm to allow a conductive scaffold to be placed in their center. (B) Photograph showing the set-up, which allows for three scaffolds to be stimulated simultaneously. (C) Schematic of the square-wave electrical pulses, depicting the duty cycle, frequency of pulses, and the range of voltages tested. (D) Schematic (left) of the scaffold with the direction of the electric stimulation shown in pink, with photomicrographs (center, right) of the cells localized in the scaffold after the duration of stimulation. Fibroblasts are stained for phalloidin (green) and Hoechst (blue). Scale bar: 110 μm. (E) Scale bar: 700 μm.

To better elucidate and understand the effects of the exogenous electrical stimulation, pulse parameters with increasing durations and different voltages were compared. As the duration of stimulation increased with pulses at 1V (Figure 5A), from 0 min (far left), 5 minutes, 15 minutes, to 30 minutes (far right), the fibroblasts in the scaffolds transitioned from random orientations to an organized arrangement of cells. Between 15 and 30 minutes, there was little improvement in the alignment of the fibroblasts. Interestingly, the cells that were exposed to stimulation were more likely to have a more elongated spindle phenotype. The orientation of the fibroblasts was quantified by comparing cell-laden scaffolds that received 0 minutes of stimulation daily, for a duration of 3 days, with cell-laden scaffolds that received 30 minutes of stimulation daily, for a duration of 3 days, with pulses of peak-to-peak voltage (Vpp) of 1V (Figure 5B). Fibroblasts were grouped and counted as being parallel to the scaffold (0°), as well as intervals of 30° orientation. The fibroblasts that received daily electrical stimulation had an increased likelihood (60%) of orientation at 0°, with very few cells perpendicular to the direction of stimulation. The cells that received no stimulation did not exhibit any significant orientation preference.

Figure 5: Quantification of fibroblast alignment after application of exogenous electrical pulses across the scaffolds.

Figure 5:

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(A) Photomicrographs of fibroblasts which experience increasing duration of electrical stimulation, from left to right. Scale bars: 160 μm. (B) Photomicrographs of fibroblasts which received no stimulation (left) or daily stimulation (30 minutes, for 3 days). On the right side, quantification of the orientation of cells after the 3-day interval, with n=5 scaffolds per condition. Scale bars: 130 μm. (C) Photomicrographs of fibroblasts, which received stimulation with amplitude of 0V, 1V, and 3V. On the right side, quantification of the orientation of cells after the stimulation was applied, with n=6 scaffolds per condition. Scale bars: 160 μm. (D) Photomicrographs of fibroblasts which received no stimulation, no stimulation but the conditioned media of stimulated fibroblasts, and cells which directly received stimulation. On the right side, quantification of the orientation cells after the different treatments, with n=6 scaffolds per condition. Scale bars: 130 μm. In all images, cells were stained for phalloidin (green) and Hoechst (blue).

Next, to evaluate the effect of voltage magnitude, pulses of increasing voltage were applied for a duration of 20 minutes (Figure 5C). Fibroblasts that experienced no exogenous electrical stimulation had no strong preference for orientation, while cells that experienced 1V were more likely to be oriented between [−30°,30°]. Cells that experienced 3V had a further increase in 0° orientation, with a substantial decrease in the other orientations. Additionally, cells that experienced 1V and 3V stimulation were elongated and had a different morphology from the fibroblasts that experienced no stimulation. After stimulation at 3V, the media from the cell cultures was collected and added to a cell-laden scaffold that received no stimulation (Figure 5D). When these cells were evaluated, there was an increased preference for the [−30°,30°] orientations. While the fibroblasts were less elongated than the cells that directly received stimulation, they were still longer and more extended than the cells that received no stimulation.

2.4. Assessment of vertical stimulation for wound healing applications

To create and test a model for wound healing, a stimulation set-up which allowed for the application of electrical stimulation in 3D was created. Petri dishes were covered with a uniform coating of conductive tape, to allow for the voltage to flow from the top plate to the bottom plate (Figure 6A, 6B). Fibroblasts were exposed to stimulation at 1V for 50 or 80 min, and the relative locations (upper, middle, and lower third) of the scaffolds were compared. For cells that received no stimulation, there were no differences in the number or morphology of the cells at either the upper or lower third (Figure 6C). Further, when quantifying the number of fibroblasts at each location, there were no statistical differences (Figure 6D). When stimulation was applied for 50 minutes, there were fewer cells in the upper third of the scaffold, with more cells in the lower third. After 80 minutes of stimulation, there was again significantly more cells in the lower third of the scaffold, and the cells were closely packed together and elongated. The remaining cells in the upper third had a small circular phenotype, which could indicate that these cells are migrating in the Z-direction with processes now extending along the Z rather than in the X-Y plane.

Figure 6: Evaluation of fibroblast migration after exogenous vertical electrical stimulation pulses in 3D.

Figure 6:

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(A) Schematic of the set-up in 3D. Copper foil (gold) uniformly covers both the top and bottom plates of a petri dish, to allow for a uniform electric field between the two plates. (B) Photograph of the set-up in 3D, shown closed (left) and intentionally misaligned, and opened (right). Scale bars: 35 mm. (C) Photomicrographs of fibroblasts, showing the upper third of the gel (left column) and the lower third of the gel (right column), with different amounts of electrical stimulation applied in the z-direction, with n=3 scaffolds per condition. Scale bars: 190 μm. (D) Quantification of the fibroblasts at each third of the gel, over the three different stimulation conditions. Mean±s.d. are plotted, with at least n=5 fields of view compared for each of the three samples per condition. Two-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) post hoc test: ****P < 0.0001, 0.001<**P<0.01, non-significant (n.s.) P > 0.05.

3. CONCLUSIONS

We describe tunable porous hydrogels which are designed to be conductive and match the mechanical properties of the dermal layer of skin. The hydrogel matrix is made from alginate, while the conductive additives are carbon nanomaterials with high aspect ratios so only that a low (<0.3% weight) content is needed to introduce conductivity. Fibroblasts can be integrated into the scaffolds with high cell viability, and the morphology and proliferation of the cells can be affected by the mechanical and electrical properties of the scaffold. Interestingly, the scaffolds tested with a lower storage modulus (G’) and a higher conductivity seemed to best support the fibroblasts. The more conductive scaffolds had a higher ratio of CNT, which increase the scaffold roughness and could facilitate the spreading of the fibroblasts, and thus promote their proliferation and migration. Further, as electrically conductive scaffolds can be used to grow 3D networks of the cells, both horizontal and vertical electrical pulses can be applied to orient or direct the fibroblasts to desired parts of the scaffold. These findings demonstrate the potential for these scaffolds to be used in a wound healing model, where the scaffold can mimic the mechanical properties of the dermis and electrical pulses can be used to guide cells to the defect or injury zone.

While this study just explores the effects of voltage amplitude on the fibroblast location, other properties of the stimulation profile such as frequency and duty cycle could also be explored. By changing these properties, the effects on the cells can be better understood. Additional cell types, including keratinocytes, which have been shown to be responsive to electrical stimulation[12,34], could be incorporated in future studies to build a more complex skin model, and the effects of electrical stimulation and scaffold stiffness and viscoelasticity on the resulting model tissue could be explored. As the application of electrical pulses does not affect the scaffold integrity, and the described set-ups are easily integrated into an incubator, the cells can be subject to long duration of electrical stimulation without affecting their viability. With many applications to tissue engineering and biohybrid electronics, these scaffolds may be translated to various in vitro and in vivo applications.

4. EXPERIMENTAL SECTION

Fabrication of porous and conductive hydrogels

To prepare the porous gels, an alginate precursor solution was made by dissolving sterile alginate powder (Protonal LF 10/60) which was coupled with the oligopeptide GGGRGDSP (Peptides International) with carbodiimide chemistry as previously described[35], in deionized water at 2% weight volume (w/v). The samples were cast, and then moved to −20°C to introduce ice crystals into the scaffold. Next, the ice was removed by placing the scaffolds on a lyophilizer. The porous structure was preserved by crosslinking the scaffolds with a 450 mM solution of calcium nitrate dissolved in 100% ethanol.

To create conductive formulations, carbon nanomaterials were mechanically suspended into the alginate matrix and then vortexed briefly, followed by a 10 minute sonication step. Both multiwalled carbon nanotubes (CNT; Nanocyl NC3100, with length 1.5 μm and diameter of 10 nm) and graphene flakes (GF; Sixonia, Germany, where flakes were 1–2 μm in the lateral dimension and the GF concentration was 2.143 mg/mL in water with no additives or surfactants) were used.

Samples that had carbon nanomaterials had both CNT and GF added. The compositions were as following: 0%, no carbon nanomaterials; 0.13% total as 0.06% CNT and 0.071% GF; 0.22% total as 0.16% CNT and 0.06% GF; 0.31% as 0.3% CNT and 0.01% GF. The ratios were chosen so that there was a higher GF-lower CNT, lower GF-higher CNT, and middle GF-middle CNT condition.

Mechanical measurements

The local properties of the scaffolds were calculated with a nanoindenter (G200 Keysight Technologies). A 400 μm spherical tip was used, and at least 5 measurements were taken per sample. After every sample, the tip was inspected and cleaned to remove and residues from the previous sample(s).

Electrical measurements

To evaluate the electrical properties of the scaffold, a custom-made 4 point probe was fabricated by soldering 4 wires to a straight-pin header, with pins spaced 2.54 mm. The probe was gently placed underneath the samples tested. Scaffolds had a diameter of 16mm and a thickness of 0.8 mm. The 4 point probe was connected to a Hioki 3544 resistance meter, and provided the sheet resistance of each scaffold. As each of the sample dimensions were known, the equivalent conductivity (S/m) was calculated using the correction factors as described by Topsøe[36]. At least 3 gels per conditions were measured, with at least 4 measurements from each gel.

Structural properties

Scanning electron microscopy (SEM) was done using a Hitachi SU8230 Field Emission microscope. The samples were dehydrated in ethanol and left to completely dry, before mounted on SEM stubs (Ted Pella) with carbon tape (Ted Pella). Next, 10 nm of Pt/Pd 80/20 (EMS 300T Dual Head Sputter Coater, Quarum/EMS) was deposited on the surface of the sample with a current controlled at 40 mA.

Cell culture maintenance and cell seeding into the gels

3T3 Fibroblasts were purchased from the American Type Culture Collection (ATCC, Manassas VA). Cells were kept in media which consisted of DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and kept in an incubator at 37°C in 5% CO2 and passaged when reaching ~70% confluency. To incorporate the cells into the scaffold, all the materials were soaked at least overnight in the appropriate media for the fibroblasts. On the day of cell seeding, the media was removed and the scaffolds were left to dry slightly for 10 minutes. A mixture of cells in media were added into the scaffold, dropwise, at a volume such that the desired number of cells (200,000) were dropped into the scaffold, drop-wise and at different locations of the scaffold to facilitate uniform seeding. The cell-laden scaffolds were placed in the incubator for 15 minutes to facilitate cell attachment, after which additional media (~400 μl) was added to the scaffolds so that they were completely submerged. Complete media changes were done every 2 days. To assess the viability of cells in the scaffold, fibroblasts were cultured for 2 days after which they were fixed and stained. For experiments on the stimulation of the cells, the duration of the experiment was 3 days. After the experiment was finished, the samples were immediately fixed and prepared for staining.

Immunohistochemistry (IHC) staining

Cell-laden scaffolds were prepared for IHC by removing the media and then rinsing the scaffolds once with sterile PBS. Afterwards, a 4% paraformaldehyde solution was added to the scaffolds and left for 15 minutes. Next, three washes with PBS were done and the samples were stored in PBS until ready for use. For staining, the samples were incubated with a 0.1% Triton-X solution for 6 minutes and then washed with PBS three times. The samples were incubated with phalloidin (Alexa Fluor 488, Thermo Fisher Scientific; 1:100 dilution) and Hoechst (33342, 20 mM solution, Thermo Fisher Scientific; 1:500 dilution) for one hour. Afterwards, the samples were rinsed two times with PBS, mounted on a glass slide, covered with a droplet of ProLong Gold Antifade (Invitrogen) and then a coverslip was placed on top. The samples were not sectioned but rather kept as a bulk gel, as the 10x objective was able to image through the entire thickness (~600 μm) of the sample.

Image acquisition

Images were collected using a Zeiss confocal, on 10x and 20x objectives. 2D images were acquired and were saved as .czi and then converted to .tiff or .png using ImageJ. Scaffolds are imaged over multiple planes, to understand how the cells are localized in the z and in various (x,y) positions.

Horizontal and vertical electrical stimulation set-up

For the horizontal electrical stimulation set-up across the scaffold, the lid of a six-well tissue culture plate was milled to have 2 holes which were spaced 20 mm. The holes were milled with a 0.6 mm bit piece so that the openings could fit platinum wires (Strem Materials, wire diameter: 0.5 mm). The wires were fed through the openings of the holes until they touched the bottom of the plate. At this point, the wires were bent 90° so that they spanned the length of the well. A 5 minute epoxy was used to glue and secure the platinum wires in place, and the entire construct was sprayed with 70% ethanol and left to completely crosslink and dry overnight under the UV of a sterile tissue culture hood. Cables with a banana clip ending were attached to a function generator, which was set with the desired frequency, voltage amplitude, duty cycle, and pulse waveform. The stimulation profile was confirmed by attaching a green LED and observing the pattern of the light pulses as the stimulation conditions were attached.

The stimulation parameters were chosen using values that were previously reported, and briefly evaluated analytically, with the following previous work[37–41]. Because the impedance of the conductive hydrogel scaffold is so low, the impedance of the spacing between the platinum wires and the conductive hydrogel scaffold dominate the system. The impedance of the cell culture media can be evaluated (~5 kΩ) as well as the double layer capacitance (~2.5 μF for this spacing and system). The current across the spacing ~21 μA which is 3 orders of magnitude higher than the current passing through a cell with a membrane potential of −40 mV. Together with previously reported studies, our analytical evaluation, and our results, we believe this is sufficient to direct the migration of cells.

For the vertical electrical stimulation set-up, small sterile tissue culture petri dishes (35 mm diameter) were opened under the hood and both the top and the bottom plates were covered with a uniform layer of copper-foil adhesive tape. The tape was applied carefully so that none of the pieces overlapped, but were touching, so that the resulting electric field would be uniform. After a uniform coating of copper-foil covered each plate, a piece of tape was placed perpendicular and on top to the coating so that all the pieces were connected. The perpendicular piece was then folded on the end, to allow a cable to clip to it.

For experimentation, the scaffold was placed in the well so that it was in the center of the two platinum wires, or simply placed between the two copper-foil coated plates. All scaffolds were cast in a 24 plate so that they were the same diameter, and were cast by volume (μl) so that their height was also the same. Stimulation was applied to the material by connecting the clip-end of the cables to their respective platinum wires, or to the folded copper-foil piece, with the anode on the left side or top, and the cathode on the right side or bottom plate. For stimulation durations of less than 30 minutes, the stimulation was done under the hood. For stimulation durations greater than 30 minutes, the plate was placed in a sterile incubator so that the fibroblasts were under the desired temperature and CO2 values.

Statistical analysis

Statistical analysis was done using Prism 9 and data were first confirmed to be normally distributed. The data was not preprocessed and is presented as mean +/− standard deviation (s.d.) as noted in the figure legends.

Supplementary Material

Supinfo

FUNDING AND ACKNOWLEDGEMENTS

The authors thank Dr Junzhe Lou for sharing fibroblasts, and Mike Tarkanian (MIT) for his help with machining the set-up for 2D stimulation of cells. Funding sources: this work was supported in part by the Center for Nanoscale Systems at Harvard University, which is a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation under award no. 1541959. This work was supported by an NSF GRFP to CMT, an NIH grant to DJM (RO1DE013033), an NIH grant to DJM (5R01DE013349), NSF-MRSEC DMR-2011754, and funding by the Wyss Institute for Biologically Inspired Engineering at Harvard University.

Footnotes

CONFLICT OF INTEREST STATEMENT

CMT and DJM have filed a patent application on viscoelastic conductive scaffolds.

DATA AVAILABILITY STATEMENT

The data that support the findings of this work are available from the corresponding author upon reasonable request.

REFERENCES

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supinfo
NIHMS1892790-supplement-Supinfo.docx (3.6MB, docx)

Data Availability Statement

The data that support the findings of this work are available from the corresponding author upon reasonable request.

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