Magnetic actuator device assisted modulation of cellular behavior and

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Magnetic actuator device assisted modulation of cellular behavior and tuning of drug release on silk platform Dimple Chouhan, Shreya Mehrotra, Omkar Majumder, and Biman B. Mandal ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00240 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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ACS Biomaterials Science & Engineering

Magnetic actuator device assisted modulation of cellular behavior and tuning of drug release on silk platform Dimple Chouhan, Shreya Mehrotra, Omkar Majumder, Biman B. Mandal* Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India

*Corresponding author: Dr. Biman B. Mandal Associate Professor Department of Biosciences & Bioengineering Indian Institute of Technology Guwahati, India Phone: +91-361-258 2225 Fax: +91 361 258 2249 (O) E-mail: [email protected]

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Abstract: Externally applied physical forces and mechanical stimulations have been found to be instructive to cells which lead to their signaling or differentiation. Further, bioreactors and functional biomaterials have been designed based on this principle to modulate cellular behavior under in vitro conditions. Herein, we have designed a magnetic actuator device (MAD) to understand the fundamental responses of two different phenomena: the effect of actuation on cardiac muscle cells and drug delivery under the influence of pulsed magnetic field. Silk fibroin (SF) based magnetically responsive matrix, developed by incorporating magnetic iron oxide nanoparticles (IONP) within silk nanofibers was actuated with MAD. The silk matrix was seeded with cells and drugs independently to study effect of physical actuation by MAD on cellular behavior and drug release properties. Neonatal rat cardiomyocytes and H9c2 cells were used for studying the former while model drug was used to observe the latter. Pulsed magnetic stimulation promoted proliferation of cells at a significantly higher rate in comparison to those under static conditions, p ≤ 0.01. For instance, a significantly higher expression of Connexin 43 gene was observed in both H9c2 and primary rat cardiomyocytes under magnetic stimulation compared to non-stimulation conditions after day 14, p ≤ 0.01. A differential drug release profile corresponding to respective actuation frequency was observed while studying drug release properties. Overall, the device can be applied as a non-invasive technique to stimulate cardiac cells grown under laboratory conditions for developing functional artificial construct coupled with additional regulated drug release properties. The study thus demonstrates versatile applications of MAD in biomedical and tissue engineering. Keywords:

Magnetic

actuation;

tissue engineering;

silk

cardiomyocytes


nanofibers;

drug delivery;

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INTRODUCTION Modulation of cellular behavior through biochemical cues and mechanical stress is well known to improve the viability and functionality of various types of cells.1,2 Several studies highlight the ability of surrounding mechanical stress and external physical forces like magnetic field in manipulating certain cellular signaling pathways.1,3 These cues are particularly applicable in tissue engineering where such mechanical stresses have been shown to activate the mechanotransduction pathway of cells. Based on these findings, several bioreactors and mechanical actuators have been developed since the last decade in order to improve cellular secretions, proliferation and maturation.4,5 In general, the idea behind designing shear stress type or perfusion bioreactors is to improve the nutrient supply to cells.6,7 Recently, the magnetic actuation technique has gained attention for its capacity to stimulate cells either by developing magnetic scaffolds or by labelling the cells with magnetic nanoparticles.3 Magnetic actuation means stimulation of cells through direct or indirect application of magnetic field in a constant (static) or oscillating (pulsed) manner.3 Such approach of applying physical stimulation during cell cultivation has been used in cardiac and neuronal tissue engineering. The external forces aid in cell development and maturation of cardiomyocytes and neurons.8,9 For instance, electrical stimulations require techniques to be employed in close proximity to cells.10 Additional critical issues generally experienced in optical and electrical based systems in turn pose crucial effects on the cells like heating, and contact electro-polarization of electrodes leading to breakdown of cell-membrane.11 Unlike direct stimulation of cells using electric diodes, strategies involving magnetic field might be considered as an alternative approach. The non-contact mode of action by electromagnets offer significant benefits through its ability to remotely control magnetic nanocarriers and their



environment in the body.12 Effect of magnetic field on cellular behavior to stimulate cardiomyocytes has been well studied in previous reports.9 Formation of cell sheet acquired by encapsulating magnetic nanoparticles in muscle cells and their direct stimulation via constant magnetic field has been reported.13 Nevertheless, this technique requires encapsulation of magnetic nanoparticles inside cells; thus limiting its applications in grafting procedures. To overcome such limitations, scientists have resorted to developing magnetic scaffolds by combining magnetic particles embedded inside the scaffold material.14,15 In our efforts, we have used pulsed magnetic field by developing a microcontroller based easy-to-use device to stimulate cardiomyocytes. The device connected with electromagnets provides magnetic field only at the desired pulse rate. By modulating the frequency of fluctuation or ‘on and off timings’ of electromagnet using ATmega16 microcontroller, pulsed magnetic field could easily be generated. The device was designed in such a way that it provides an alternate magnetic field externally at the required frequency. Using a microcontroller based circuit has additional advantage of providing flexibility of programmable adaption tunable for specific actuation. In addition, the response time is reduced owing to the faster speed of execution with a microcontroller integrated circuit when compared to electro-mechanical devices.16 The novelty in our approach lies in the potential of the device to be able to tune frequency of actuation as per requirement which is also of great importance in drug delivery applications. Tuning the delivery rate of drugs by modulating physical and chemical properties of drug carriers is an area of intense research.17,18 Thus, modulation of drug delivery profile at different actuation frequencies through the developed magnetic actuator was also studied in the present work. Herein, we fabricated silk based nanofibrous matrix containing iron oxide nanoparticles (IONP) to develop


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magnetic matrix. IONP were chosen as the magnetic entity owing to its biocompatibility and superparamagnetic behavior.3 Notably, IONP have been widely reported to label various cells like neuronal and mesenchymal stem cells and does not alter cellular proliferation, viability and differentiation potential of cells.19,20 Furthermore, IONP are considered commonly to construct magnetic scaffolds for tissue engineering applications and proven for its safety and biocompatibility.14,15 In the present study, we developed magnetic nanofibrous matrix in order to explore various applications of the device pertaining to improved surface properties of nanofibers in both cell culture and drug delivery applications.21,22 Silk fibroin (SF) isolated from Bombyx mori cocoons was chosen as the base material due to its biocompatibility and ease in material processing.23,24 SF being a protein polymer, possesses tunable mechanical strength and can be used to fabricate versatile formats such as thin films, hydrogels, nanofibrous matrix and porous scaffolds.25–28 Silk based artificial organs have been developed ranging from soft tissues like cardiac patch, skin graft, pancreas to hard tissues like bone, cartilage and intervertebral disc.23,28–31 In our approach, magnetization of the silk matrix was achieved through addition of iron nanoparticles by co-spinning them using electrospinning technique. The silk based nanofibrous matrix thus obtained was used as a magnetically responsive substrate to investigate various applications of the magnetic actuator device (MAD). The study focuses on designing a miniaturized microcontroller based electronic device that can exert programmable magnetic pulsed actuation. In the present work, efficacy of the developed device was explored with two different applications; at tuning the drug release profile and stimulating cardiac myoblast cells. Biophysical forces have been established to stimulate cardiomyocytes and hence, the effectiveness of MAD was monitored using these cells. To establish a model for cellular stimulation, these cells were cultured under magnetic actuation



with the future prospect of developing artificial cardiac patch at laboratory scale. We have also delved into the substrate’s ability under magnetic actuation in modulating growth and gene expression of cardiomyocytes within short period of culture conditions. Through this we can state that such an actuator possesses potential in applications of bioreactor systems wherein autologous cells can be cultured and stimulated in short-term culture conditions. The current study demonstrates advanced engineering technique with an aim to improve scope of regenerative medicine and tissue engineering. MATERIALS AND METHODS Design and development of magnetic actuator device (MAD) The actuator device was developed based on microcontroller based circuit unit connected with electromagnets. The whole device primarily consisted of a controlling system (ATmega16), a motor driving unit (L293d), electromagnets and a rigid enclosure box to house the system as shown in schematic diagram (Figure 1a). An AVR 40 Pin Development Board (Robokits, India) loaded with a microcontroller ATmega16 was the chief controlling unit of the circuit. The ATmega16 was programmed to operate the electromagnets (Electromagnet, Type 58, 24VDC, Stephenson Gobin procured from element14) with specific pulse rates for four different frequencies. Connection between ATmega16 and electromagnets was set through L293d Dual Motor Driver H-BRIDGE Module. The motor drivers were connected to the output of board through the in-built push button response switches and the LED based display to indicate the corresponding frequency of the electromagnets. The device enclosure box was composed of a clear plastic material (acrylic, SK Instruments, India) to prevent corrosion of internal components and to withstand UV sterilization process. The motor drivers connected to the electromagnets facilitated ‘on and off’ mechanism which provided magnetic field only at the required time points. The period of switching decided


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the frequency by changing the duration of ‘on-off’ cycles. This differential approach helped to acclimate the frequency required to accomplish optimum actuation. Commercially available 60 mm petri dishes were used for cell culture and were conveniently placed on the electromagnets. The sterilization process involved wiping the system with 70 % ethanol followed by a UV lamp exposure of 20 minutes. In order to address scaling and reproducibility, two different electromagnets with the same control program were connected via corresponding motor drivers. In the design, a distance was assigned between the electromagnets so that they do not interfere beyond their zone of magnetic influence. This non-invasive actuation method contributed towards making the device simple, robust and user-friendly without compromising the overall sterility of the same. Fabrication of magnetic nanofibrous matrix using silk as biomaterial. Silk fibroin (SF) as a biomaterial was used to fabricate magnetic matrices using electrospinning technique. SF was isolated from the B. mori silk cocoons according to the previously established protocol.33 Briefly, the cocoons were cut into small pieces and degummed using 0.02 M sodium carbonate (Merck, India) to obtain silk fibers. The silk fibers were then dissolved in 9.3 M lithium bromide (LiBr) solution (Sigma Aldrich, USA) at 60 °C for 4 h and dialyzed against water using a 12 kDa cellulose dialysis membrane (Sigma Aldrich, USA) for 48 h at room temperature (RT). The concentration of the obtained SF solution was 8 % (w/v) as measured by gravimetric method. Further, to concentrate the SF solution, it was dialyzed against poly(ethylene glycol) (PEG) solution (25 % w/v) using 3 kDa snake skin dialysis membrane (Thermo Fisher Scientific, USA) for 12 h at 4 °C. The final concentration of PEG dialyzed SF thus obtained was 20 % (w/v). In order to fabricate magnetic silk nanofibrous matrix, poly(ethylene oxide) (PEO) (high molecular weight 9,00,00 Mv) was used as the sacrificial material for electrospinning. Magnetite Iron



(II/III) oxide nanoparticle (IONP) (particle size < 50 nm, Sigma-Aldrich, USA) was mixed in PEO (5 % w/v) solution and sonicated using ultrasonicator (Vibra Cell, Sonics) at 25 % amplitude for 10 min prior to addition of the SF solution. The sonication step was applied to ensure proper dispersion of the IONP in the blend and hence prevent their agglomeration once mixed with SF.

The SF/PEO/IONP blend solution was filled in a syringe (10 mL) and

electrospun fibers were generated using the electrospinning unit Super ES1 (E-spin nanotech, India) at the following settings: voltage = 15 ± 2 kV, flow rate = 1.200 ± 0.100 mL/h, tip-tocollector distance (d) = 15 cm, and rotating speed of the drum collector = 500 rpm. The electrospun matrices were collected on an aluminum foil to ensure conductivity between the collector and the tip of needle. Subsequently, the matrices were gently removed from the foil as the matrices were self-supporting. Both the matrices were fabricated by spinning 10 mL solution which resulted in thickness of 450 ± 50 µm as measured by a coating thickness gauge meter. Two types of nanofibrous matrices were developed: SF blended with PEO containing 5 % IONP (w/v) and SF blended with PEO without IONP. β-sheets were induced in the nanofibrous matrices through ethanol vapor treatment for 6 h using vacuum desiccator followed by an hour long 70 % ethanol exposure. Post β-sheet induction, the matrices were washed with sterile water multiple times for 3 days to remove the PEO.34,35 The silk matrix (SF) without the incorporation of IONP was used as control in the subsequent experiments. Morphology of magnetic nanofibrous matrix. Morphology of the nanofibers in the electrospun matrices was observed using field emission scanning electron microscopy (FESEM; Sigma 300, Zeiss). Prior to imaging, the matrices were dried and coated with a thin gold layer using a sputter coater. Morphology of as spun matrices and post PEO leaching was observed to ensure the retention of nanofibrous architecture. To further look into the IONP distribution in the


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nanofibers, analysis was performed by high resolution transmission electron microscope (HRTEM (JEOL, JEMCXII, operating voltage 200 kV). Characterization of magnetic nanofibrous matrix. Fourier Transform Infrared Spectroscopy (FTIR) of the matrices was carried out using Spectrum two FTIR spectroscopy (PerkinElmer) in the region of 4000-400 cm-1. The sample was prepared as pellets using spectroscopic grade KBr and spectrum was attained by accumulation of 32 scans with a resolution of 4 cm-1. Functional groups of both SF and IONP were detected using the spectra obtained by FTIR. Magnetic hysteresis loops of the silk nanofibrous matrix containing IONP was measured using a vibrating sample magnetometer (VSM, Lakeshore model 7410). The VSM was equipped with a sample holder to record the magnetization of the samples. All analysis was carried out at magnetic field ranging from -15000 to 15000 Oe and ramp rate of 112.78 Oe/sec. The results of magnetization, coercivity and retentivity values were reported as average ± standard deviation (S.D.) (n = 3). Commercially available IONP (Sigma-Aldrich, USA) were also analyzed under similar conditions for comparison. Degradation and swelling properties of silk nanofibrous matrices. Integral stability of the nanofibrous matrices was analyzed by examining the degradation rate of matrices under in vitro conditions. For degradation studies, the matrices were immersed in sterile phosphate buffer saline (PBS, pH7.4) containing protease XIV (extracted from S. griseus, Sigma-Aldrich, USA; 1 U mg-1) for a period of 21 days. The protease solution was changed every 3 days to maintain its proteolytic activity. The degraded matrices were then dried completely and their weight were measured at specific time points using an electronic balance (Sartorious, AG Germany). The integral stability of matrices was examined through a similar experiment without using any additional proteases in PBS. The entire experiment was performed in sterile environment at 37



°C. The results were reported as average ± standard deviation (S.D.) (n = 3). Degradation profile was plotted by calculating the change in mass using the standard formula: Percent mass remaining = (Mass at time (t)/initial mass) × 100……………………. (1) In order to study the swelling properties of nanofibrous matrices, the weight of dried and swollen matrices were compared at defined time points (t). The dried matrices were weighed and immersed in PBS (pH 7.4) at 37 0C. Weight of the swollen matrices were measured at predefined time points till saturation level was attained. The results were reported as average ± standard deviation (S.D.) (n = 3). The swelling ratio was calculated using the following equation: Swelling ratio (%) = ((mass of swollen matrix - mass of dry matrix)/mass of dry matrix) × 100 ..… (2) Fabrication of magnetic nanofibrous matrices with drug encapsulation and drug release profile at different actuation frequencies. To fabricate drug loaded nanofibrous matrices, Ciprofloxacin HCl (Himedia, India) was used to establish the drug release prototype. In order to avoid drug loss during ethanol treatment and subsequent washing with water for PEO removal, drug incorporation onto the matrices was performed by coating method. For this, desired amount of drug solution was coated onto the processed magnetic matrices instead of cospinning the drug while electrospinning. The matrices (2 cm diameter) were coated with total 100 µL of drug solution (30 mg/mL) of which 3 mg of drug was adsorbed on each matrix. The drug coated matrices were then subjected to release experiment at different magnetic actuation frequencies. To study this, four different frequencies were chosen to actuate the electromagnet attached to the device: 1. Actuation at very fast speed where the duration of device ‘on’ (ton) is 1 second (ton = 1 s) and duration of device ‘off’ (toff) is 1 second (toff = 1 s) 2. Actuation at an intermediate speed (ton = 15 s and toff = 15 s) 3. Actuation at slow speed (ton = 30 s and toff = 30 s)


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and 4. No actuation, i.e. the device is off during the whole experiment (toff = ∞). The drug released from the coated magnetic matrices was quantified by submerging the coated matrices in 3 mL PBS (pH 7.4) at 37 °C. 100 µL of the releasate was taken out and replaced with 100 µL of fresh PBS at pre-specified time-points (viz., 1, 3, 6, 12, 18, 24, 36 and 48 h). Percentage of drug released at various time points was determined by measuring absorbance at 270 nm using a microplate reader (Tecan Infinite Pro M200) and comparing the concentration with standard curve of the drug. The results were reported as average ± standard deviation (S.D.) (n = 3). The following formula was used to examine drug release: Drug release (%) = (Mt / M) × 100…………………………………………………………..(3) where Mt was the amount of drug released at time t and M was the theoretical value of drug present on the nanofibrous matrix. In vitro cytocompatibility of magnetic nanofibrous matrices. To examine biocompatibility of developed matrices under in vitro conditions, the matrices were cultured with primary human dermal fibroblast (HDF) cells purchased from Himedia, India. Both matrices (SF and SF-IONP) were sterilized through autoclaving for 20 min and conditioned overnight in media prior to cell seeding. Circular matrices (diameter 1.5 cm) were used for the proliferation assay in 24-well cell culture plates in static condition (without MAD stimulation). Cells were seeded at a density of 1 x 104 cell / cm2 in all the groups. Proliferation of cells cultured on the matrices was evaluated by MTT assay at specific time-points (day 1, 7 and 14) on both SF and SF-IONP matrices. Fresh media was replaced every alternate day during the culture. Separate cultures were prepared altogether for different time points as the MTT assay is a terminal based assay. Briefly, the cultured matrices were incubated with 1:10 ratio of 3-(4,5-Dimethylthiazol-2yl)-2,5-Diphenyltetrazolium Bromide (MTT) reagent (Sigma, USA) and serum-free DMEM



medium for 3 h at 37 °C and 5% CO2. Post removal of the media containing MTT, dimethyl sulfoxide (DMSO; Sigma, USA) was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a multiplate reader and cell proliferation index was calculated by comparing the optical density (O.D.) obtained at pre-determined time points relative to the O.D. obtained on day 1. The results were reported as average ± standard deviation (S.D.) (n = 3). In vitro immune response towards nanofibrous matrices. In vitro macrophage response was examined by culturing RAW 264.7 mice macrophage cells (NCCS, India) at a density of 5 × 105 cells/cm2 and subsequently examining tumor necrosis factor alpha (TNF-α) secretions by Enzyme-linked immune sorbent assay (ELISA). In order to compare the amount of TNF-α secretion from cells cultured on various nanofibrous matrices, 1000 ng/mL lipopolysaccharide (LPS, from Escherichia coli, Sigma Aldrich, USA) was taken as a positive control and tissue culture place (TCP) was taken as a negative control. TNF-α secretions were quantified from the cultured media after 24 h using mouse TNF-α ELISA kit (Life technologies, sensitivity < 3 pg/mL) according to manufacturer’s protocol. The results were reported as average ± standard deviation (S.D.) (n = 3). Cellular proliferation cultured on magnetic nanofibrous matrices under MAD. In order to investigate the proliferation profile of HDF cells under magnetic actuation conditions, alamar blue test was performed at pre-defined time-points. Briefly, HDF cells seeded at a density of 10,000 cell / cm2 on silk matrices were maintained in the culture conditions for 14 days. Reduction of alamar blue dye was analyzed at pre-defined time points, viz., 1, 3, 7, 10 and 14 days following manufacturer’s protocol. Briefly, the spent media was discarded and alamar dye (diluted 1:10 in complete DMEM medium) was added to the culture and incubated at 37 °C and 5 % CO2 for 3 h. Subsequently, the media containing reduced alamar dye was taken and


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examined for absorbance at 570 nm and 600 nm using a microplate reader. Fresh media was added to the wells after alamar blue assay to continue the culture for 14 days. Magnetic actuation of frequency (actuation twice per second) was provided to the IONP containing SF matrices during the experiment. Isolation of primary rat cardiomyocytes and their culture under magnetic actuation conditions. Protocols approved by ethical committee were used to execute the animal experiments for the isolation of neonatal rat cardiomyocytes (NRCMs). The isolation method was similar to the previously described protocol.27 In brief, 2-3 day old neonatal Wistar rats were dissected to isolate rat hearts. The heart tissue was further dissected to separate the ventricles, which were washed thoroughly using glucose rich KH buffer (6.89 g/L NaCl, 0.358 g/L KCl, 0.285 g/L K2HPO4, 0.3 g/L MgSO4 , 250 mM HEPES buffer and 1.98 g/L glucose) to remove any traces of blood coagulates. The ventricles were additionally cut into smaller pieces, washed thoroughly and subjected to collagenase type II (Gibco, U.S.A.) digestion to separate the associated extracellular matrix from the cells. The digested lysate was then centrifuged to obtain the pellet, trypsinized (using Trypsin/EDTA) to obtain single cells and was seeded into the flask. After initial attachment of non-myocytes, the supernatant was collected, centrifuged and replated into a new flask for cardiomyocyte enrichment (by preventing the growth of non-myocytes). The isolated cardiomyocytes were grown in cardiomyocyte growth medium comprising of Dulbecco’s modified eagle media (DMEM, Gibco, U.S.A.) supplemented with 10% Fetal Bovine Serum (FBS, Invitrogen, U.S.A.), 2 mM L-glutamine (Sigma-Aldrich, U.S.A.) and 100 U/mg/mL Pen/Strep (Himedia, India). Cells were cultured at 37 °C in a 5% CO2 incubator. For, in vitro experiments primary cardiomyocytes (P2) were cultured on the prepared magnetic matrices and transferred over the magnetic actuator in cell incubator maintained at 37 °C and



95/5% O2/CO2. Magnetic actuation of frequency (actuation twice per second) was provided to SF-IONP matrices during the whole experiment. Culture and differentiation of H9c2 cells under magnetic actuation conditions. Ventricular myoblast H9c2 (derived from BD1X rat), were procured from National Centre of Cell Sciences, Pune, India and grown in growth medium (GM) comprising of DMEM supplemented with 10 % FBS and 100 U/mg/mL Pen/Strep. However, the differentiation of H9c2 into cardiomyocyte lineage was carried out by culturing them in differentiation medium (DM) comprising of DMEM supplemented with 1 % FBS and 50 nM all-trans Retinoic acid (RA, Sigma-Aldrich, U.S.A.). Fresh RA was added daily to the DM with media changes every two days. Cells were cultured at 37 °C in a 5 % CO2 incubator. Magnetic actuation of frequency (actuation twice per second) was provided to the SF-IONP matrices during the whole experiment. Proliferation and live dead assay of cultured cardiomyocytes. The proliferation rate of undifferentiated H9c2 cells and neonatal rat cardiomyocytes (NRCMs) seeded on the SF-IONP matrices was evaluated to determine compatibility of the cardiac cells with the IONP containing matrices. Conventionally used method of alamar blue dye reduction was applied to examine cell proliferation rates. Undifferentiated H9c2 and neonatal rat cardiomyocytes (NRCMs) were seeded at a density of 5 × 104 cells /cm2 and 1x 105 cell/cm2 respectively. Alamar blue assay was performed on 1, 3, 7 and 10 days following manufacturer’s protocol (Invitrogen, USA). Briefly, the spent media was discarded and alamar dye (diluted 1:10 in complete DMEM medium) was added to the culture and incubated at 37 °C and 5 % CO2 for 3 h. Subsequently, the media containing reduced alamar dye was taken and examined for absorbance at 570 nm and 600 nm using a microplate reader. The culture was replenished with fresh media and maintained at the


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same culturing conditions. The viability of cultured H9c2 cells and neonatal rat cardiomyocytes on the matrices was also evaluated using a live/dead viability kit (Sigma-Aldrich, USA) following manufacturer’s protocol. Similar cell seeding was performed as described above on day 7, the spent media was discarded. The cells were then incubated with 4 mM calcein-AM (Sigma-Aldrich, USA) and 2 mM ethidium homodimer (Sigma-Aldrich, USA) for 10–15 min in dark conditions. The cells were visualized using a fluorescence microscope (EVOS FLc, Life Technologies, USA). Analysis of gene expression. Gene expression study was performed for both differentiated H9c2 cells and NRCMs cultured on nanofibrous matrices to evaluate the effect of magnetic actuation on cultured cardiomyocytes. Magnetic actuation of defined frequency (actuation rate twice per second) was provided to the SF-IONP matrices during the entire experiment. For analysis, the cultured matrices were chopped using scissors in Trizol reagent (Sigma-Aldrich, USA). Conventional Trizol method was performed for RNA isolation followed by quantification using Nanodrop (Eppendorf). cDNA was prepared from the obtained mRNA using a high capacity reverse transcription kit (Applied Biosystems) in a PCR thermal cycler (Takara) in accordance with the manufacturer’s protocol. Real time PCR (RT-PCR) was performed using the SYBR Green dye (Invitrogen, USA) in a 7500 real time PCR system (Applied Biosystems). Primers for connexin 43 (F-5’-AGGAGTTCCACCAACTTTGGC-3’ and R-5’-TGGAGTAGGCTTGGACCTTGTC-3’),

cardiac

GATCTCGCCCCTTATCTCAGTGTCCTCA-3’ GATCTGAGGACACTGAGATAAGGGGCGA-3’) dehydrogenase

(GAPDH)

Troponin

I

and and

(F-5’R-5’-

glyceraldehyde-3-phosphate-

(F-5’-ATGCTGGTGCTGAGTATGTC-3’

and

R-5’-

AGTTGTCATATTTCTCGTGG-3’) were used for gene expression analysis. The obtained



expression of the cardiac specific genes was normalized to that of the house-keeping gene, GAPDH. Analysis included triplicates (n = 3) from each group. Immunocytochemistry. In order to evaluate the effect of magnetic actuation (under actuation twice per second) on NRCMs, immunocytochemistry was performed for studying expression of cardiac specific marker proteins (connexin 43 and sarcomeric alpha actinin). NRCMs were seeded onto the SF-IONP matrices at a density of 1 × 105 cells / cm2 on nanofibrous matrices and were cultured for 7 days under the above described actuation conditions. After 7 days, the cultured matrices were carefully washed with PBS (pH 7.4) and cell fixation was performed subsequently by incubating them in 4 % paraformaldehyde for 2 hours at room temperature. Prior to staining, cells were permeabilized using 0.1 % Triton X-100 (SigmaAldrich, USA) for 30 min and blocked with blocking buffer (1 % BSA, 0.1% Triton X-100) for 10 hours. The cells were then incubated with primary antibodies mouse monoclonal sarcomeric alpha actinin (1:50, Abcam, UK) and rabbit polyclonal connexin 43 (1:100, Abcam, UK) for 8 h at 4 °C. Immune complexes were further detected by adding Alexa 488 labelled goat anti-mouse secondary antibody and Alexa 594 labelled goat anti-rabbit IgG secondary antibody (1: 200, Abcam) for 3 h. For counterstaining the nucleus, the cells were incubated in DAPI for 1.5 h. Fluorescence images were obtained using a confocal microscope (LSM 880, Zeiss). Statistical analysis. All the experiments were carried out for n = 3 samples unless otherwise specified. Data were expressed as mean ± standard deviation. Data analysis was done using statistical software OriginPro 8 (Origin lab Corporation, USA) at both significant (* p ≤ 0.05) and highly significant (** p ≤ 0.01) levels. The significance level was measured by comparing the data between groups and within groups by performing one-way analysis of variance (ANOVA) followed by Tukey’s test.


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RESULTS AND DISCUSSION Development of magnetic actuator device (MAD). In order to study the effect of magnetic actuation on biological systems, magnetic actuator device (MAD) has been designed and fabricated in the present work. The microcontroller circuit was well connected with electromagnets via L293d motor driver (Figure 1). The microcontroller programmed with dual output system was successfully connected to two independent L293d motor drivers. Each electromagnet was separately connected to the L293d motor driver. As a result, the whole set up could be easily accommodated into the device enclosure box with only the electromagnets protruding outside. Petri dishes of diameter 60 mm could be placed on top of each electromagnet directly. The device once put inside the incubator for cell stimulation did not hamper sterility of the culture. The in-built four input system of ATmega16 microcontroller connected with four different switches offered four different frequencies. Thus, tuning of output frequency was possible by changing the ‘on and off’ time period in the program fed to the microcontroller. Hence, this magnetic actuator device showcases simple and robust design. This can also serve as a working model for stimulating cells without disturbing the culture conditions of mammalian cell incubator. The magnetic actuator device has been fabricated to understand the fundamental responses of two different phenomena: tunable drug delivery and actuation of cardiomyocytes under the influence of intermittent magnetic field.



Figure 1. (A) The schematic image shows design of the magnetic actuator consisting of ATmega16 based microprocessor system connected with electromagnets via L293d driver motors. (B) Schematic representation depicting applications of magnetic actuation device (MAD) in stimulation of cell differentiation and for frequency dependent drug delivery systems. (C) Photograph of MAD showing encased circuit board in a transparent box with two external electromagnets connected with the system. Morphology of IONP impregnated silk nanofibrous matrix. Smooth nanofibers from SF-PEO and SF-PEO-IONP blends were generated by electrospinning. Blending PEO material


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as a sacrificial polymer has already been well established for generation of smooth and bead-less SF nanofibers in previous studies.35 In addition, PEO also helped in homogenous distribution of IONP in the blend solution. Sonication of PEO-IONP blend prior to blending with SF prevented settling down of IONP in the blend solution as represented in the schematic image (Figure 2A.i.) The electrospun fibers of both SF-PEO and SF-PEO-IONP appeared to be randomly distributed in the nonwoven matrices. Micrographs from FESEM analysis of the electrospun fibers demonstrated nanofibrous architecture of the matrices (Figure 2B). The blend of 20 % SF and 5 % PEO + 5 % IONP mixture generated nanofibers of size in range of 100 to 300 nm diameter. The nanofibrous architecture was maintained even after PEO leaching was carried out from both the matrices. This suggested structural retention of the silk fibroin nanofibers. However, IONP could not be visualized by FESEM images because the IONP were coated by SF polymer as observed by TEM analysis (Figure 2C). TEM analysis demonstrated presence of IONP well distributed within the silk nanofiber (Figure S1, Supporting Information). This validated stable impregnation of IONP within silk nanofibers even after PEO leaching. The IONP were ranging between 10 to 20 nm in diameter and coated by silk biopolymer. The nanofibrous architecture of electrospun matrix has also been reported to provide additional functional advantage to cell culture when compared to flat films or microfibers.36 SF-IONP nanofibrous matrix also showed attraction towards magnetic material when placed manually suggesting magnetization in the SFIONP matrix (Figure 2A.ii.). Thus, silk based nanofibrous matrix incorporated with magnetic particle could be a suitable platform to culture and stimulate cells using the developed magnetic actuator device.



Figure 2. (A) Schematic representation of (i) fabrication process of silk based magnetic nanofibrous matrix formed by blending iron oxide nanoparticles (IONP) (5 % w/v) and poly(ethylene oxide) (PEO) (5 % w/v) with 20 % (w/v) silk fibroin (SF) solution through electrospinning. The IONP were first mixed with PEO and sonicated for proper dispersion prior to blending with SF. (ii) representative image of the fabricated matrices showing magnetic property of SF-IONP matrix. (B) Field emission scanning electron microscopy (FESEM) images shows morphology of the nanofibers in various matrices: (i) SF-PEO nanofibers, (ii) SF-PEOIONP matrix containing nanoparticles of iron oxide, (iii and iv) shows morphology of respective nanofibrous matrices after PEO leaching indicating retention of nanofibrous architecture. (C)


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Highly magnified transmission electron microscopy (TEM) images of single nanofiber of SFIONP showing distribution of IONP inside the silk nanofiber. Characterization of magnetic silk matrix. The FTIR spectra recorded for pristine IONP, only SF matrix and SF-IONP nanocomposite matrix showed characteristic bands of both SF and IONP in the respective materials (Figure 3A). The spectra of pristine IONP showed a major band ranging from 500 to 750 cm-1 which are generally attributed to the Fe-O bonds confirming their octahedral and tetrahedral sites.14 The characteristic peaks of SF were observed from 1130 cm-1 to 1650 cm-1 corresponding to the amide bonds. Sharp peak at 1630 cm-1 associated with C=O stretching (amide I) is related to the β-sheet conformation.35 Further, peaks around 1450 and 1286 cm-1 indicative of amide II and amide III regions were visible in case of SF matrix. Similar type of amide peaks were also shown by SF-IONP nanocomposite matrix suggesting stable SF structures, especially β-sheet conformation in the matrices. The structure of silk samples (both SF and SF-IONP) demonstrated silk type II, which is the typical silk structure (β sheet).24,35 The FTIR spectra also supported the fact that PEO was leached out from both the types of silk matrices as the results obtained were found to be at par with previous studies.37 SFIONP also demonstrated slight peak shift at the band ranging from 500 to 602 cm-1 depicting FeO bonds due to incorporation of IONP in the silk nanofibers. In addition, the band ranging from 3000 to 3750 cm-1 in case of SF-IONP nanocomposite showed two different peaks characteristic to both IONP and SF. This further validate stable interactions between the two moieties in the nanocomposite. To further examine the magnetic behavior of SF-IONP nanocomposite matrix, VSM study was performed (Figure 3B and 3C). It is evident from the narrow hysteresis loop that SFIONP matrix demonstrated properties of soft ferromagnetic material. The magnetization (Ms)



value of SF-IONP (5.83 ± 1.04 emu/g) was low in comparison to that of 100 % IONP (28.99 ± 0.82 emu/g) as only 5 % IONP was incorporated in SF-IONP matrix. However, the magnetization achieved by SF-IONP showed sufficient magnetic activity as observed from the hysteresis curve. SF-IONP exhibited higher magnetization on increasing the magnetic field and vice versa (linearity between X and Y axes) till the saturation point which was achieved at 1000 Oe magnetic field (Figure S2, Supporting Information). This further validated the magnetic moment dependence of SF-IONP matrix. The average magnetization of SF-IONP (5.83 ± 1.04 emu/g) was also observed to be in the range of previously reported magnetic scaffolds.15,38 Magnetic silk porous scaffolds demonstrated magnetization value of 2.7 and 13 emu/g depending on the amount of magnetic particle present which proved to be sufficient.38 Hence, developed SF-IONP matrix which also showed similar magnetization is expected to prove its potential as a magnetic matrix. Moreover, there was no significant difference in the coercivity of both IONP and SF-IONP matrix suggesting good magnetic behavior when placed under magnetic field. The remnant magnetization (Mr) value of SF-IONP (0.74 ± 0.2 emu/g) was significantly lower to IONP suggesting very low magnetism left in the nanocomposite matrix once the magnetic field was eliminated. Lower remnant magnetization (Mr) is an essential parameter for applications in magnetic actuation applications. This indicated that the SF-IONP matrix would behave as magnet only in the presence of magnetic field. The matrix would no longer show magnetic behavior after removal of surrounding magnetic field. Thus, the SF-IONP nanocomposite could serve as a platform for actuating mammalian cells using the developed MAD.


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Figure 3. Physical characterization of the magnetic SF-IONP matrix by (A) Fourier transform infrared spectra of pristine iron oxide nanoparticles (IONP), only silk fibroin (SF) nanofibrous matrix and SF-IONP magnetic matrix. (B and C) Vibrating sample magnetometer (VSM) analysis shows hysteresis loop and magnetic characterization of SF-IONP matrix in comparison with pristine IONP demonstrating magnetic behavior of SF-IONP matrix conferred as a result of incorporation of IONP. Integral stability of magnetic nanofibrous matrix and swelling capacity. Integral stability of matrix is considered to be a necessary parameter for long term cell culture studies. The incubation of nanofibrous matrices under the solutions with and without the presence of protease revealed biodegradability and stability respectively (Figure 4A). In the protease free PBS, both SF and SF-IONP matrices showed 100 % mass retention showing their stability owing to the β sheet structures. In contrast, weight of the matrices was reduced in the presence of protease solution. SF, being a protein biopolymer demonstrated linear degradation rate in terms



of weight ratio in the protease solution. Due to its controllable biodegradability, silk based scaffolds have been widely used for the development of various organs.23 The highly organized β-sheet crystal regions are responsible for silk’s rigidity and stability for prolonged implantation.39 Degradation study of silk porous scaffolds conducted under in vivo system further revealed stability of the scaffolds even after one year of subcutaneous implantation.40 However, SF protein is an enzymatically degradable polymer and the degradation rate of silk based scaffolds depends on various factors.41 The nanocomposite matrix of SF-IONP lost 30 to 33 % mass in 21 days suggesting biodegradability by the protease action. The SF matrix showed significantly higher degradation as it lost 35 to 40 % mass after 21 days, p ≤ 0.01. The comparatively lower degradation rate of SF-IONP matrix might be attributed to the nondegradable nature of IONP present in the composite matrix. Nevertheless, the study suggested higher integral stability of silk based magnetic matrix in protease free environment. Optimum degradation rate in protease rich environment demonstrated its capacity to be an ideal substrate for tissue engineering applications. Further, observation of swelling behavior revealed high water uptake capacity of the silk matrices (Figure 4B). Silk matrix encapsulating IONP demonstrated significantly lower swelling ratio in comparison to pristine SF matrix, p ≤ 0.01. This was probably due to the presence of IONP within silk nanofibers lowering the absorption capacity. However, value of a swelling ratio greater than 200 suggested an overall good swelling property adequate for culturing mammalian cells.42 Higher swelling ratio of SF matrix might also attribute to higher degradation rate. Such property may lead to increased absorption of proteases in the matrix thus allowing enhanced rate of enzymatic degradation.


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Drug release profile at different actuation frequencies. In tissue engineering and regenerative medicine, on-demand drug delivery is a fundamental prerequisite. In order to examine the effect of varied magnetic pulse on the release profile, drug release behavior was studied under different magnetic actuation frequencies (Figure 4C). A model antibiotic drug coated over SF-IONP nanofibrous matrix was used for release study. The study was conducted so as to validate proof of concept for applications of the developed device in drug delivery. Drug release was found to be dependent on actuation frequency i.e. the number of pulses provided per second. At the highest frequency (ton/off = 1 s), the matrix was given a single magnetic actuation every second. This contributed to overall increased rate of drug release on higher actuation frequency. Similarly, the release rate was found to be declining at lower frequency, for instance a frequency of ton/off = 30 s showing a time lag of 30 seconds between the actuations or ‘on’ and ‘off’ states of the microcontroller. Frequency of ton/off = 30 s depicts single stimulation at a gap of 30 seconds; whereas frequency of ton/off = 1 s depicts 30 stimulations in 30 seconds. The initial burst release was also significantly higher in case of high actuation frequency (ton/off = 1 s) when compared with low speed actuations, p ≤ 0.01. This indicated greater amounts of drug being diffused out due to frequent alternating pulses of magnetic field. Due to recurrent ‘on and off’ states of electromagnets per second, the device could induce more force on drug coated matrix leading to its release at higher rate. On further observation of the drug release profile at different actuation frequencies, it was found to be sustained while being dependent on the actuation frequency. After 6 h of release study, drug reservoirs actuated per second showed more than 50 % increment in drug release in comparison to non-actuated matrices. Therefore, the saturation time of drug release also varied depending on the actuation frequency. Results of drug release



observed under various actuation frequencies of electromagnets has been depicted by the schematic image (Figure 4D). The study thus reports development of a magnetically controlled drug delivery device tuned for on-demand drug release. Detailed analysis at various frequencies provide an estimate of drug release profile which can be further tuned as per requirement. Hence, the MAD can also be used as a drug delivery stimulator acting from outside the body. The versatile approach demonstrated in the present work might be beneficial to various drug delivery applications.

Figure 4. Physical characterization of the nanofibrous matrices (A) Degradation profile of SF and SF-IONP matrices under in vitro conditions with and without additional protease XIV treatment observed for 21 days. The study represents integral stability of the matrices in absence of protease showing no significant mass change. (B) Swelling ratio of SF and SF-IONP shows


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high water uptake capacity of both the matrices particularly that of the SF when compared to SFIONP. (C) Release profile of Ciprofloxacin drug from coated magnetic SF-IONP matrix through application of a range of frequencies of actuation by magnetic actuator device (MAD). Highest release was observed when the frequency was set to 1 second in the on/off cycle (ton/off = 1 s). (D) Schematic representation of the results obtained from release experiment. Drug release profile showing higher drug release at high actuation frequency (ton/off = 1 s) and lower release at lower frequency. Biocompatibility of magnetic nanofibrous matrix. Biocompatibility of SF-IONP matrix is a very important property to look into prior to developing cell culture substrates for actuation studies. To examine cellular compatibility under in vitro conditions, we cultured HDF cells on the matrices in static condition (without MAD stimulation). The proliferation index of cells in both SF and SF-IONP matrices was found to be close to those on TCP as determined by MTT assay (Figure 5A). Proliferation of cultured cells with respect to time corroborated good biocompatibility of the nanofibrous matrices. MTT assay measures the metabolic activity of cultured cells on matrices and is also considered as an indirect way to examine growth of cells.34 Both types of silk matrices (SF and SF-IONP) were found to be compatible for culturing cells. This also ascertained the fact that IONP embedded within silk nanofibers does not hamper its cytocompatibility property. To further examine the response of immune cells under in vitro conditions, macrophage cells were cultured on nanofibrous matrices for 7 days. Secretions of TNF-α cytokine from the cultured macrophage cells (RAW 264.7) was quantified on day 1 and day 7 (Figure 5B). There was no significant difference among the silk samples and TCP on both time points (day 1 and 7). LPS was taken as the positive control and it induced significantly higher secretion of TNF-α on day 1 and day 7 revealing immunogenic response towards LPS.



TNF-α secretion from the LPS containing wells (2316.31 ± 25

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pg/mL) was found to be

significantly higher than secretions from other samples on day 7, p ≤ 0.01. SF matrix showed TNF α secretion (~1650 ± 44 pg/mL) almost equivalent to that of SF-IONP (1727 ± 30 pg/mL) on day 7. Increased TNF α secretions during in vitro assays suggest high inflammatory response and vice versa.31 Thus, SF and SF-IONP silk based matrices showing less TNF α secretion exhibited non-inflammatory property during in vitro cell culture. Biocompatibility of SF has been well proven for tissue engineering application in various studies.23,24,26 SF, as a biomaterial has also been approved by the U.S. Food and Drug Administration (FDA) for biomedical applications.43 Moreover, magnetic particles have also been reported as non-toxic compounds as studied by various cellular assays.14,44,38 Such magnetic particles have also been used for activating stem cells and labelling mammalian cells in cell tracking systems.45,46 Thus, SF-IONP nanocomposite could prove to be a compatible matrix without effecting the cellular viability and proliferation. Effect of magnetic actuation on HDF cells by MAD. To evaluate the proliferation rate of cells under magnetic field offered by our developed device, alamar blue assay was performed (Figure 5C). SF-IONP matrix cultured with HDF cells placed over MAD under magnetic actuations twice per second were compared with SF matrix under non-stimulated conditions. The difference in the proliferation rate of cells cultured on SF and SF-IONP matrices was less pronounced until day 7. However, the proliferation rate of HDF on SF- IONP matrix was found to be significantly higher on day 10 and 14 in comparison to those on SF matrix, p ≤ 0.01. Higher proliferation on IONP impregnated matrices also vouched for its biocompatibility under alternating magnetic field environment. The observation established the fact that the magnetic effect produced by MAD was sufficient to actuate cells. Effect of magnetic field for biomedical


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applications and tissue regeneration have been studied in various reports.47,48 Magnetic field induced cell sheet construction, magnetic cell seeding and cell expansion procedures have been developed using magnetic nanoparticles.49,50 The composite poly(D, L-lactide) (PLA) films of magnetic particle along with hydroxyapatite nanoparticle induced faster differentiation of osteoblast cells in one of the studies.51 In other studies, weak magnetic fields promoted healing of bone fracture and bone ingrowth.52,53 Effect of magnetic field for wound healing promotion under in vivo conditions have also been well explored in a previous study.54 Such studies have inspired the development of magnetic responsive scaffolds in various biomedical applications.55– 57

Hence, the preliminary study conducted on fibroblast cells under pulsed stimulation of

magnetic field offers clues on substantial benefits of actuation by MAD.



Figure 5. Biocompatibility assessment of SF-IONP matrix under in vitro conditions. (A) MTT assay of HDF cultured nanofibrous matrices show significant growth of cells on all the samples with respect to time. (B) Macrophage response under in vitro conditions show significantly lower tumor necrosis factor alpha (TNF-α) secretion on the matrices in comparison to positive control lipopolysaccharide (LPS at 1000 ng concentration) on both day 1 and 7 hence demonstrating no additional immune response due to iron oxide nanoparticles (IONP) incorporation in silk nanofibers. (C) Proliferation of human dermal fibroblast (HDF) cells cultured on matrices under actuation by magnetic actuator device (MAD) for 14 days showing significantly higher proliferation profile on SF-IONP magnetic matrix on day 10 and day 14, p ≤ 0.01. Proliferation and viability of cultured cardiomyocytes. To further investigate the modulation of cell behavior by MAD, we cultured cardiac muscle cells on magnetic silk platform under pulsed magnetic actuations. External magnetic field is considered to be highly beneficial to muscle type cells due to improvement in their contraction mechanism.3 Therefore, as a case study, effect of actuations were examined for cardiomyocytes with the hypothesis of developing an artificial cardiac patch. Cellular studies aimed for cardiac tissue engineering are generally described by increased cellularity and elongated morphology of the cardiomyocytes.27 In our studies, alamar blue and viability test were performed to evaluate potential of the fabricated matrices for culturing cardiomyocytes. Assessment of cellular viability via live dead assay at day 7 revealed high cellular attachment of NRCMs and H9c2 cells onto the SF and SF-IONP matrices (Figure 6A). Elongated morphology was exhibited by the cardiomyocytes growing on the nanofibrous silk matrices. Alamar blue assay attested the superior proliferative capacity (p ≤ 0.01) of both H9c2 cells (Figure 6B) and NRCMs (Figure 6C) growing on magnetically stimulated SF-IONP matrices. Several studies in the past have enumerated the effect of external


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forces on cell adhesion which involves the integrin and the focal-adhesion kinase (FAK) pathways.58,59 Such conditions activate the FAK pathways and their downstream signaling (talin and vinculin) that results in cytoskeletal reorganization and attachment.58 Therefore, it could be speculated that the superior proliferative capacity of the cardiomyocytes growing on the stimulated SF-IONP would have resulted in activating the integrin/FAK pathways thus resulting in greater cell attachment in comparison to only SF matrices. The results also implied that stimulated matrices had an immediate effect in cell attachment and proliferation.

Figure 6. Cell viability analysis (A) Live dead analysis of H9c2 and neonatal rat cardiomyocytes growing on electrospun matrices. Scale bar- 400 µm. Cell Proliferation analysis using alamar blue assay for (B) H9c2 cells and (C) Neonatal rat cardiomyocytes (NRCMs) cultured on nanofibrous matrices under actuation by magnetic actuator device (MAD). Effect of magnetic actuation on cardiomyocytes: gene expression analysis and immunostaining. Previous studies on the role of external stimulations in maturation of



cardiomyocytes have attested the phenotypic transition of the neonatal cardiomyocytes into adult-like cells.60–62 In this regard, gene expression of cardiac specific markers connexin 43 and cardiac Troponin I was studied for differentiated H9c2 cells growing on SF and SF-IONP nanofibrous matrices under continuous magnetic stimulation to ascertain cardiomyocyte maturation. The frequency of stimulation was set so as to achieve magnetic actuation twice per second. This alternate stimulation was provided for a period of 14 days during the culture conditions. Analysis using a comparative 2-∆∆Ct method revealed higher expression (~1.3 folds, p ≤ 0.01) of connexin 43 gene at day 14 for differentiated H9c2 cells growing under actuation conditions in comparison to those growing in non-actuated matrices (Figure 7A). Higher expression of connexin 43 implies greater intercellular coupling within the differentiated cells which would help promote synchronous excitation.63 Expression of cardiac troponin I was also found to be higher for 14 day culture in comparison to day 7 (Figure 7B), confirming the maturation of the seeded cultures growing on the nanofibrous matrices. Higher troponin I expression of H9c2 cells growing on SF mats without actuation in comparison to SF-IONP with actuation is probably due to the presence of greater number of differentiated cells at initial stage under static conditions compared to actuated conditions. Although, differentiated H9c2 cells and neonatal cardiomyocytes have similar responses, H9c2 cells do not exhibit any beating characteristics.64 Therefore, further gene expression studies were performed using NRCMs which exhibited beating activity post isolation. In order to exclusively understand the effect of magnetic actuation on the maturation of cardiomyocytes, the expression of connexin 43 and cardiac troponin I with respect to housekeeping gene GAPDH was studied for NRCMs growing on SF-IONP matrices both in the presence and absence of external magnetic stimulations using 2-∆Ct method (Figure 7C and 7D).


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Higher expression of both connexin 43 (p ≤ 0.01) and cardiac troponin I (p ≤ 0.05) was observed for NRCMs growing on magnetically stimulated matrices in comparison to the unstimulated matrices. Troponin I is known to play an important role in the maintenance of the sarcomeric structure and cellular (sarcomeric) contractions. Pronounced expression of Troponin I gene for cardiomyocytes cultured on stimulated matrices substantiate the development of sarcomeric structures in NRCMs in the presence of external magnetic force. These gene expression results further attested that an external magnetic stimulation would result in greater maturation of cardiomyocytes in comparison to the non-stimulated culture matrices.

Figure 7. Gene expression analysis of cardiac specific markers: Real time polymerase chain reaction analysis for (A, B) H9c2 cells seeded on SF-IONP and SF nanofibrous matrices depicting culture conditions with and without actuation by magnetic actuator device (MAD)



respectively. (C, D) Neonatal rat cardiomyocytes (NRCMs) seeded on the SF-IONP matrices in the presence and absence of actuation by MAD. Previous studies have indicated the capability of magnetic actuation itself to cause alterations in the actin-myosin tension levels and in cytoskeleton reorganization.3 Also, such low frequency actuations are known to play a protective role in H/R injury. In this regard, SF-IONP nanofibrous matrices seeded with NRCMs were further analyzed via immunocytochemistry to realize the effect of magnetic stimulation (magnetic actuation twice per second for a period of 7 days) in the organization and expression of the cytoskeletal proteins. Confocal image analysis on staining the cultured matrices with sarcomeric alpha actinin and connexin 43 revealed the presence of mature sarcomeric structures in NRCMs growing on stimulated SF-IONP matrices in comparison to non-stimulated ones (Figure 8A). Presence of mature sarcomeric striations are the characteristics of adult like cardiac phenotype and indicate the cardiomyocyte maturation on magnetic actuation.65 Connexin 43 expression revealed the cardiomyocytes growing on stimulated matrices to be better coupled to each other resulting in transfer of beating stimulations among the cardiomyocytes. The confocal images were further analyzed using ImageJ to infer the effect of external magnetic actuation on sarcomeric structures (Figure 8B and 8C). The presence of alternate stimulation for a period of 7 days resulted in a more organized structure with well-spaced bands as opposed to non-stimulated samples. However, no significant difference was found in the actinin area/cell. Cardiomyocytes growing on non-stimulated matrices exhibited globular shapes in comparison to the stimulated ones, attesting the role of the magnetic actuation in cell maturation and cytoskeletal organization.


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Figure 8. Immunostaining analysis (A) Immunostaining of cardiac specific markers for neonatal rat cardiomyocytes seeded on SF-IONP matrices in the (i) absence and (ii) presence of actuation by magnetic actuator device (MAD). Scale bar-10 µm. (B) analysis of sarcomeric length and (C) sarcomeric area using imageJ. Developing a functional cardiac patch is a complex task as several stimulations both biochemical and external may be required to maintain its functional maturation. In addition, maintenance of beating cardiac cells under in vitro conditions is a herculean task. Magnetic field has been applied to develop oriented dense muscle sheets, strings, and rings from mouse myoblasts in previous studies.3,13 Strings of muscle bundles were produced by culturing magnetically labelled cells under steady immobile magnet.13 Culturing cells on magnetic matrix without labelling the cells and actuating them in a dynamic system might provide an additional



advantage. Through our studies, we have been able to link relationships between magnetic conditional cues and functional response in cardiomyocytes maturation. The current study exhibits tremendous potential of the MAD in modulating behavior of cardiomyocytes. However, further intervention is required for studying their effect on other responses of the cardiomyocytes or other type of muscle cells. Conclusion It is evident from the study that a magnetic actuator developed using microcontroller and electromagnets could stimulate moieties present in the magnetic matrix. Tunable drug delivery was achieved by applying different frequencies to magnetically responsive drug reservoir. A prime consideration while developing the device was to provide magnetic actuation to cells via non-contact mode. The compact and rigid design of the instrument made it possible to culture cells on a magnetic substrate that can be placed in a petri dish above the electromagnets without compromising culture conditions. Results obtained from the study provided a proof of concept in applications of magnetic actuator to accomplish both on-demand drug delivery and stimulate biological systems. Future applications of MAD include stimulation of certain types of muscle cells while developing artificial muscles, or cardiac patch under in vitro laboratory conditions. Response of muscle cells under varied actuation frequency and several actuation modes might also be an interesting topic to look into and can be performed using this device. It can also be applied in various drug delivery platforms and to study self-healing structures in response to fluctuating magnetic field. The non-invasive actuation model reported in this study not only helped in stimulation of cells seeded on magnetic matrix but also could render tunable release properties of drugs coated on the same. The current findings thus provide insights


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towards developing an easy and versatile approach to stimulate cells via magneto-mechanical actuation.

Acknowledgements BBM

greatly

acknowledges

Department

of

Biotechnology

(DBT,

Grant

#

BT/PR25081/NER/95/1000/2017, BT/548/NE/U-Excel/2014) and Department of Science and Technology (DST), Government of India for generous funding. BBM wishes to thank Dr. Samit K. Nandi (WBUAFS) for providing primary cardiac tissues. Dr. Rocktotpal Konwarh is acknowledged for helping with TEM images. DC wishes to thank Mr. Namit Dey for his generous help and valuable suggestions. Central Instruments Facility and Department of Biosciences and Bioengineering, IITG are highly acknowledged for providing high-end instruments.

SUPPORTING INFORMATION

Figure S1. TEM images of IONP embedded in the silk nanofiber.

Figure S2. Magnified hysteresis curve of SF-IONP matrix.

References

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Magnetic actuator device assisted modulation of cellular behavior and tuning of drug release on silk platform Dimple Chouhan, Shreya Mehrotra, Omkar Majumder, Biman B. Mandal* Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India

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Magnetic actuator device assisted modulation of cellular behavior and

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