An ink-jet printed electrical stimulation platform for muscle tissue regeneration

Author’s Accepted Manuscript An ink-jet printed electrical stimulation platform for muscle tissue regeneration Gabriele Maria Fortunato, Carmelo De Maria, David Eglin, Tiziano Serra, Giovanni Vozzi www.elsevier.com

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Received date: 29 August 2018 Revised date: 28 September 2018 Accepted date: 1 October 2018 Cite this article as: Gabriele Maria Fortunato, Carmelo De Maria, David Eglin, Tiziano Serra and Giovanni Vozzi, An ink-jet printed electrical stimulation platform for muscle tissue regeneration, Bioprinting, https://doi.org/10.1016/j.bprint.2018.e00035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An ink-jet printed electrical stimulation platform for muscle tissue regeneration Gabriele Maria Fortunato1,2, Carmelo De Maria1, David Eglin2, Tiziano Serra2, Giovanni Vozzi1 1

Research Centre ‘E. Piaggio’ and Dept. of Ingegneria dell’Informazione of University of Pisa, Pisa, Italy

2

AO Research Institute Davos, Davos, Switzerland

E-Mail adresses: [email protected] (G Vozzi) [email protected] (GM Fortunato)

Abstract Conducting polymeric materials have been used to modulate response of cells seeded on their surfaces. However, there is still major improvement to be made related to their biocompatibility, conductivity, stability in biological milieu, and processability toward truly tissue engineered functional device. In this work, conductive polymer, poly(3,4-ethylene-dioxythiophene):polystyrene-sulfonate (PEDOT:PSS), and its possible applications in tissue engineering were explored. In particular PEDOT:PSS solution was inkjet printed onto a gelatin substrate for obtaining a conductive structure. Mechanical and electrical characterizations, structural stability by swelling and degradation tests were carried out on different PEDOTbased samples obtained by varying the number of printed PEDOT layers from 5 to 50 on gelatin substrate. Biocompatibility of substrates was investigated on C2C12 myoblasts, through metabolic activity assay and imaging analysis during a 7-days culture period, to assess cell morphology, differentiation and alignment. The results of this first part allowed to proceed with the second part of the study in which these substrates were used for the design of an electrical stimulation device, with the aim of providing the external stimulus (3V amplitude square wave at 1 and 2 Hz frequency) to guide myotubes alignment and enhance differentiation, having in this way promising applications in the field of muscle tissue engineering.

Keywords: PEDOT; inkjet printing; muscle cells; electrical stimulation device; cell alignment, cell differentiation

1 - Introduction Conductive polymers (CPs) have been one of the most attractive topics of biomedical research in recent years. CP-based materials can be effectively used as tissue scaffolds for the replacement or restoration of damaged or malfunctioning tissues since many of them respond to electrical stimulation [1]. The use of conductive polymers in tissue engineering and regenerative medicine is very promising due to the combination of good electrical and optical properties (similar to metals and inorganic semiconductors) with their mechanical properties. In fact, these materials provide better mechanical compatibility and structural tunability with cells and organs finding a variety of applications in bone, skeletal muscle, nerve, cardiac and skin tissue engineering [2]. CPs can be fabricated in several ways to produce scaffolds or substrate for tissue engineering: pure polymer films [3–5], blends or composite films [6][7], copolymer films [8][9], nanofibers 1

[10], hydrogels [11] and 3D scaffolds [12][13]. The Poly(3,4-ethylenedioxythiophene) (PEDOT) is one of the most promising conductive polymer. PEDOT is a Polythiophene derivative, characterized by high electrical conductivity, optical transparency in visible light range and good electrochromic characteristics [14]. It is a chemically stable, conjugated polymer insoluble in water that is of considerable interest for a variety of applications including coatings for interfacing electronic biomedical devices with living tissue thanks to its intrinsic conductivity [16-18]. The solubility problem was circumvented by using poly(styrene sulfonate) (PSS), a water-soluble polyelectrolyte, as the charge-balancing dopant during polymerization to yield PEDOT:PSS. The result is a water-soluble polyelectrolyte system with good film-forming properties, high conductivity (about 10 S/cm), high visible light transmissivity, and excellent stability [18]. Thanks to its excellent properties of conductivity and biocompatibility, PEDOT:PSS resulted a suitable conductive polymer to be used in this work. PEDOT and other CPs were used in several works in combination with electrical stimulation to enhance cell alignment and differentiation. Cross-linked PEDOT:PSS spin-coated films were used for the culture of neural stem cells exposed to pulsed stimulation obtaining a better alignment [19][20]. Polypyrrole films were used for PC-12 cell culture obtaining a better differentiation under electrical stimulation [21]. GelMA hydrogels doped with carbon nanotubes (CNTs) combined with electrical stimulation enhanced C2C12 cell line differentiation [22]. The aim of this study was to use CP-based substrates to control muscle cell alignment and differentiation. PEDOT-based substrates were fabricated and characterized. Inkjet printing was used to fabricate different PEDOT patterns on gelatin substrates, modifying the print design and conductive material concentration, to adjust the electrical characteristics of the substrates by varying the conductivity. After electrical and mechanical properties, and stability evaluation, biocompatibility and muscle cells response were assessed. Finally, these substrates were used for the design of an electrical stimulation device: C2C12 cells alignment and differentiation were evaluated under electrical stimulation.

2 - Materials and methods 2.1 Materials PEDOT:PSS aqueous dispersion (Baytron® P from Bayer, Leverkusen, Germany) was used in this work. Baytron® P is a water solution of 1.3 % w/v conjugated polymer PEDOT:PSS (or PEDOT in the remaining manuscript text). Gelatin from porcine skin type A was purchased from Sigma-Aldrich, Italy, and (3glycidoxypropyl)-trimethoxysilane (GPTMS) from Alfa Aesar, Ward Hill, US.

2.2 Methods 2.2.1 PEDOT-gelatin samples Samples used for electrical characterization were obtained by mixing PEDOT solution with 5% w/v gelatin solution in Milli-Q® water containing 3.68 % v/v GPTMS , at 1:0.1, 0.2, 0.3, 0.4 PEDOT-gelatin (P-G) volume ratios (with higher gelatin content solution was inhomogeneous). The mixtures were stirred on a 2

heated plate at 50°C for 30 min, casted in petri dish (3 ml solution) and dried for 48 hr at room temperature to complete crosslinking by GPTMS, obtaining a film, and then rehydrated for 30 min before use. Samples used for the others characterizations were produced using an inkjet printing fabrication technique, deposing 5, 10, 20, 30, 40 and 50 layers of PEDOT on a thin pre-cross-linked gelatin substrate [23–25]. Briefly, 2 ml of gelatin solution as described above was placed in petri dishes of 4 cm diameter and put in the fridge at 4 °C for about 1 min. Cooling permitted to have a faster physical gel formation. A 3D thermal inkjet printer 'Penelope', based on HP technology, was used for printing PEDOT [26] (Fig. 1A). After printing, the samples were dried for 48 hr at room temperature to complete crosslinking of gelatin, and then rehydrated for 30 min before use.

2.2.2 Electrical characterization Electrical characterization was performed by impedance measurement and subsequent fitting of experimental data. Each specimen was equipped with copper contacts (for inkjet printed samples a 3D printed support with copper needles inserted on it was needed) and impedance was evaluated using an impedance analyzer Agilent E4980A with a logarithmically variable frequency in the range 20Hz - 2MHz. Using Matlab® System Identification Toolbox a fitting transfer function with finite number of poles and zeros was evaluated for each dataset, by minimizing the weighted least squares error. A circuit model was then associated to the fitting function and lumped circuital parameters were identified. Tests were performed in triplicate at different time-points up to 21 days in aqueous solution (see the section 2.2.4).

2.2.3 Electromechanical characterization Mechanical properties of inkjet printed samples were determined by a tensile test (Zwick-Roell Z005 ProLine, deformation rate 1%/min until failure) and at the same time the dependence of electrical properties on strain rate was evaluated by measuring impedance at each 1% strain (stepwise load application). Sample size were measured using a digital calliper, and the elastic modulus was evaluated by linear regression of the first linear part of the stress strain curve obtained from tensile test. Tests were performed in triplicate.

2.2.4 Swelling test Structural stability of substrates was proved by a 28-days swelling test followed by weight loss evaluation (degradation) after freeze-drying. The evaluation was done over a 28-days period to demonstrate the stability also in the case of future studies with other cell types requiring longer culture periods (e.g. hMSCs). Six mm diameter printed samples were immersed in phosphate saline buffer (PBS) at 37°C and weighted at intervals. Tests were performed in triplicate and pure gelatin substrate was used as control.

2.2.5 Wettability and roughness Wettability was determined by contact angle test (Kruss® Drop Shape Analysis System DSA 10) in dry and wet conditions. For each sample, 5-10-20-30-40-50 PEDOT layers and gelatin control, 9 measurements were 3

acquired. Roughness was evaluated by profilometry (FRT TM white light profilometer) evaluating Ra (arithmetic average value of filtered roughness profile determined from deviations about the center line) and Rz (average distance between the highest peak and lowest valley in each sampling length of the tested profile).

2.3 Cell culture Cell interaction and biocompatibility of fabricated samples were evaluated by cell culture of C2C12 myoblast cell line for 7 days [27]. Six mm diameter samples of gelatin, 20 and 50 printed PEDOT layers on gelatin substrate were used for cell seeding (

cells/mm2). Cells were also seeded on plastic tissue

culture plate (TCP) for control. Two different protocols were carried out in parallel by adding or not differentiation medium (Horse Serum (HS) added) on day 2. Culture medium was prepared with Dulbecco’s Modified Eagle’s Medium (DMEM high glucose (4.5 g/l) powder 13.38 g/l (Gibco, USA), Sodium bicarbonate (NaHCO3) 3.7 g/l (Sigma-Aldrich, USA) and Sodium pyruvate 0.11 g/l (Sigma-Aldrich, USA)) + 10% v/v FBS (Fetal Bovine Serum, Gibco, USA) or 2% HS (Horse Serum, Gibco, USA) + 1% v/v PEN/STREP (Penicillin-Streptomycin, Gibco, USA). The cell culture experiment was repeated three times.

2.3.1 Metabolic activity and imaging Metabolic activity was evaluated on day 1, 3 and 7 by CellTiter-Blue® assay. Cell morphology, differentiation and alignment were assessed by cell imaging using an EVOS FL Auto2 cell imaging system (ThermoFisher, Waltham, USA), and analysed using Fiji® software. Cell imaging analysis was carried out considering 3 different images acquired randomly for each sample (gelatin (GEL), gelatin with 20 and 50 printed layers (GEL-20, GEL-50), TCP (tissue culture plate)) with and without differentiation medium. Cell morphological parameters investigated on day 1 were cell area, circularity and aspect ratio. After fixation with 4% buffered formalin and permeabilization with 0.2% v/v Triton™X-100 in PBS, cell cytoplasm actin filaments staining was performed using Phalloidin-TRITC (Sigma-Aldrich, USA), and nucleic acid staining using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, USA). C2C12 differentiation was evaluated by fusion index (number of nuclei in myotubes (>2) as a percentage of total nuclei) and maturation index (myotubes number with more than 5 nuclei) [28]. Alignment was investigated by fast Fourier transform (FFT) analysis of fluorescence images [29].

2.4 Electrical stimulation device Electrical stimulation device prototype was designed for parallel electrical stimulation of three samples. It consists of a support for glass slides connected through stainless steel electrodes to the electronic components (Fig. 1D): 2 3V batteries; an adjustable frequency pulse generator module (NE555); a LED to control that current is passing through samples. For an easy handling of the device and at the same time to guarantee its sterility, a box and two covers (for glass slides and electronic parts) were designed. Each part of the device was printed in ABS using a 3D printer (Airwolf3DTM Axiom Dual Direct Drive printer). 4

Conductive samples were fabricated on a glass slide in order to have a better interface with electrodes for electrical stimulation (ES). A support was 3D printed for casting the gelatin substrate and PEDOT was subsequently inkjet printed on top (Fig. 1B). A cylindrical silicone cage was built using a 3D bioprinter (3DDiscoveryTM Biosafety, regenHU) on the top of the sample to be able to seed cells [20]. The stability of the cage was verified (after 5 days drying at 70°C) over a period of 7 days with PBS at 37°C. Inkjet printed PEDOT samples (50 layers only) were prepared for cell seeding and subsequent electrical stimulation (Fig. 1C). C2C12 cells were seeded in silicone wells (

cells/mm2) and stimulated from day 3 to 7 at 3V/cm, 1-2

Hz, 0.5 duty cycle, 6 h/day. Cultures with and without differentiation medium and with no electrical stimulation were also carried out. Metabolic activity (day 1, 3, 7) and cell imaging on day 7 were performed as reported in section 2.3.1 [31].

2.5 Statistical analysis All data are presented as mean ± standard deviation (error bars in all graphs). Statistical analyses were conducted using Graphpad Prism® software. Differences between samples were determined from one-way or two-ways ANOVA and were considered statistically significant when p < 0.05. When differences were detected differences between groups were determined by Tukey’s multiple comparisons test. 3 – Results

3.1 Samples characterization Results about electrical and mechanical properties, swelling, wettability and roughness are reported in the following sections.

3.1.1 Electrical characterization The best fitting resulted from a 2-poles and 1-zero transfer function (eq. 1):

( )

(eq. 1)

A circuit model was associated to the transfer function obtained from the fitting (Fig. 2A), and lumped circuit parameters R1, R2, C1 and C2 were calculated. R1 value remained constant (3-5% maximum variation) for different PEDOT-Gelatin composites with a variable content of gelatin (Fig. 2B), so it was associated to PEDOT. The same analysis was carried out on inkjet printed specimens obtaining similar results. Impedance measurement of inkjet printed rehydrated samples (in deionized water) was carried out over a 21-days period showing an excellent stability in aqueous environment (Fig. 2C).

5

3.1.2 Electromechanical characterization Tensile test showed that mechanical properties are not influenced by the number of PEDOT layers on the gelatin substrate, as elastic moduli are not statistically different for samples with different number of PEDOT layers. The electrical impedance is not altered by strain rate, as the circuital parameters remain constant with increasing strain (3-5% maximum variation) (Fig. 2D). 3.1.3 Swelling test Swelling results showed similar behaviour from 30 min to 28 days of immersion in PBS at 37°C: weight swelling 40% (Fig. 2G), area 3-5%. Degradation was not significant: weight loss of samples was 4-5% after freeze-drying (Fig. 2E).

3.1.4 Wettability and roughness Contact angle measurement highlighted a better hydrophilicity with printed PEDOT (Fig. 2F), while profilometry test showed a decreasing roughness (Ra and Rz) with increasing number of printed PEDOT layers (Fig. 2G-H).

3.2 Cell culture Metabolic activity showed an increasing trend from day 1 to 7, indicating a good proliferation, with no statistical significant differences between 20 and 50 printed layers samples (Fig. 4A). In Fig. 3 some samples with differentiation medium are presented. From day 1 images, cell area, circularity and aspect ratio were evaluated. Circularity was 0.3 for each sample, indicating a good cell elongation and adhesion. Differentiation was evaluated on day 7. A two-ways ANOVA on the Fusion Index showed significant influence of both PEDOT content (p<0.0001) and differentiation medium HS (p<0.0001). A slight increase in the differentiation of C2C12 into myotubes (without differentiation medium) was observed for 50 layers sample compared to 20 layers one (Tukey’s multiple comparisons: p < 0.02) (Fig. 4B). This result can be explained considering the substrate conductivity: with 50 layers sample resistivity is much lower and therefore, the interaction between the cells (communication through electrical signals) could be facilitated, allowing a better differentiation [30]. Maturation index showed similar results: better differentiation on 20 and 50 layers samples respect to gelatin control (Fig. 4C). Cell alignment was also evaluated on day 7 by FFT analysis. From Ratio to Mean Orthogonal Angle (RMOA) quantification, it resulted a better alignment with increasing PEDOT content (Fig. 4D), indicated by the two-ways ANOVA (p<0.0001: Differentiation medium has not a statistically significant effect on alignment).

3.3 Electrical stimulation device Results about metabolic activity and cell imaging of electrically stimulated cells are reported in this section. An example of images acquired on day 7 is showed in Fig. 5. Metabolic activity increased during 7-days culture with no statistical significant differences between stimulated and not stimulated samples (Fig. 6A).

6

Differentiation and alignment were evaluated on 3 pictures for each sample (1 Hz, 2 Hz and without electrical stimulation). Fusion index and maturation index were determined (Fig. 6B-C). 1 Hz samples showed a good enhancement in differentiation (higher values of maturation and fusion indexes), especially considering cultures with no differentiation medium. A two-ways ANOVA showed, for both factors, stimulation and differentiation medium a significant influence (p<0.0001). FFT analysis was carried out for alignment quantification. Myotubes resulted aligned to voltage direction (angle of 180° for stimulated samples). 1 Hz stimulation showed better results also in this case respect to 2 Hz and no stimulation (Fig. 6D). A two-ways ANOVA showed a significant influence of electrical stimulation (p<0.0001) and not significant role of differentiation medium. 4 – Discussion In this work we presented the fabrication of biocompatible substrates obtained by inkjet printing of PEDOT:PSS aqueous dispersion on a thin cross-linked gelatin substrate. First of all, the stability of substrates was proved by demonstrating unaltered intrinsic electric properties and also a near-absent degradation in a physiological environment. Then, biocompatibility was demonstrated by culturing C2C12 cells. For 20 and 50 printed PEDOT layers on gelatin substrate, metabolic activity, differentiation and alignment resulted better compared to pure gelatin. This can be explained by topographical effect: wettability and roughness, higher in printed samples, play a crucial role in cell adhesion and proliferation [30–32]. Gelatin resulted more hydrophobic compared to PEDOT samples while cells well adhere onto surfaces with contact angles of 60-70°. Moreover, gelatin resulted to have a smooth surface while cells better proliferate onto rough surfaces [34]. In the end, an enhanced alignment could be due to PEDOT conductivity that allows a better cell interaction and exchange of electrical signals: alignment is better for 50 layers sample so, increases as PEDOT content increase. Several works in recent years showed the possibility to use anisotropy of scaffolds and substrates to improve cell alignment. For example, GelMA-CNTs hybrid hydrogels were used in combination to electrical stimulation to obtain aligned myotubes [22]. However CNTs safety issues in the human body have not been adequately addressed, as most in vivo toxicity tests were conducted over a relatively short time, and long term safety has to be demonstrated [35]. Some works about long-term effects of PEDOT-based materials, instead, showed good cell interaction and biocompatibility [36][37]. In the second part of the work, PEDOT substrates were used for the design of an electrical stimulation device. Samples with 50 layers of PEDOT were chosen in this phase due to physical and biological reasons. First of all, in fact, these samples, having a higher content of conductive polymer, offered a lower resistance to the passage of electrical current and therefore the risk of overheating of the substrate, which can be harmful to the cells, was lower. Then, better results about differentiation and alignment of myotubes were obtained with 50 layers substrates in the first part of the work. The objective was to exploit the anisotropy of electrical stimulation to guide cell alignment. A very important result in this step was the alignment of myotubes that plays a crucial role in muscle tissue engineering, for the generation of muscle strength and the functionality of tissue [38]. To enhance this behaviour it is possible to fabricate scaffolds with aligned 7

patterns [39] or apply a mechanical or electrical stimulation on cultured myoblasts that can also influence gene regulation, protein expression and accumulation and even fibres alignment [40]. In this study, we showed the possibility to achieve myotubes alignment through electrical stimulation. In previous works electrodes were put inside cell culture medium risking thereby to influence cell alignment by the presence of ions [22][28]. In this work, instead, electrodes were placed outside the silicone well that contains cells, thus only using the conductivity of the substrate for cell alignment. In particular, with 1 Hz electrical stimulation, a better differentiation and alignment (alignment angle 180°) was obtained. The improvement of differentiation was particularly evident in the absence of differentiation medium HS. Thus, the fabricated conductive substrates can be used in combination with an external electrical stimulation to guide cell alignment and at the same time achieve good myoblasts differentiation without HS.

Conclusion Here we showed a method to fabricate biocompatible PEDOT-based substrates used for the design of an electrostimulation device that opens the way to number of possibilities for studying cell behaviour. Our conductive substrates showed stability (unaltered electrical properties in an aqueous environment, nearabsent degradation) and biocompatibility (good metabolic activity, adhesion, differentiation, alignment) having in this way the potential to be used as implantable templates. The designed device proved the possibility to guide cell alignment and enhance myotubes differentiation through electrical stimulation, having therefore promising future applications in the field of muscle tissue engineering. At the current stage, the electrical stimulation device could be used to study the behaviour of muscle cells subjected to different electrical stimuli by varying the combinations of frequency and voltage amplitude. Future work could include the assessment of co-culturing system to create heterogeneous multicellular biological tissue where anisotropy plays a crucial role, such as vascularized musculoskeletal tissue with pre-organized vessel networks or complex neuromuscular junctions.

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Figure 1 Fabrication of PEDOT-based substrates: A) Inkjet printing of PEDOT on cross-linked gelatin substrate; Electrical stimulation device prototype: B) CAD file for gelatin casting support C) 50 printed PEDOT layers on gelatin substrate sample with silicone cage on top - D) CAD design electrical stimulation device prototype Figure 2 A) 2-poles 1-zero transfer function with related circuit model (R1 represents PEDOT content) for 5, 10 and 20 PEDOT layers samples - B) R1 and R2 trend with increasing gelatin content: R1 is practically constant - C) R1 values of inkjet printed samples over 3 weeks: constant trend - D) Impedance measurement carried out while tensile test running: constant circuital parameters with increasing strain (R1 is showed) E) Weight loss evaluation: degradation is not significant (5 up to 50 printed - F) Contact angle of different samples: 5 up to 50 printed PEDOT layers and gelatin control (Tukey’s multiple comparisons - ****: p<0.0001) - PEDOT layers and gelatin control) - G-H) Roughness parameters Ra and Rz for different samples (0=gelatin, 5, 20 and 50 PEDOT layers) Figure 3 Cell imaging, staining with DAPI (nuclei) and Phalloidin TRITC (cytoplasm). Images for different samples (cultures with differentiation medium +HS): gelatin control, gelatin + 20 and 50 printed PEDOT layers and TCP control (scale bar is 200 μm) Figure 4 A) Metabolic activity C2C12 cell culture (CellTiter® blu assay) - B) Differentiation analysis: A) Fusion index (Tukey’s multiple comparisons - *: p<0.05) – C) Maturation index (Tukey’s multiple comparisons - ***: p<0.0002, ****:p<0.0001 - D) Cell alignment quantification (Tukey’s multiple comparisons - *: p<0.05) - E) Original image with clearly visible myotubes alignment angle - F) FFT with oval profile for radial sum intensity - G) Radial sum intensity with highlighted angle of interest Figure 5 Day 7 images, staining with DAPI (nuclei) and Phalloidin TRITC (cytoplasm) and use of the electrical stimulation device: A) 1 Hz electrical stimulation +HS - B) 2 Hz electrical stimulation + HS - C) No electrical stimulation +HS - D) 1 Hz electrical stimulation -HS - E) 2 Hz electrical stimulation -HS F) No electrical stimulation –HS (scale bar is 200 μm) Figure 6 Electrical stimulation device: A) Metabolic activity C2C12 cell culture (fluorescence measurement) - Differentiation analysis: B) Fusion index - C) Maturation index (Tukey’s multiple comparisons - *: p<0.05; **: p<0.002; ****: p<0.0001) - D) Cell alignment quantification (ES device): A) RMOA quantification (Tukey’s multiple comparisons - *: p<0.02; **: p<0.01; ****: p<0.0001) - E) FFT with oval profile for radial sum intensity - F) Radial sum intensity with highlighted angle of interest

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