Biocompatible Elastic Conductive Films Significantly Enhanced

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Biocompatible elastic conductive films significantly enhanced myogenic differentiation of myoblast for skeletal muscle regeneration Ruonan Dong, Xin Zhao, Baolin Guo, and Peter X Ma Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00749 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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Biocompatible elastic conductive films significantly enhanced myogenic differentiation of myoblast for skeletal muscle regeneration

Ruonan Dong a, Xin Zhao a, Baolin Guo a,*, Peter X. Ma a,b,c,d,e,* a

Frontier Institute of Science and Technology, and State Key Laboratory for

Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an, 710049, China b

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI

48109, USA c

Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor,

MI 48109, USA d

Macromolecular Science and Engineering Center, University of Michigan, Ann

Arbor, MI 48109, USA e

Department of Materials Science and Engineering, University of Michigan, Ann

Arbor, MI 48109, USA * To whom correspondence should be addressed. Tel.:+86-29-83395361. Fax: +86-29-83395131. E-mail: [email protected], [email protected].

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Abstract: The key factor in skeletal muscle tissue engineering is regeneration of the functional skeletal muscles. Materials that could promote the myoblast proliferation and myogenic differentiation are promising candidates in skeletal muscle tissue engineering.

Herein,

we

developed

an

elastic

conductive

poly(ethylene

glycol)-co-poly(glycerol sebacate) (PEGS) grafted aniline pentamer (AP) copolymer that could promote the formation of myotubes by differentiating the C2C12 myoblast cells. The results of hydration behavior and water contact angle suggested that by adjusting the poly(ethylene glycol) (PEG) and AP content, this film showed a proper surface hydrophilicity for cell attachment. Additionally, these films showed tunable conductivity and mechanical properties that can be altered by changing the AP content. The maximum conductivity of the films was 1.84×10-4 S/cm and the Young’s modulus of these films ranged from 14.58±1.35 MPa to 24.62±0.61MPa. Our findings indicate that the PEGS-AP films promote the proliferation and myogenic differentiation of C2C12 cells, suggesting that they are promising biomaterials for skeletal muscle tissue engineering.

Keywords: skeletal muscle tissue engineering, conductive polymers, elastomer, hydrophilicity, aniline oligomer

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1. Introduction

Skeletal muscles play an integral role in generating force and facilitating the voluntary movement,1 but have limited ability to regenerate after injury related to both trauma and disease.2 In recent years, tissue engineering has emerged as a promising method of repairing damaged and dysfunctional skeletal muscle. Of particular interest is the development of a biomaterial capable of acting as a temporary replacement for the extracellular matrix, which is integral to the proliferation and differentiation of the certain satellite cells,3 without which the regeneration of functional skeletal muscle tissue is impossible.

1

After injury, the satellite cells reenter the cell cycle, inducing

the proliferation and fusion of myoblasts to form myotubes, thereby regenerating the muscle fibers that are the structural base of skeletal muscle tissue. Materials used in tissue engineering of skeletal muscle should be biocompatible, biodegradable and be able to support muscular tissue formation, including the localization, proliferation and differentiation of cells.4 Of the many approaches used to promote the proliferation and/or differentiation of muscular cells, two of the most promising are the application of electrical and mechanical stimuli.

Electrical stimuli had been proven to have a positive effect on developing highly differentiated and functional skeletal muscle tissue.5, 6 Therefore, conducting materials are a good choice as the matrix for the electrical signal delivery for cell culture.7 Electroactive or conducting substrates have also been proven to have significantly promote the formation of muscular tissues,8 benefiting both cell proliferation and 3


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differentiation.2,

9, 10

There are several ways to endow the materials with

electroactivity, such as using the nano-reinforcement and grafting of conductive polymers to the non-conducting materials. Unfortunately, nano-reinforcement with materials such as carbon, carbon nanotubes, graphene or gold particles suffers from premature degradation, which may be problematic in vivo. Additionally, the materials for nano-reinforcement are not soluble, making it difficult to homogenously distribute them in a two phase system.11 Graft polymers, show good biocompatibility, biodegradability and they are soluble in many organic solvents, making them easy to process.

12

Polyaniline is a conducting polymer that is widely used in the tissue

engineering field, owing to its tunable conductivity, good biocompatibility and environmental stability.11,

13-15

However, its utility is limited, as it is difficult to

process and is non-degradable.9,

12

Fortunately, aniline oligomers, which have a

similar electroactivity as well as oxidation and reduction transition chemistry to polyaniline, showed better biocompatibility, higher solubility and easier processability than polyaniline.12, 16-18 Aniline pentamer (AP), terminated with carboxyl groups in both ends, is easily introduced into the polymer main chain by grafting or blocking to endow the materials with electroactivity, leading to the wide utility of AP in areas where the conductive/ electroactive materials are desired.19-22

Mechanical stimulation is another promising method to improve myoblast proliferation and differentiation. Previous studies have proven that by applying mechanical strain experienced by native skeletal muscles during growth and

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organogenesis, functional skeletal muscle tissue could be formed.23, 24 For this reason, it is highly desirable to develop biomaterials that can withstand the mechanical load experienced by skeletal muscle tissue during formation. Muscles and tendons are elastic tissues, which prefer elastic tissue engineering matrices, leading to a tendency within the field of using elastomers as the material for the skeletal muscle engineering. Poly(glycerol sebacate) (PGS) has been proposed as a promising matrix in soft tissue engineering,25-27 such as the cardiac tissue engineering28,

29

and neuron tissue

engineering.30, 31 However, because of its poor water uptake ability, the utility of this material is limited.32, 33 Poly(ethylene glycol) (PEG) which has a good hydrophilicity and biocompatibility was introduced into the PGS by Patel et al.33 to enhance its hydrophilicity. They showed that the poly(ethylene glycol)-co-poly(glycerol sebacate) (PEGS) had a controllable degradation behavior. However, different PEG concentration led to different surface hydrophilicity which had a significant effect on the cell attachment. Patel et al.33 investigated the effect of PEGS films with different PEG concentration on the proliferation of NIH 3T3 fibroblast cells, but the effect on the myoblast cells localization, proliferation and differentiation remained to be studied. Furthermore, PEGS is biological inert34, and it is desirable to endow PEGS with biological activity, to promote the interaction between cells and materials and to further control the cellular activities.

In this study, we developed a series of conductive elastic copolymers composed of PEGS and AP with favorable mechanical properties for skeletal muscle tissue

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engineering. Films with different PEG concentration were developed and their hydration properties, together with the cell attachment behavior on the films, were studied in order to choose the most suitable PEG concentration for the films. AP endowed the films with good electroactivity and conductivity which could be confirmed by the UV-vis spectra, cyclic voltammetry (CV) curve and conductivity results. C2C12 mouse myoblast cells were seeded on the films, and the results showed that the effect on promoting the cell proliferation and myogenic differentiation are obvious. Thus, these conducting materials have good biocompatibility that has the potential to promote the skeletal muscle tissue formation and could be a promising candidate as a scaffold for skeletal muscle tissue engineering.

2. Materials and Methods

2.1 Materials Poly(ethylene glycol) with a molecular weight of 2000 mol-1 was dried under reduced pressure before use. Stannous octoate [Sn(Oct)2, 95%] from Aldrich was dried over molecular sieves and stored at a N2 atmosphere before use. Glycerol, sebacic acid, N-phenyl-1,4-phenylenediamine,

p-phenylenediamine,

succinic

anhydride,

ammonium peroxodisulfate, anhydrous tetrahydrofuran (THF), camphorsulfonic acid (CSA), dimethyl sulfoxide (DMSO), hexamethylene diisocyanate (HDI), phosphate buffer solution (PBS) were all purchased from Alderich or J&K Scientific Ltd. Other organic solvents were analytical grade. Aniline pentamer (AP) was synthesized as reported.35 6


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2.2 Synthesis of poly(ethylene glycol)-co-poly(glycerol sebacate)-g-aniline pentamer (PEGS-AP)

2.2.1 Synthesis of poly(ethylene glycol)-co-poly(glycerol sebacate) (PEGS) copolymer

The poly(ethylene glycol)-co-poly(glycerol sebacate) copolymer was prepared based on the method Patel et al.33 reported with modification. First of all, 2 g (1 mmol) of poly(ethylene glycol) and 5.55 g (27.5 mmol) of sebacic acid were stirred at 130 oC for 5 h under the nitrogen atmosphere. Then the pressure was reduced from the standard atmospheric pressure to 5 kPa for 24 h. Subsequently, 2.44 g (26.5 mmol) of glycerol was dropped into the system under nitrogen atmosphere and stirred for 6 h, followed with vacuum for another 48 h. Then the product was dissolved in 10 mL of THF, and precipitated in diethyl ether. The dissolution and precipitation processes were repeated for 3 times. After filtered and dried in the vacuum drying oven for 48 h, white solid product was obtained with yield of about 80%. Gel permeation chromatography (GPC) was used to determine the molecular weight of PEGS polymer. According to the result, the molecular weight (Mw) of PEGS was about 5000 Da.

2.2.2

Synthesis

of

poly(ethylene

glycol)-co-poly(glycerol

sebacate)-g-aniline

pentamer (PEGS-AP) copolymer

The PEGS-AP copolymer was synthesized by esterification reaction of hydroxyl group in PEGS and carboxyl group in AP. PEGS-AP copolymers with different AP 7


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content were obtained and the feed ratio of AP ranged from 6% to 18%. Taken the PEGS-AP6 (AP weight ratio in the copolymer is 6%) as an example, 1000 mg of PEGS and 63 mg of AP were first mixed together, melted at 130 oC and stirred for 12 h at standard atmospheric pressure for further reaction. Then the pressure was reduced to 5 kPa for 72 h. Finally, the product was dissolved into 5 mL of THF and precipitated with diethyl ether. After filtered, the final product was dried in the vacuum drying oven for 48 h and dark blue powder of PEGS-AP copolymers was obtained.

2.2.3 Preparation of PEGS-AP film

Table 1. The feed ratio of CSA in different PEGS-AP films Code of PEGS-AP films

PEGS-AP (g)

CSA (mg)

PEGS-AP0

0.6

0.0

PEGS-AP6

0.6

20.4

PEGS-AP12

0.6

55.8

PEGS-AP18

0.6

76.2

The films of PEGS-AP were prepared using HDI as the crosslinking agent. First, 0.6 g of PEGS-AP copolymers and an appropriate amount of CSA (Table 1) were dissolved into 5 mL of anhydrous THF under nitrogen atmosphere. The amount of CSA that added to each sample was shown in Table1. When the solid dissolved absolutely, 150 µL of HDI was added to the solution and further stirred for 2 h under nitrogen atmosphere to obtain a homogeneous solution. Then, 1.8 µL of Sn(Oct)2 was dropped into this system, and mixed homogeneously for about 2 min. Finally, the solution was 8


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poured into a Teflon culture dish, and the film was formed after laying on a horizontal platform at 55 oC for 24 h.

2.3 Characterization

The molecular weight and the molecular weight distribution of PEGS polymer were determined by gel permeation chromatography system (GPC, Agilent 1260) using Tetrahydrofuran (THF) as the eluting solvent with a flow rate of 1.0 mL/min. The 1H nuclear magnetic resonance (1HNMR) spectra of PEGS and PEGS-AP were recorded using a Bruker Avance 400 MHz NMR instrument with DMSO-d6 as solvent at room temperature. The FT-IR spectra of PEG, PEGS, PEGS-AP18 polymer and PEGS-AP18 film were obtained using a Nicolet 6700 FT-IR spectrometer (Thermo Scientific Instrument) with a resolution of 4 cm-1.

2.4 Electroactivity of the PEGS-AP polymer

The AP content and electroactivity of the PEGS-AP polymer were determined using the UV-vis spectrophotometer. AP and PEGS-AP polymer were dissolved in a mixed solvent of DMSO and deionized water with the ratio of 1:1. The UV–vis spectra of AP and PEGS-AP polymer at undoped state were evaluated using these solutions. And the spectra of the doped co-polymers were tested after adding appropriate amount of camphorsulfonic acid (CSA) into these solutions. The wavelength of scanning ranged from 1000 nm to 260 nm.

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An Electrochemical Workstation (CHI 660D) was used to conduct the cyclic voltammetry (CV) of AP and PEGS-AP polymers. This electrochemical workstation employed a three electrode system with a platinum disk as working electrode, a platinum wire as auxiliary electrode and a Ag/AgCl as reference electrode and the cyclic voltammetry was conducted with a scanning rate of 50 mV/s.

2.5 Conductivity of the PEGS-AP films

The conductivity of the PEGS-AP films was tested using the True RMS OLED Multimeter (U1273A, Agilent). Briefly, the PEGS-AP films were clamped well between two pieces of copper sheet with a certain area, and then the resistance (R) of each film was read from the multimeter. The conductivity of these films was obtained using the equation below.

σ=S/RL

where σ stands for conductivity of the PEGS-AP films; L means the thickness of the films; R is the resistance of films that could be read directly on the machine and S is the area of the films that between the two pieces of copper sheets.

2.6 Hydrophilicity and degradability of the PEGS-AP films

2.6.1 Hydration properties of PEGS-AP films

The hydration properties of the PEGS-AP films were conducted by testing the hydration kinetic and the water contact angle of the films. The hydration kinetic of the 10


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PEGS-AP films was tested in a physiological condition (37 oC) in Dulbecco’s phosphate buffer saline (DPBS). Briefly, all the samples in the same shape and size were put into the DPBS solution and taken out at regular time intervals. After blotted with the filter paper to remove the excess water on the surface, the samples were weighted. The hydration degree can be represented in the form

Hydration degree (%) = × 100% hydration degree (100%) = (wt-w0)/w0 × 100%,

where the wt showed the weight of the sample at fully hydrated state and the w0 was the original weight of the films.

The water contact angles of the films were measured using a static water contact angle measurements (CAMs). A picture was taken after one drop of Mili-Q water was dropped on the surface of the film for 30 seconds, and the contact angle of the film was obtained after the images were analyzed using a Kino software.

2.6.2 In vitro enzymatic degradation of the PEGS-AP films

The in vitro enzymatic degradation test was performed in the presence of proteinase K (Sigma) as we previously reported.9, 10 In brief, the films were cut into rectangle shape, and then the initial weights of the samples were weighed (W1). After that, each of the samples was immersed into 5 mL of Tris/HCl buffer, which contained 0.2 mg/mL proteinase K and 0.02 wt % of sodium azide. Then, the samples were placed at 37 °C on a shaker with a shaking speed of 100 rpm. The buffer and proteinase K were replaced every 24 h to maintain the activity. After reaching the pre-set time 11


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point, the samples were taken out, washed with deionized water and then dried at 60 °C for 24 h in oven. The remaining weights of the samples were weighed (W2). The weight

loss

was

calculated

by

the

following

formulation:

Weight

loss

(%)=(W1-W2)/W1*100%. 2.7 Mechanical properties of the PEGS-AP films

The mechanical properties of the PEGS-AP films were judged by the Young’s modulus, stress-strain behavior and the elongation at break, which were tested using the mechanical testing machine and the TestWorks4 software (Criterion Model 43, MTS, MN) at room temperature with a humidity of 50%. The films were cut into 6-mm-wide pieces and clamped in the fixtures of the machine whose gap is 2 mm. The thickness of each film was around 0.13 mm measured before the stress-strain test by a thickness meter. With the testing rate of 10 mm/min, the films were stretched. Applied force (N) and cross section area (mm2) were used to determine the stress– strain curves and the tensile stress (MPa). By calculating film length in tensile test, the elongation at break was obtained. The Young’s modulus of film was determined from the slope of stress– strain curve at 5% of strain. The mean value and standard variation were obtained after testing no less than 3 species per sample.

2.8 Cell culture and cell seeding

Considering the long-term goal of regenerating the skeletal muscle, C2C12 mouse myoblast cells were used. C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sangon 12


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Biotech), 1.0×105 UL-1 penicillin (Gibco) and 100 mg L-1streptomycin (Gibco) at 37oC in a humidified atmosphere with 5% CO2. When confluence reached 90%, C2C12 cells were passaged as 1:2 by digestion with 0.25% trypsin (GIBCO). All the films, with a thickness of about 100 µm, were sterilized with 75% ethanol for 2 h, rinsed in DPBS for 3 times in order to remove the residual ethanol and dried at room temperature. Then, without any coating or other treatment, the films were seeded with C2C12 cells (at a passage of 5) in a certain density for different tests.

2.9 Viability and proliferation of C2C12 cells on the films

C2C12 mouse myoblast cells were cultured on the films and their viability was measured using the Live/Dead assay after cell attaching. Briefly, C2C12 cells with a density of 6000 cell/cm2 were seeded onto the sterilized films directly. The Live/Dead assay was carried out after 24 h when the attachment was completed. Briefly, the plate with cells was washed for 3 times with PBS for 5 min each before adding the Live/Dead kit (Molecular Probes). Then the cells were treated with Ethidium homodimer-1 (0.5 µM) and calcein AM (0.25 µM) (Life Technologies) for 45 min and were observed using an inverted fluorescent microscope (IX53, Olympus).

The proliferation of the cells at 1, 2 and 3 days were conducted using the Alamar Blue assay. On day 1, day 2 and day 3, the culture medium was removed from each well and 250 µL of Alamar blue solution (10% of Alamarblue in culture medium) was added. After 4 h, 100 µL of the solution from each well was transferred into a 96 well

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black plate (Constar) to do the fluorescent intensity test using a microreader (SpectraMax i3, Molecular Devices, USA).

2.10 Differentiation of the C2C12 cells on the films With a density of 8000 cells/cm2, the C2C12 cells were seeded on the sterilized films and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sangon Biotech), 1.0×105 UL-1 penicillin (Gibco) and 100 mg L-1streptomycin (Gibco) at 37 oC in a humidified atmosphere with 5% CO2. After the cell confluence reached 80%, the culture medium was switched into the differentiation medium (DMEM with 2% horse serum). The differentiation medium was renewed every other day until the myotubes were observed.

After 7 days of differentiation, samples were fixed with 2.5% glutaraldehyde for 15 min at room temperature and then washed with PBS for three times. Each sample was then added with 0.1% Triton X-100 and permeabilized at room temperature for 45 min. After rinsing in PBS for 3 times, the samples were blocked with 1% BSA for 1.5 h at room temperature. The rabbit-anti-mouse skeletal myosin heavy chain (MHC) antibody was added to each sample as the primary antibody at 4 oC overnight. Rinsed with PBS for 3 times, each sample was added with Alexa flour 488 conjugated secondary antibody (Molecular Probes) and incubated for 2 h at room temperature. DAPI was used as the nuclear markers. After the staining, the samples were observed under an inverted fluorescent microscope (IX53, Olympus) with the microscopy objective of 20X. The numbers of myotubes were counted, and the diameter and the 14


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length of the myotubes were calculated using the Image J software. The myotube maturation index was computed as the percentage of myotubes with more than 5 nuclei in all the myotubes.

The qRT-PCR was used to measure the gene expression of the differentiated cells. The RNA of the cells on different films was extracted using Trizol (Invitrogen) and the reverse transcription was proceeded using a reverse transcription reagent kit (Roche). Then the real-time PCR was conducted following a certain protocol and the process was performed using an Applied Biosystems 7500 Fast Real-time PCR system. The temperature profiles are as follows: 50 oC for 2 min, 90 oC for 2 min, then 90 oC for 3 s followed by 30 s at 60 oC. Two different genes, Myogenin (MyoG) and Troponin T (TNNT) were detected along with the housekeeping gene (glutaraldehyde phosphate dehydrogenase (GAPDH) to assess the myogenic differentiation. The relative gene expression fold changes were calculated bythe ∆∆Ct method using the PEGS-AP0 group as a control. The sequences of forward and reverse primers of these three genes are in Table S1 in supporting information.

2.11 Statistics

All the measurements were tested on 3 samples for each condition, and for the cell proliferation and gene expression parts, not only 3 independent replicates were used, but also 3 times of each replicate was tested to reduce the random error. The data were expressed as mean ± standard deviation. Student t-test was used to analyze the statistical significance, and the difference was considered statistically when p < 0.05. 15


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3 Results and discussion

3.1 Synthesis and characterization of PEGS-AP copolymers

PEGS films were developed as a highly elastomeric material with a support for protein absorption and cell proliferation.33 PEG was a polymer that has a high hydrophilicity and showed the anti-attachment ability,

36

and we introduced PEG in

PGS polymer by the esterification reaction (Figure 1 (a)). However, the introduction of a large amount of the PEG into the PGS polymers may lead to problems with cell attachment. Therefore, we adjusted the PEG content in the PEGS polymer to obtain a film with a suitable surface hydrophilicity. The water contact angles of PEGS films with 20%, 40% and 60% PEG are 65o, 34o and 12o, respectively. It has been demonstrated that when the water contact angle is about 40o to 80o, the cells showed a maximum attachment on the surfaces.

37

Meanwhile, the cell attachment of C2C12

cells on the PEGS films indicated that on the 20% PEG group, cells showed the best attachment behavior (data as shown in Figure S1 in supporting information). Thus, we chose the group of PEGS films with 20% PEG to carry out the further research.

AP was introduced into the PEGS polymers to obtain an electroactive and conductive polymer which may promote the cell proliferation and differentiation. The PEGS-AP polymer was synthesized by esterification of PEGS and AP in a vacuum condition 16


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with nitrogen protection, as shown in Figure1. With different feed ratio of PEGS and AP, PEGS-AP polymers with different AP contents were obtained. Table 2 showed the feed ratio of AP in PEGS-AP copolymers and the actual content of AP in samples. With enhanced the feed ratio of AP, the AP content in PEGS-AP copolymers increased. The molecular structures of PEGS and PEGS-AP copolymers were confirmed by the 1HNMR spectroscopy (Figure 2a and 2b). The peaks at 1.23, 1.50 and 2.27 ppm belonged to the methylene (a, b, c) of sebacic acid in the PEGS polymer; the signal of methylene protons (d) of PEG segment appeared at 3.50 ppm; the peaks from 3.85-4.27 ppm and 4.70-5.26 ppm were assigned to protons in the glycerol (e, f). All these peaks appeared in 1H NMR spectra of PEGS can be observed in that of PEGS-AP copolymer, and the peaks at 2.73 ppm and from 6.90-7.25 ppm were present due to the protons of methylene and benzoic ring in AP (g, AP), respectively, suggesting that the AP fragment was successfully grafted onto the PEGS polymer chain.

The PEGS-AP films were formed by reaction of the isocyanate group in HDI and the residual hydroxyl in the backbone of PEGS-AP copolymer. The formation of PEGS-AP films was confirmed by FT-IR analysis (Figure 2c). Compared with FT-IR spectrum of PEG2000, the PEGS polymer’ spectrum showed an obvious peak at 1735 cm-1 corresponding to the C=O stretching from the ester groups. Meanwhile, the broad band at 3370 cm-1 due to the free hydroxyl group (-OH stretch) can also be seen in the PEGS polymer. This result agreed well with the FT-IR spectra of the PEGS

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polymer reported by Wu et al.32 Two new peaks at 1510 cm-1 and 1610 cm-1 which are characteristic of the benzene ring appeared in the PEGS-AP polymer, respectively, demonstrating that the AP fragment was grafted onto PEGS. In the PEGS-AP film, the peak at 3370 cm-1 owing to the rest hydroxyl group in the PEGS-AP polymer exhibited a sharp decrease. This was caused by the reaction of the isocyanate group (-NCO) in HDI and the hydroxyl group (-OH) in PEGS-AP, indicating the successful formation of PEGS-AP film. Furthermore, the peaks at 1150 cm-1 (-COO) increased after the crosslinking process, due to the reaction of the isocyanate group in HDI and the rest hydroxyl group in PEGS. An image of the different types of films was shown in Figure S2 in supporting information.

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Figure 1. Synthesis scheme of the PEGS-AP polymer and films. (a) Synthesis of PEGS copolymer. (b) Synthesis of PEGS-AP copolymer. (c) Preparation of PEGS-AP crosslinked copolymer films.

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Figure 2. 1H NMR spectra of (a) PEGS copolymer and (b) PEGS-AP copolymer. (c) FT-IR spectra of PEG2000 polymer, PEGS copolymer, PEGS-AP18 copolymer and PEGS-AP18 film. 20


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Biomacromolecules

Table 2. Feed ratio of AP in PEGS-AP copolymer and actual content of AP in samples by UV-vis test

Samples

PEGS (g)

AP (g)

Actual wt% of AP in samples

Conductivity -4

(×10 S/cm)

PEGS-AP0 copolymer

1

0.000

0

--

PEGS-AP6 copolymer

1

0.063

3.4

0.57

PEGS-AP12 copolymer

1

0.126

9.3

1.74

PEGS-AP18 copolymer

1

0.189

12.7

1.84

3.2 Electroactivity of the PEGS-AP polymers

Aniline oligomers have been demonstrated to improve the electroactivity and conductivity of the materials.38, 39 In our work, aniline pentamer (AP) was introduced into the PEGS polymer in order to obtain an electroactive elastomer for skeletal muscle tissue engineering. UV-vis and CV were used to characterize the electrochemical properties of PEGS-AP copolymer. The UV-vis spectrum of undoped and doped PEGS-AP copolymer and AP oligomer in Figure 3a showed that PEGS-AP copolymer exhibited a similar oxidation process as the AP oligomer (Figure 3a). Briefly, the PEGS-AP copolymer exhibited two absorption peaks at ~330 nm and ~610 nm, owing to the π-π* transition in the benzene unit and the excitonic transition from the benzoid ring to the quinoid ring. After doping with camphorsulfonic acid (CSA) a peak at ~430 nm appeared owing to the formation of delocalized polarons, and a new peak at ~830 nm indicated the generation of emeraldine salt and the ability of conducting electrons in PEGS-AP polymers. This agreed well with the results of 21


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the AP oligomer’s UV spectra of Wu et al.,31 showing a good electroactivity of the PEGS-AP copolymer. Besides, from the UV-vis spectra, the AP concentrations in the polymers were calculated. As shown in Table 2, PEGS-AP6, PEGS-AP12 and PEGS-AP18 showed an AP content of 3.4%, 9.3% and 12.7%, respectively.

CV was further carried out to confirm the electrochemical property of the PEGS-AP copolymers (Figure 3b). As shown in Figure 3b, PEGS-AP copolymer showed three pairs of reversible redox peaks at 0.26, 0.45, and 0.56 V. The first oxidation peak appeared at ∼0.26 V, due to the redox process of aniline pentamer from the “leucoemeraldine” to the ‘‘emeraldine I” form, and the second oxidation peak appeared at ∼0.45 V, assigned to the transition from the ‘‘emeraldine I” to the ‘‘emeraldine II” state.35 The oxidation peak at∼0.56V was attributed to the transition from the “emeraldine II” state to the “pernigraniline” oxidation state.31

Figure 3. (a) UV-vis spectra of AP, AP doped with CSA, PEGS-AP and PEGS-AP doped with CSA; (b) CV of PEGS-AP copolymer.

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3.3 Conductivity of the PEGS-AP films

Studies have demonstrated that using the electroactive materials as the matrix, myoblast cells showed a better proliferation and differentiation behavior.9,

10

Meanwhile, by applying electrical stimuli, a more highly differentiated and more functional skeletal muscle tissue was formed. The conductive matrix that could transmit the electrical signal is a good option to use in skeletal muscle tissue formation. Our PEGS-AP films showed a good conductivity compared with the PEGS groups as shown in Table 2. The film without AP showed the lowest conductivity (whose resistance is too high that could not be detected). And with the increase of the AP content, the conductivity has an increase in order of magnitude (~10-4 S/cm). The conductivity of the PEGS-AP films enhanced when the AP content increased (from 0.57×10-4 S/cm to 1.84×10-4 S/cm). This increase agreed well with our expectation that the AP fragment in the films would give the film a good conductivity when doped. These conductive films will be used for C2C12 myoblast culturing. Therefore, the conductivities of the PEGS-AP films after immersing in the cell culture medium (DMEM+10% FBS) for 5 days was further tested. After immersing in the fluid for 5 days, the conductivity of the materials showed a significant increase (about ten times higher than the dried ones as shown in Table S2 in supporting information), indicating that within the fluid environment, these films could also serve as a great conductive matrix for cells to survive, proliferate and differentiate. 23


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3.4 Hydrophilicity and degradability of the PEGS-AP films

The hydration degree of the PEGS-AP films with different AP content and time was shown in Figure 4a and Figure S3. The hydration process of all the samples was completed in no more than 30 min. It ranged from 33.7% to 14.0%, and decreased with the increase of AP content. PEG showed a good hydrophilicity which could enhance the water uptake capability of the materials.40 Meanwhile, the AP segment is a hydrophobic part in the material that would reduce the hydration degree. Therefore, when we used the films with same PEG concentration, the AP content mostly affected the hydrophilicity. Thus, the hydration degree of the PEGS-AP films dropped (from 33.7% to 14%) when the AP segment increased (from 0 to 12.7%).

Figure 4. (a) Hydration degree and (b) water contact angle of PEGS-AP films with different PEGS and AP content.

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The water contact angle of the surface was an important parameter to affect the cell behavior.41 Contact angle of 40o to 80o on a surface has been demonstrated to enhance the cell attachment efficiently.37 Fortunately, the contact angles of the films varied from 64o (PEGS-AP0) to 76o (PEGS-AP18), offering the cells a good condition for adhesion (Figure 4b). With the AP content increasing, the contact angles of the PEGS-AP films increased owing to the hydrophobicity of AP. The combination of those three components to obtain a suitable hydrophilicity of the materials’ surface will enhance the cell attachment.

Proteases show the activity to cleave ester bonds and urethane bonds in addition to amide bonds 9, 42, 43. Protease is almost present everywhere in the human body, such as blood serum and leukocytes. Thus, we used protease K solution to mimic the in vivo environment to evaluate the degradation capacity of the PEGS-AP films. As it is shown in Figure S4, all the four films showed nearly linear degradation profiles. Moreover, PEGS-AP0 film showed the highest weight loss ratio of 72% within 84 h. With the introduction and increase of AP content in the PEGS-AP films, the three AP-contained films’ degradation rates gradually decreased, which presented weight loss varying from 40%, 37% to 27% after 84 h incubation with the proteinase K. Actually, the introduction of AP would decrease the films’ hydration ability and increase the films’ crosslinking density, which could hinder the enzyme’s diffusing into the film network leading to reduced degradation speed. All the above results demonstrated that these PEGS-AP films could be degraded by protease and presented

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adjustable degradation rate by changing the AP content, providing a favorable property for their in vivo application.

3.5 Mechanical properties of PEGS-AP films Elastomers showed a wide application in soft tissue engineering.44 The PEGS-AP films have good mechanical properties, making it a promising candidate for soft tissue engineering, especially for the skeletal muscle tissue engineering. The Young’s modulus of these films ranged from 14.58±1.35 MPa to (24.62±0.61) MPa, and showed an increase when the content of AP was enhanced. Compared with the thermally cross-linked PEGS films reported by Patel et al.33 (1.59±0.055 MPa), the PEGS-AP films that cross-linked with HDI performed a higher Young’s modulus. Besides, with the addition of AP, the Young’s modulus of the films enhanced significantly, owing to the strong π-π interaction between the AP segments. The elongation of the film with no AP content was 49.5% and when AP was introduced in the film, the elongation reached up to 113.3% (p

Biocompatible Elastic Conductive Films Significantly Enhanced

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