1 Introduction

With the discovery of “bioelectricity” in late 1970’s there have been attempts in the field of tissue regeneration to incorporate the effect of various electric parameters in order to achieve enhanced regeneration and growth of the tissue [1]. Incorporation of such parameters for the in vitro studies and their results extrapolated and used for the real in vivo applications could facilitate improved results in the regeneration process. The role of bioelectricity has been well studied in various physiological processes like muscle contraction, nerve conduction, cell alignment and division, etc. [2, 3]. So there have been methods wherein direct electric current/electric field is applied to various cells like fibroblast [4], neural cells [5], cardiac cells [6], etc. and further their effect is monitored. This has shown to enhance the rate of proliferation in various cell types but the effect is not localized and might spread to the nearby cells which cannot be desirable. So in order to overcome this disadvantage, one approach can be to use scaffolds made up of conducting materials [7]. Piezoelectric materials (PZ) [8]—capable of transient surface charges, metal electrodes [9], etc. are some of the materials used in the biological field for generating electric fields but they show some limitations, like the external control over the stimulation are limited in case of PZ materials [8], whereas, in the case of metals, apart from the leaching effect, metals lack biological interface between the electrode and the cells which play an important role [9]. So there has been search for various alternatives for conducting materials and among them conducting polymers have witnessed a great impetus in the biomedical area because of their biocompatibility and ease of synthesis.

Conducting polymers are organic polymers capable of conducting electricity because of their alternate single and double bond [10]. Doping these polymers with the different molecules like sodium dodecyl sulphate [11], tosylate [12], biological moieties like neurotrophins [13], have improved their conductivity and compatibility with the cells. Although addition of some biological moieties [14] have caused alteration in the pyrrole chemistry and thus would play a role in the change in conduction of electricity and cell adhesion, etc. Conducting polymers are synthesized either by chemical oxidation or electrochemical process. Chemical process as compared to electrochemical process is easy, cost effective and results in high yield of conducting material [15]. Despite their potential usage in tissue engineering either as an electrode or sheet (prepared by electropolymerization) for the growth of cells, their degradation still remains a matter of concern. It has been reported that if these conducting polymers are incorporated with natural, biodegradable polymers (in accordance with the percolation threshold value) the resulting composite is not only conducting but also can be made degradable [16, 17]. Different formats of the conducting polymers together with biodegradable polymers have been synthesized by methods like, electrospinning [18], solvent extraction [19], etc. Recently it has been shown that there is great potential of the cryogelation technology for the synthesis of macroporous gels referred to as “cryogels” in the field of tissue engineering [20]. Cryogels are highly interconnected porous gels, formed at sub-zero temperature, which facilitates proper exchange of nutrients and waste, thus providing the adhered cells a conducive microenvironment for their growth [21]. Cryogels made up of both natural or synthetic polymers have been used as a three-dimensional (3-D) support for the cultivation of different types of cells like, chondrocytes [22], cardiomyocytes [23], fibroblasts [24], etc. The mechanical properties of the cryogels can be tailored as per the requirement by changing the concentration of the polymers or the crosslinkers used [25]. Of the known natural polymers, chitosan and gelatin has gained a lot of attention in the field of biomedical applications because of their biocompatible and biodegradable nature [21]. Chitosan, a cationic polysaccharide is a copolymer of glucosamine and N-acetyl glucosamine linked by β-1, 4 glycosidic bonds [26]. Apart from tissue engineering applications chitosan has been extensively used for drug delivery, gene delivery [27], enzyme immobilization, etc. [28]. Gelatin, the denatured form of collagen has RGD motifs which interact with the integrins of the cells and results in adherence of the cells on the matrix. For the synthesis of such porous conductive scaffold, polypyrrole as the conducting polymer is used as it has shown to be biocompatible with the mammalian cells [29].

In this study we describe the synthesis of conducting cryogel scaffold which would not only have the beneficial properties of cryogel but will also be conductive in nature. The study further presents the interaction of the excitable cells like neural and cardiac cells with the synthesized conducting cryogel scaffolds. Moreover the scaffolds are thought to help the stimulation of the cells in a localized manner and help in their faster proliferation and regeneration. The cell seeded scaffolds will be subjected to electrical conductance to stimulate the cells and enhance their cell proliferation property.

2 Materials and methods

2.1 Materials

Chitosan (low viscosity), gelatin (from cold water fish skin, MW: ~60,000), propidium iodide (PI), dulbecco’s modified medium (DMEM) and penicillin–streptomycin antibiotic were bought from Sigma Chemical Co. (St. Louis, MO, USA). Foetal bovine serum (FBS) was procured from HyClone (Utah, USA). Sodium dodecyl sulfate (SDS) and ammonium persulfate (APS) were procured from Merck Chemical Co. (Mumbai, India). Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde were purchased from s.d. fine-chemicals limited (Mumbai, India). Iron chloride (FeCl3) was bought from Hi-Media (Mumbai, India). Pyrrole was procured from Spectrochem (Andhra Pradesh, India).

2.2 Synthesis of polypyrrole

Polypyrrole (PPy) was synthesized using different combinations of detergent and oxidizing agents. Pyrrole (0.13 M) was added to 0.1 M CTAB or 0.1 M SDS solution placed on a magnetic stirrer at 4 °C. This solution was stirred for 10 min followed by the addition of APS (0.03 M). This solution mixture was stirred for 3 h at 4 °C. Similarly 0.4 M pyrrole was added to 0.1 M CTAB or 0.1 M SDS solution with FeCl3 as the oxidizing agent where the ratio of FeCl3/pyrrole was 2.33 [30]. The prepared polypyrrole solution is then dialyzed for 48 h against deionized water. The synthesized PPy solution (5 ml) after dialysis was air dried and the yield was determined.

2.3 Synthesis of conducting scaffold

A homogenous solution of 0.3 % chitosan and 1.6 % gelatin in 2 % acetic acid was prepared. After that the dialyzed polypyrrole solution in the ratio 1:1 was mixed with this homogenous chitosan–gelatin solution. The conductivity of the control (chitosan–gelatin) as well as the conducting solutions (chitosan–gelatin–polypyrrole) was analyzed by liquid conductometer. Glutaraldehyde (0.2 %) was added as the crosslinker. The solution was vortexed and poured in the moulds (2–5 ml plastic syringes) and kept at −12 °C for 12–16 h in the cryostat. After this the gels were thawed at room temperature. The synthesized cryogels were dried by lyophilization and kept for further use.

2.4 Measurement of swelling ratio and swelling kinetics

The characterization of the prepared monolith cryogels was done by cutting them in sections of 13 mm in diameter and 5 mm in thickness. The samples were used in triplicates for all the characterization procedures. The prepared cryogel scaffolds were dried and weighed. Further, these samples were immersed in phosphate buffer saline (PBS) (pH −7.4) for regular interval of time and weighed after removing the excess water with filter paper till an equilibrium weight was reached.

$$ {\text{SR }} = \, \left( {{\text{W}}_{\text{S}} -{\text{ W}}_{\text{d}} } \right) \, /{\text{ W}}_{\text{d}} $$

where Ws and Wd is the weight of the swollen gel and dried gel, respectively.

Swelling kinetics was measured using gravimetric method. This is a measure of the uptake capacity of the solvent (PBS) by the cryogels. Water uptake percentage was calculated as follows:

$$ {\text{Wu }}\% \, = \, 100 \, \times \, \left[ {\left( {{\text{W}}_{\text{t}} \, -{\text{ W}}_{\text{d}} } \right)} \right] \, /{\text{ W}}_{\text{eq}} $$

Where Wt is the weight of the cryogel at specific time interval, Wd is the dry weight of the cryogel, Weq is the weight of the wet gel at equilibrium.

2.5 Flow rate analysis

The resistance to the flow of the solvent in a cryogel was measured by passing the solvent through the cryogel at varying flow rates using a peristaltic pump (2 bars). The swollen gel was placed in the syringe mould such that there is no leakage from the sides of the syringe. Both the openings of the syringe were connected to the pump through rubber tubings. The free flow of the solvent was noted at varying flow rates ranging from 1 to 8 ml min−1 till back pressure was observed. The pump settings were calibrated before running the experiment without column being connected [21].

The crosslinking of chitosan with gelatin and the incorporation of polypyrrole in the hybrid material were studied by Fourier transform infrared spectroscopy (FTIR). It was done using Perkin Elemer Paragon 1000 spectrophotometer (USA). The microstructural analysis of the scaffolds was done using scanning electron microscopy (SEM). The samples were dried before coating for better resolution and imaging. Gold coating was done using a sputter coater (Vacuum Tech, Bangalore, India). FEI Quanta 200 at 20 kV and spot size of 3.5 mm in high vacuum was used for SEM analysis. Thermogravimetric analysis (TGA) analysis of the samples was done to study the change in weight of the gels with temperature after incorporation of the conducting material. The analysis was performed in an inert atmosphere of nitrogen by increasing the temperature from 10 to 400 °C at the rate of 10 °C min−1.

2.6 Rheology measurements

The samples were analysed for their mechanical properties by studying change in flow and deformation under controlled conditions. Sections (5 mm in height and 8 mm in diameter) were cut and placed on the holder plate of 12.5 mm in diameter with a constant force of 1 N s−1 for 15 min at a frequency of 1 Hz. All the conditions were kept same for all the samples and the flow and deformations were measured at 37 °C. The analysis was done for all the samples in the dry and wet state where the dry state was achieved by lyophilizing the sample and the wet state was obtained by saturating the cryogels in PBS till equilibrium was reached.

2.7 Degradation analysis

The synthesized cryogel scaffolds were studied for their degradation capacity for a period of 1 month. The dried samples (13 × 5 mm) were weighed and then sterilized by passing through a gradient of ethanol (20, 40, 60, 70 and 100 %). The scaffolds were then transferred in sterile conditions to 15 ml tubes containing 0.1 M sterile PBS. The degradation studies were performed at 37 °C under sterile conditions. The samples were then collected at regular intervals (1, 2, 3 and 4 weeks) and were washed with de-ionized water. The samples were dried and then weighed again to examine the change in the weight of the sample. The degree of degradation was thus determined by

$$ {\text{Degree of degradation}}\,\% \,= {\text{ Initial weight }}-{\text{ final weight }}/{\text{ initial weight }} \times \, 100 $$

2.8 In vitro biocompatibility study

In order to check the cell material interaction MTT assay was done using NIH3T3 as the model cell line. The cryogel samples were sterilized by giving them gradient ethanol (20, 40, 70 and 100 %) wash. After that the scaffolds were washed with PBS three times for 15 min in sterile non-treated 24 well plates. Further the gel samples were equilibrated with DMEM media + 10 % FBS. The media was removed and the samples were dried for an hour before seeding the samples with NIH3T3 with the cell number of 1 × 105. The experiment was carried out for 7 days.

2.9 In vitro electrical stimulation

In order to provide electrical stimulation to the synthesized scaffolds seeded with cells, we developed an indigenous electrical stimulation setup. Stainless steel electrodes, semi-circular in structure were cut such that they closely fitted the 24 well plate wall on both the sides and had a gap of 13 mm left between the two electrodes. Each well had one electrode connected to the anode while the other to the cathode so that the circuit is complete. The scaffolds were placed such that no gap was left between the electrode and the scaffold.

The scaffolds seeded with cells were placed in between the electrodes and different voltages were applied for different time duration depending on the cell line used. The two cell lines used were neuro2a (neural cell line) and C2C12 (myoblast cell line). The voltage was varied from 30 to 100 mV for 2 h for neuro2a. In the case of C2C12 it was varied from 100 mV to 2 V for 1 min. The control was also maintained in the same conditions except that it was not provided with the stimulation. The experiment was performed in triplicates. After seeding the neuro2a and C2C12 cells, they were allowed to adhere onto the scaffolds for a period of 24 and 72 h, respectively [30, 31]. Then the electrical stimulation was provided as per the specifications mentioned before. In order to measure the effect of addition of conducting polymer into the scaffolds, electrical stimulation was provided to the cryogel scaffold as well as the PPy incorporated cryogels. All the stimulation experiments were carried out on SF (SDS/FeCl3) scaffolds. The sterilized scaffolds were placed in between the electrodes with their wires connected to the power supply.

2.10 Statistical analysis

The experiments were performed in triplicates and the data are presented as the average of all the samples analyzed. The average values of data were tested using Student’s t test.

3 Results and discussion

3.1 Synthesis of polypyrrole

Synthesis of the conducting polymer from its monomer was done by microemulsion polymerization using low temperature. It has been shown that microemulsion polymerization results in synthesis of the polymer with higher yield and conductivity [32]. The surfactant acts as the microreactor vessel leading to the formation of particles in the range of nanometers. Low temperature inhibits the surfactant mobility thus leading to more organized polypyrrole structures which significantly affects the conductivity of the polymer [33].

Pyrrole oxidation by APS or FeCl3 results in the formation of radical cations which further form radical oligomers and propagates the reaction in the surfactant formed micelles [34]. Polypyrrole was synthesized using different emulsifiers and oxidizing agents as the conductivity and the structure of the synthesized polymer is affected by varying these parameters. The morphology of the synthesized polymer depends on the template used, like CTAB/APS resulted in nano-fibers, CTAB/FeCl3 in sphere like morphology, no particular structure was obtained by using SDS/APS whereas SDS/FeCl3 resulted in PPy films. The result of these nano-structures were due to the interaction between the cationic and anionic surfactant with the anionic oxidants (SO4 − of APS and Cl− of FeCl3), wherein CTAB resulted in more organized structures because of interaction with their counter-ions of the oxidant which got disrupted in the case of the anionic SDS. The critical micelle concentration of CTAB and SDS is 0.87 and 8.2 mM, respectively in deionized water at room temperature [35].

The purpose to synthesize these various combinations was to see the effect of the charge produced by these cationic and anionic surfactant and different oxidizing agents on cells. The other important parameter was to check the effect of these nano-structured PPy particles on the chitosan–gelatin scaffolds. It has been studied that the addition of the fillers in the polymer affects the mechanical strength and thermal stability [36]. Thus we synthesized polypyrrole by various methods in order to achieve better results after incorporating them in the neat polymer.

3.2 Synthesis of conducting scaffold

Although polypyrrole is reported to be biocompatible with the mammalian cells, the degradation of the polymer in its specific use is still a limitation. Therefore efforts are being made in order to make it degradable in nature. It has been reported that conjugating PPy with ester linkages enables the degradation of this polymer [37]. It has also been reported that incorporation of 3–6 % PPy in chitosan results in degradable composite [17]. Polypyrrole particles in the range of 50–200 nm, if released after the degradation, are small enough to be removed from the circulatory system of the body [38]. In this study we tried to synthesize the conducting scaffold by incorporating dialysed polypyrrole in chitosan–gelatin blend. The digital image of the gels produced in this way is shown in Fig. 1.

Fig. 1
figure 1

Photographic image of synthesized conducting and nonconducting cryogel scaffolds. Digital image showing (a) chitosan–gelatin cryogel scaffold (control) in the dry and (b) wet state, (c) polypyrrole (PPy) incorporated cryogel scaffold in the dry and (d) wet state. Both are represented in their monolith and disc format, respectively

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Dialysis of the synthesized PPy solution enables the removal of detergent and unreacted small monomers, otherwise the detergent forms a coating around the polymer solution as has been reported earlier [39]. Moreover microemulsion polymerization results in PPy particles in the required nanometer range. Chitosan being brittle in nature does not provide the adequate mechanical properties required for its applications in soft tissue regeneration. Therefore addition of gelatin not only provides the RGD motifs required for the interaction and adherence of cells but also provides better mechanical stability as already reported [40]. The scaffolds synthesized by the cryogelation technology results in macroporous scaffolds with interconnected pores required for convective mass transport. The cryoconcentration of the polymers during cryogelation enables tight association between the chitosan–gelain (CG) and PPy.

The various possible interactions that occur among the (–COOH and NH2) groups of gelatin and –NH2 group of chitosan polymeric chains in the presence of glutaraldehyde (–CHO group) are:

$$ {\text{Gelatin }}-{\text{NH}}_{2} + {\text{ CHO }} - \, \left( {\text{glutaraldehyde}} \right) \, - {\text{ CHO }} + {\text{ NH}}_{2} \;\left( {\text{chitosan}} \right) $$
(i)
$$ {\text{Chiotsan }}-{\text{NH}}_{2} + {\text{ CHO }} - \, \left( {\text{glutaraldehyde}} \right) \, -{\text{ CHO }} + {\text{ NH}}_{2} \;\left( {\text{ chitosan}} \right) $$
(ii)
$$ {\text{Gelatin }}-{\text{ COOH }} + {\text{ NH}}_{2} -{\text{ chitosan}} $$
(iii)
$$ {\text{Gelatin }}-{\text{COOH }} + {\text{ NH}}_{2} -{\text{ gelatin}} $$
(iv)

Reaction (i) and (ii) are being more favourable because of the added crosslinking reagent. PPy, as discussed, would be overall neutral in charge because of the incorporation of dopants like SO4 2−, Cl−1 ions and would get incorporated in the chitosan–gelatin polyelectrolyte complex forming an interpenetrating network.

The amount of PPy synthesized by various procedures was calculated and it varied for different combinations of oxidizing agents and surfactant used. As a result different concentration of PPy (in 5 ml) was incorporated in the chitosan–gelatin solution used for the synthesis of the conducting cryogel scaffold. The concentrations obtained are 2, 0.6, 4 and 1 %, when different combinations of surfactant and oxidizing agents like SDS/FeCl3, SDS/APS, CTAB/FeCl3, CTAB/APS were used, respectively.

The conductivity in the control and conducting solutions in different combinations used for making cryogels were measured and is reported in Table 1.

Table 1 The conductivity of the polymer solution mixtures in different combinations
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3.3 Microstructural analysis by SEM

The microstructural analysis of the synthesized CTAB/APS cryogels showed the presence of heterogenous pores in the range from 20 to 80 μm with smooth wall surface. The SEM micrograph also shows the presence of pores in the wall of the gels which increased the overall porosity and thus the swelling ratio and swelling kinetics of these gels compared to the other synthesized gels (as discussed before). SA gels also showed comparatively uniform pore size distribution similar to CTAB/APS scaffolds wherein the polypyrrole could be observed as nodules on the surface of the pore walls. The SF scaffold clearly showed rough walls with PPy clearly impregnated on the surface resulting in PPy network throughout the gel. The pores in the SF gels were more uniform and in the range of 60–80 μm. Cryogel scaffolds without PPy showed homogenous pore size distribution with smooth walls as shown in Fig. 2.

Fig. 2
figure 2

Scanning electron micrographic (SEM) analysis. SEM micrography was performed to study the pore size and the distribution of polypyrrole (PPy) in the gels synthesized using (a) CTAB/APS (CA): (b) SDS/APS (SA) and (c) SDS/FeCl3 (SF) as the surfactant and oxidizing agent, respectively. All the samples were analyzed at × 500 (20 μm). The presence of PPy as a thin film could be seen throughout the gel

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3.4 Measurement of swelling ratio and swelling kinetics

The swelling ratio of the polypyrrole incorporated cryogel was higher compared to the non-conducting cryogel scaffold as shown in Fig. 3a. The presence of polypyrrole in the CG solution as fillers led to an increase in the porosity of the gel thus allowing more absorption of water and a higher swelling ratio as shown in Fig. 1. Moreover the hygroscopic nature of PPy might also play an important role [41].

Fig. 3
figure 3

Swelling ratio and swelling kinetics of the cryogel scaffold. (a) Swelling ratio of (CG) chitosan–gelatin gels (square with vertical fill) and gels incorporated with PPy synthesized using CTAB/APS (CA) (square with horizontal fill) SDS/APS (SA) (square with diagonal rectangle) and SDS/FeCl3 (SF) (square with dots) as the surfactant and oxidizing agent, respectively. (b) The water uptake percentage was measured at regular interval of time for all the synthesized gels till equilibrium was reached. CG (black diamond) CA (black square) SA (black up-pointing triangle) and SF (black circle) gels. All the experiments were performed in triplicates. Other details are described in “Materials and methods” section

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The swelling kinetics of all the gels was almost similar and they all swelled to 90 % of their original weight except SA (SDS/APS) (Fig. 3b). This might be because the different morphology of the synthesized nano-structures influenced the properties of the synthesized gel. It has been reported that these nano-fillers not only affect the mechanical properties but also the water absorbing capacity of the polymer [42].

3.5 Flow rate analysis

The flow rate of all the cryogel samples incorporated with polypyrrole as well as cryogels without polypyrrole was about 8 ml min−1 at a pressure of 2 bars. This indicates the presence of proper interconnectivity within the synthesized cryogels which can be useful property of the material for the proper exchange of nutrients and waste especially when the cells are seeded for a longer duration.

3.6 FTIR analysis

In order to check the crosslinking between the chitosan and gelatin as well as the incorporation of the polypyrrole particles, FTIR analysis was performed (Fig. 4). The peak at 1,550 cm−1 in the Fig. 4 corresponds to the presence of amide bond between the polymer and cross-linker which was found to be present in all the synthesized gels, whereas 3,500 cm−1 represents the hydrogen bonded –OH groups. The band for the C–C stretching vibration of the pyrrole ring is present at 1,552 cm−1 in the presence of SDS (PPy-SDS) and FeCl3 (PPy-Cl). The characteristic polypyrrole peaks at 1,560 and 1,480 cm−1 were due to ring-stretching modes of the symmetric and antisymmetric, respectively. The doping state of polypyrrole was defined by the peaks around 1,195 and 920 cm−1. N–H and C–H stretching vibrations were attributed to bands at 3,000–3,500 cm−1, respectively. The two peaks around 2,924 and 2,854 cm−1 represents the stretching mode of methyl and methylene groups. It is indicative of the complete removal of the surfactants from the polypyrrole nano-structures. Thus it represents the pure form of PPy [43].The shift in the two peaks observed in Fig. 4 in CA and SA system could be due to the presence of different surfactant molecules.

Fig. 4
figure 4

Fourier transform infrared (FTIR) analysis. FTIR analysis of the polypyrrole (PPy) incorporated cryogel scaffolds performed in the range from 0 to 4,000 cm−1. Chitosan–gelatin gels incorporated with polypyrrole (PPy) synthesized using CTAB/APS (CA): SDS/APS (SA) and SDS/FeCl3 (SF) as the surfactant and oxidizing agent, respectively

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3.7 Thermogravimetric analysis (TGA)

The hybrid gels prepared lost their molecular weight initially as shown in Fig. 5 because of the loss of water mainly from gelatin and PPy as gelatin has the capacity to absorb lots of water in its polymeric network. Moreover PPy being hygroscopic in nature also looses residual water around this temperature. Then there was a continuous weight loss from 250 to 450 °C. Although SDS decomposes slightly faster than the others because of its lower decomposition temperature [43], the synthesized cryogel samples containing SDS had a higher decomposition temperature. This might be because of strong ionic bonds formed between anionic surfactant (SDS) and pyrrole radical cations (formed during oxidation) compared to the cationic surfactant (CTAB).

Fig. 5
figure 5

Thermogravimetric analysis (TGA) was done to determine the percentage change in weight of the conducting cryogel scaffold by varying the temperature from 0 to 450 °C. Chitosan–gelatin gels incorporated with polypyrrole (PPy) synthesized using CTAB/APS (CA): SDS/APS (SA) and SDS/FeCl3 (SF) as the surfactant and oxidizing agent, respectively

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3.8 Rheology analysis

Rheology is a parameter used to study the visco-elastic behavior of materials when they are subjected to stress conditions. Therefore it is defined as the study of deformation and flow of materials [23]. It basically analyses the viscous and the elastic properties of the material. The term “loss modulus” (G’’) and “storage modulus” (G’) in rheology defines the viscous and elastic properties of the material and are represented in terms of pascal (Pa). It is one of the best tools for the characterization of the material properties and these are presented in Fig. 6.

Fig. 6
figure 6figure 6

Rheology studies were performed to compare the visco-elastic properties of the chitosan–gelatin (CG) scaffold with that of the conducting scaffold in the same conditions (37 °C and 12.5 mm plate). For the study, samples were analysed both in the dry state (upper panel—a, b, c, d) and wet state (lower panel—e, f, g, h). Storage modulus, loss modulus, phase angle are represented by (black up-pointing triangle), (black square), (black plus), respectively. CG-chitosan–gelatin gels (a, e) and CG incorporated with polypyrrole (PPy) synthesized using CTAB/APS (b, f), SDS/APS (c, g), and SDS/FeCl3 (d, h) as the surfactant and oxidizing agent, respectively

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All the polymeric scaffolds showed a general trend and there was a decrease in the storage and loss modulus in all the scaffolds upon hydration. In this study we tried to analyze the change in the visco-elastic properties of the sample when these cryogel scaffolds were subjected to shear stress over a period of time. It has been observed that few non crosslinked polymers, over the time, undergo deformation and begin to flow in the presence of this stress. Thus scaffolds were checked for their mechanical strength in the dry and in the wet (as in the in vitro/in vivo) state. The storage modulus of the CA (CTAB/APS) scaffold was lower compared to the neat polymer (CG) whereas it was similar for the SA and higher for SF scaffolds. The incorporation of the polypyrrole nano-fillers into the scaffold showed to affect the mechanical properties of the synthesized scaffolds. With the change in phase angle by approximately 2.5° for SF and 1.5° for the other scaffolds the scaffolds showed good visco-elastic behavior. The increase in the stress–strain moduli in the case of SF scaffolds could indirectly suggest the incorporation of a larger amount of nano-fillers (when the same amount of PPy solution was added) or the network formation/structure of PPy in the SF scaffolds influenced these properties. However, CA showed the lowest modulus and it could also be seen by its structural morphology. Thus it can be inferred that these scaffolds when subjected to shear stress for 15 min under controlled conditions by varying the phase angle did not undergo much deformation and survived the stress.

3.9 Degradation analysis

Degradation studies clarified that the presence of PPy in the composites didn’t affect the degradation rate of the gels. All the PPy incorporated gels as well as the non conducting polymer gels degraded to around 12 % of their original weight within 1 month of duration. This was desirable as the slow degradation rate will not disrupt the incorporated PPy network and thus maintain the conducting properties of the synthesized cryogel for longer duration.

3.10 In vitro biocompatibility study

To check the biocompatibility of the synthesized gels, model cell line NIH3T3 was used. The cells were seeded on the scaffolds as well as the tissue culture plate which was used as the control for the experiment. The experiment was carried out for a week and there was no inhibition in proliferation or cytotoxicity observed on these gels as there was a continuous increase in the cell number on the scaffolds. Compared to 2-D the cell number was higher in the scaffolds because of the availability of a larger surface area compared to the tissue culture plates (Fig. 7).

Fig. 7
figure 7

In vitro biocompatibility of the conducting cryogel materials. MTT assay was performed to check the proliferation of the NIH3T3 cells seeded on these scaffolds. The absorbance was measured at 570 nm. All the experiments were performed in triplicates. CG-chitosan–gelatin gels and CG incorporated with polypyrrole (PPy) synthesized using CTAB/APS (CA), SDS/APS (SA) and SDS/FeCl3 (SF) as the surfactant and oxidizing agent, respectively. All the experiments were performed in triplicates and the Student ‘t’ test was performed to obtain the P value. P < 0.05 CTAB/APS (square with vertical fill): SDS/APS (square with horizontal fill): SDS/FeCl3 (square with orthogonal crosshatch fill): CG (square with upper left to lower right fill): 2D (square with upper right to lower left fill)

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3.11 In vitro electrical stimulation

SF scaffolds were chosen for cell studies because of their better in vitro results as well as mechanical properties. Moreover the high yield and uniform distribution of the PPy film throughout the gel was one of the major considerations for the selection of the scaffold.

In order to check the effect of incorporation of conducting polymer both the neat polymer (CG) and the conducting cryogel (SF) were seeded with the neuro2a cells. After allowing the cells to adhere for 24 h the cell seeded scaffolds were electrically stimulated using 100 mV for 2 h. Both the scaffolds when electrically stimulated showed an enhancement in their proliferation compared to their un-stimulated counterparts. There was a 30 % increase in cell number (Fig. 8a) in the SF scaffolds as is also earlier shown that electrical stimulation increased the cell proliferation of neural cells whereas in the neat polymer an enhancement of only 10 % was observed [44]. Although the exact mechanism for this increase in cell number by electrical stimulation is not reported, there are few proposed theories. Electrical stimulation might cause depolarization of the membrane and thus influx of ions which in return influence the release of transmitters and signaling molecules which might be the cause of enhancement in the cell proliferation. CG scaffolds on stimulation did show a minor increased proliferation which might be because of the formation of a polyelectrolyte complex between chitosan and gelatin (as both are polyelectrolytes) which helps in the conduction of electricity and thus this increase. However, both the stimulated CG and SF scaffolds showed better results compared to their respective controls. In the case of neuro2a cell line seeded on SF scaffold and stimulated by various voltages, 100 mV showed better results compared to the control (SF scaffold without electrical stimulation) although others were more or less similar to the control. In order to check if the 2 h stimulation of the neuro2a cell line in every 24 h (100 mV) was harsh for the cells we stimulated the cells after every 72 h and checked for the proliferation by MTT assay. After this when we compared the 24 h stimulation data with 72 h data the proliferation rate was found to decrease continuously as shown in Fig. 8b, which needs to be further studied in order to understand the mechanism behind this effect.

Fig. 8
figure 8

(a) Voltage optimization for neural cell line (Neuro2a) (square with vertical fill) 30 mV: (square with horizontal fill) 50 mV: (square with upper left to lower right fill) 70 mV: (square with upper right to lower left fill) 100 mV: (square with dots fill) control. In order to assess the proliferation rate of the electrically stimulated neuro2a cell line seeded on stimulated and unstimulated (control) SF scaffolds, MTT assay was done. As compared to the control, 100 mV showed better proliferation rate. The cells were stimulated after 24 h of seeding on the scaffolds. CG-chitosan–gelatin cryogels and SF-chitosan–gelatin gels incorporated with polypyrrole synthesized using SDS/FeCl3 as the surfactant and oxidizing agent, respectively. (b) In vitro electrical stimulation (square with vertical fill) SF unstimulated: (square with horizontal fill) CG unstimulated: (square with orthogonal crosshatch fill) SF 24 h: (square with upper left to lower right fill) CG 24 h: (square with dots fill) PPy 72 h: (square with diagonal crosshatch containing black small square) CG 72 h. MTT assay was done on neuro2a cells seeded on stimulated and unstimulated CG and SF scaffolds. SF and CG unstimulated scaffolds showed decreased proliferation rate when compared to their stimulated counterparts. Stimulation was provided after the cells were allowed to adhere onto the scaffolds for 24 h. However, their proliferation rate decreased when the stimulation was given at a regular interval of every 72 h. All the experiments were performed in triplicates and the Student ‘t’ test was performed to obtain the P value. P < 0.05

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Similarly another cell line C2C12 was chosen for stimulation studies and after the cells were allowed to adhere for 72 h, cell proliferation was assayed (Fig. 9). In this case, cells stimulated with 2 V showed higher rate of proliferation as compared to the others. Further to check the cell material interaction and homogenous cell distribution on the newly synthesized conducting scaffold SEM and fluorescence microscopy was done (Fig. 10).

Fig. 9
figure 9

Voltage optimization for C2C12 cell line. MTT assay was done to assess the proliferation rate of C2C12 cells after allowing them to adhere on the SF scaffolds for a period of 72 h. Various range of voltages varying from 100 mV to 2 V was provided in order to stimulate the cells. Higher rate of proliferation was shown at 2 V as compared to the others. Control (square with horizontal fill): 100 mV (square with upper left to lower right fill): 300 mV (square with upper right to lower left fill): 500 mV (square with orthogonal crosshatch fill): 700 mV (square with dots fill): 1 V (square with diagonal crosshatch fill): 2 V (black square). The control used was unstimulated SF scaffolds. Chitosan–gelatin gels incorporated with polypyrrole synthesized using SDS/FeCl3 as the surfactant and oxidizing agent, respectively (SF). All the experiments were performed in triplicates and the Student ‘t’ test was performed to obtain the P value. P < 0.05

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Fig. 10
figure 10

Cell material interaction. (a) Neuro2a cells seeded on the stimulated scaffolds after 1 week, (b) higher magnification of neuro2a cells seeded on stimulated SF scaffolds with proper spreading, (c) C2C12 cells seeded on stimulated SF scaffolds after 5 days, (d) propidium iodide staining of neural cells on stimulated SF scaffold. SF-conducting cryogel synthesized using SDS/FeCl3 as the surfactant and oxidizing agent, respectively

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In conclusion in this work we have synthesized conducting scaffold which is biocompatible and degradable in nature. Moreover at the same time the cryoconcentration process in cryogelation enabled the presence of conductive properties with incorporation of lesser amount of PPy. As PPy is very stable and does not degrade easily, usage of lesser amount is always better. Thus the synthesized conducting cryogel scaffold incorporates the beneficial properties of cryogel as well as the conducting properties which allow controlled electrical stimulation of the cells seeded on them.