Effect of patterned polyacrylamide hydrogel on morphology and orientation of cultured NRVMs AbstractWe recently demonstrated that patterned Parylene C films could be effectively used as a mask for directly copolymerizing proteins on polyacrylamide hydrogel (PAm). In this work, we have proved the applicability of this technique for studying the effect such platforms render on neonatal rat ventricular myocytes (NRVMs). Firstly, we have characterised topographically and mechanically the scaffolds in liquid at the nano-scale level. We thus establish that such platforms have physical properties that closely mimics the in vivo extracellular environment of cells. We have then studied the cell morphology and physiology by comparing cultures on flat uniformly-covered and collagen-patterned scaffolds. We show that micro-patterns promote the elongation of cells along the principal axis of the ridges coated with collagen. In several cases, cells also tend to create bridges across the grooves. We have finally studied cell contraction, monitoring Ca2+ cycling at a certain stimulation. Cells seeded on patterned scaffolds present significant responses in comparison to the isotropic ones. IntroductionThe native cardiac tissue is characterised by a high anisotropic architecture that is supported by the parallel alignment of cells. The extracellular matrix (ECM) of the myocardium provides structural and biochemical cues helping cell organisation in a contractile tissue. The anisotropic architecture facilitates the preferential propagation of the electrical signal in one direction1 and increases the conduction velocity along the tissue2. A wide range of soft materials, such as elastomers or hydrogels, was exploited to date for synthesising the artificial ECM, with hydrogels gaining significant interest in cardiac tissue engineering3. These materials have characteristic properties and particularly their ability to reversibly tailor their mechanical properties (0.1鈥?00鈥塳Pa) by varying mainly the chemical composition4,5. Also, they can be dynamically stimulated by a variety of stimuli, such as electric field, pH, and light, and thus provide excellent prospects for controlling the dynamic extracellular environment that cells feel in vivo6,7.Polyacrylamide (PAm) has been one of the most exploited gels for studying the mechanotransduction of cardiomyocytes8 because it is easy to make and control in stiffness. NRVMs maturation on these materials has shown a significant dependency on the hydrogel stiffness9. Cells on soft PAm substrates (1鈥?0鈥塳Pa) exhibit a mature phenotype, a well-organised cytoskeleton and a reasonable excitation threshold9,10. The magnitude of calcium stored in the sarcoplasmic reticulum and the expression of the sarcomeric calcium pump (SERCA2) were found to be greater on substrates with the elastic modulus of 10鈥塳Pa (calcium storage of almost 0.08鈥塵M) in comparison to cells seeded on 1鈥塳Pa substrates9 (calcium storage of almost 0.03鈥塵M). Topography has a large influence on cell alignment; cardiac cells are sensitive to both nano- and micro-scale topographies11,12. Heidi Au et al. have demonstrated that cell alignment was greater on 0.5鈥壜祄 deep grooves than 3鈥壜祄 deep grooves13. Other studies demonstrated an improving into the alignment of cell-seeded on patterned polyethylene glycol (PEG) hydrogels (800鈥塶m width grooves and ridges) in comparison to unpatterned constructs14.Nevertheless, hydrogels have interesting properties for application in cell culturing, cells typically adhere poorly to them, and the functionalization with adhesion proteins is necessary15. Patterning synthetic hydrogels are not straightforward. In the particular case of PAm, two main techniques are generally employed for patterning biomolecules: (1) covalently attaching proteins on activated regions5 and (2) copolymerizing of ECM proteins directly onto the gel16 via microcontact printing (碌CP). The latter involves the preparation of an elastomeric stamp generally made of polydimethylsiloxane (PDMS) that is directly printed atop of PAm pre-polymer solution. Di Benedetto et al.17 have demonstrated that this technique is possible to transfer the anisotropic pattern on PAm and have an anisotropic replica with grooves and ridges wide from 2 to 100鈥壜祄. However, compared to PDMS, Parylene C is typically preferred for patterning PAm hydrogels for two main reasons. Firstly, Parylene C mould preparation does not require complicated microfabrication steps. Secondly, Parylene C has a lower oxygen permeability than PDMS18, and this allows an efficient preparation of PAm, whose polymerisation is inhibited by the presence of free oxygen radicals. A photoresist lift-off patterning method was also recently reported for patterning biomolecules on PAm with high fidelity15. Moreover, while this technique allows for the control of the shape and size of epithelial cells, it remains as non-trivial.In that direction, we recently proposed a simple and versatile method for patterning PAm hydrogel scaffolds either topographically and biochemically using Parylene C masks19. Herein, we leverage this approach and demonstrate its use in NRVM culturing. We particularly focus on studying cell elongation, morphology, and orientation on flat PAm uniformly-covered with collagen (FPC) and grooved collagen-patterned PAm (GPC). Overall, we demonstrate that the patterns on GPC scaffolds promote significantly cellular alignment, while an increase of the calcium activity is also recorded.Materials and MethodsCollagen IV preparation5鈥塵g of type IV human placenta collagen (Sigma-Aldrich, C7521) was reconstituted in 5鈥塵L of sterile 1 X phosphate buffered saline (PBS) and let mix on a roller shaker for 3鈥塰ours. The solution was then split into five aliquots of 1鈥塵L and stored at 鈭?0鈥壜癈.Copolymerization of collagen IV on a polyacrylamide gelFlat polyacrylamide covered with collagen (FPC) and grooved collagen-patterned polyacrylamide (GPC) were compared to demonstrate that the technique showed in a previous work19 can be applied to mimic in the vitro cardiac extracellular environment and promote NRVM alignment. In this work, we describe only the fabrication of FPC since the fabrication of GPC was explained in19. The spacing value of GPC (width of the ridges and grooves and depth of the grooves) was chosen as the optimum pattern for NRVMs based on the literature (i.e., 10鈥壜祄 width and 1鈥壜祄 deep grooves)20.Briefly, this method relies on the copolymerization of collagen IV into the gel during the direct contact of the acrylamide precursor mix with a protein-activated Parylene C films. Glass coverslips (13鈥塵m in diameter) were washed in acetone, isopropanol, and deionised water and then dried using nitrogen gun. Parylene C films (6鈥?鈥壜祄 thick) were then deposited on the coverslips (Fig.聽1a) by chemical vapour deposition using a commercially available parylene coater (PDS2010). The silane 3-trimethoxysilylpropyl methacrylate (A174) was used to improve the adhesion of the Parylene C film on the glass. Samples were treated with O2 plasma for 1鈥塵in and 40鈥塻 using an inductively coupled plasma reactor (ICP, OPT 100 ICP 380, Oxford Instruments Plasma Technology) (Fig.聽1b). Plasma was generated at a pressure of 1.33鈥塒a, flow 100 sccm, 1000鈥塛 source power, and 20鈥塛 bias generator power. The time of exposure to plasma was identical to the one used for preparing the patterned Parylene C masks with hydrophobic/hydrophilic regions19. 10鈥壜祃 of protein solution was then spread on the activated Parylene C films and masks were let dry under a fume hood for 30鈥塵in (Fig.聽1c).Figure 1Sketch of the functionalization of flat polyacrylamide hydrogel with collagen (FPC) and cell culture application. Parylene C is placed on glass coverslips (a), treated with oxygen plasma (b) and uniformly coated with collagen (c). PAm pre-polymer solution is gently pipetted on activated glass coverslips and Parylene C mask is put in contact with the solution (e). After hydrogel polymerisation, the mask is removed (f), and FPC samples are stored in PBS (g). In (h) and (i) top and section views of FPC and GPC are shown with cells randomly distributed in FPC and aligned in GPC.Full size image5鈥塵L polyacrylamide hydrogel precursor with a total polymer content of 8.48% (w/v) and crosslinker concentration of 5.66% (w/w) was prepared. The solution was degassed for 1鈥塰 to remove all the dissolved gas that could limit the free radical polymerisation. To initiate gelation, 3鈥壩糒 of 10% w/v ammonium persulfate (APS, Sigma-Aldrich, A3678) was added to 300鈥壩糒 of gel precursor solution followed by 0.3鈥壩糒 of N, N, N鈥? N鈥?Tetramethylethylenediamine accelerator (TEMED, Sigma-Aldrich, T9281). A 60鈥壩糒 of gel precursor mix was gently pipetted on activated glass coverslips5 and sandwiched with protein-functionalized Parylene C masks (Fig.聽1e). Gels were then left to polymerise at room temperature for one h (Fig.聽1f). After polymerisation, masks were then removed and the gels were stored in PBS at 4鈥壜癈 (Fig.聽1g). Constructs were directly transferred in sterile tissue culture petri dish covered with cell culturing medium (Fig.聽1h,i).Contact angle measurementsParylene C (6鈥?鈥壜祄) was placed as described previously on glass coverslips of 13鈥塵m of diameter. PDMS was instead spin coated at 6000鈥塺pm (~ 8鈥壜祄) on glass coverslip of 13鈥塵m of diameter and cured in an oven at 75鈥壜癈 for 1鈥塰our. Sessile deionised water 10鈥壜祃 droplets were gradually engaged on the surface of Parylene C and PDMS to measure the static contact angle. A Drop Shape Analysis System (DSA 30 Kruss Co., Germany) was employed for the calculation of the contact angle. A polynomial function was fitted to the two-3-phase sections of the profile in the region of the baseline. One measurement per three samples per each category was performed, and the average values were extrapolated. Samples were blown with nitrogen gun before each measurement. Samples were stored at room temperature (22鈥壜癈) and low humidity level (35%).Topography characterisationThe topography of flat polyacrylamide was visualised using environmental variable-pressure (VP) scanning electron microscopy (SEM) (Zeiss EVO 50XVP). Almost 100鈥壜祄 thick PAm was fixed on glass coverslips as described above and it was hydrated overnight in deionised water. Samples were speckled gently with Kimwipe and loaded atop SEM stubs using carbon tape. VPSE detector at 20鈥塳V acceleration voltage was used. Measurements were taken in a reasonable time to preserve the gradual loss of water of the gel over time. The topography of Parylene C mask and PAm was evaluated using a stylus profiler (KLA-Tencor) and by using an optical microscope (Zeiss Axio Lab) at 50x magnification. Three measurements on three samples were taken for the statistical analysis.Mechanical characterisation at the nano-scaleFlat polyacrylamide samples were prepared using the polymerisation between two activated coverslips as described elsewhere5. They were stored either in PBS or water at 4鈥壜癈 before the nanoindentation tests. Our experimental setup for measuring the mechanical properties is a hybrid system made of a commercial head (SMENA, NT-MDT) with home-built electronics. Hydrogels were tested both in water and in PBS.A spherical tip of 5鈥壜祄 of diameter (sQUBE, CP-CONT-BSG-A) was used to minimise the indentation. Cantilever stiffness calibration was done following the work of Sader et al.21, and it was estimated to be 0.2鈥塏/m with an error of 10%. Samples were placed in a petri dish of 35鈥塵m of diameter filled with liquid. The approaching/retraction rate used was 400鈥塶m/s.Since the hydrogels showed a negligible adhesion, we have analysed the force-distance curves using the Hertz model. Young鈥檚 modulus can be thus deduced from the equation聽1 as follows22:$${F}_{N}=frac{4Esqrt{R{delta }^{3}}}{3(1-{v}^{2})}$$ where FN is the maximum force applied, E is Young鈥檚 modulus, R is the radius of the spherical tip, the indentation depth is 未鈥?鈥?z 鈭?d) where z is the piezo position and d is the cantilever deflection. 谓 is the Poisson鈥檚 ratio equal to 0.4823.NRVM isolation and cultureThe study with NRVMs was exempted from formal ethics review. NRVMs were isolated from Sprague-Dawley rats two days after birth in compliance with Schedule 1 methods (Scientific Procedures) Act 1986. The isolation technique has been described previously11,24. Briefly, hexempteart ventricles were minced and enzymatically digested. After the digestion, the tissue was centrifuged to produce a cell suspension which was filtered to remove any non-digested tissue. Cells were then suspended again in 25鈥塵l NRVM medium (67% Dulbecco鈥檚 modified Eagle medium (DMEM), 16% Medium 199, 10% Horse serum (Gibco), 4% foetal bovine serum (FBS) (Gibco), 2% HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) buffer and 1% penicillin-streptomycin). The suspension was then plated in T75 flasks for 1鈥塰our at 37鈥壜癈 to remove fibroblasts. NRVMs were collected and counted using a hemocytometer. NRVMs were plated on FPC and GPC substrates, and they were successively incubated (37鈥壜癈, 5% CO2). The NRVM medium was replaced every 2鈥? days. All experiments were performed 3鈥? days post seeding.ImmunostainingMonoclonal anti-伪-actinin antibody (Sigma, A7811), deoxyribonucleic acid labeled with fluorescent 4鈥?6-diamidino-2-phenylindole (DAPI) (Invitrogen) staining were used to quantify cell alignment. Fluorescent images were obtained using an inverted Zeiss LSM-780 confocal microscope with x40 oil-immersion lens (Carl Zeiss).Quantification of cellular alignment, morphology, and orientationEach cell was approximately fitted with an ellipse whose minor (Dmin) and the major axis (Dmax) was defined as the length and width of individual myocyte. Dmax of each cell was selected relative to the horizontal axis of the image field. Elongation was described through the elongation factor (Dmax/Dmin 鈭?1) that describes the extent of the equimomental ellipse lengthened or stretched out25. The values of the elongation factor were determined from each scaffold on almost n鈥?鈥?5 cells randomly selected from each sample.Cell area and circularity were analysed to study cell morphology using dedicated software, ImageJ (http://rsb.info.nih.gov/ij). Circularity is defined in the equation聽2鈥?sup>26:$$Circularity=frac{4pi A}{{P}^{2}}$$ where P is the perimeter and A is the cell area, quantified using ImageJ. Circularity index equal to 1 indicates a non-elongated cell.The cell orientation angle (胃) was measured manually using an ImageJ software package (http://rsb.info.nih.gov/ij) as the lack of deviation between the major elliptical axis and the reference axis of a single cell. For alignment of cells on grooved substrates, the direction of the pattern (preferential mean axis) was considered as a reference axis, whereas for alignment of cells on the flat substrates an arbitrary axis of alignment was chosen. The minimum alignment value close to 10掳 denotes parallel alignment with the direction of the pattern, and the maximum value close to 90掳 represents perpendicular alignment. Cells with an orientation angle of less than 10掳 were counted as aligned cells27. Orientation angles are reported in polar plots obtained using ready-made routines in MATLAB.Nuclei orientation was quantified as described previously via conversion of the DAPI channel images into binary images using ImageJ11 to recognise any ellipse (nuclei) present with the size set to 10鈥?0鈥壜祄. Alignment was defined as the lack of deviation in the axis of an individual nucleus from the mean axis of all individual nuclei11. The ellipses were only counted if they were between the set range to exclude non-nuclei or composite structures from the analysis. At least 90 cells were randomly selected per each category of scaffolds to determine cell shape and orientation. Each analysis was performed on fluorescence images (three images per each scaffold) at the same magnification.Ca2+ transient measurementsFluo-4-acetoxymethyl ester (fluo-4 AM) (Invitrogen, ThermoFisher Scientific) was used to visualise the intracellular calcium transients. NRVMs were loaded with 4鈥壩糒 fluo-4 AM (prepared as 50鈥壜礸 Fluo-4 AM dissolved in 50鈥壜礚 DMSO) in 1鈥塵L DMEM and placed in the incubator at 37鈥壜癈 for 20鈥塵in11,20. The DMEM was refreshed, and the cells were returned to the incubator for a further 20鈥塵inutes for de-esterification of the dye. The constructs were mounted on the stage of an upright Nikon Eclipse FN1 microscope or an inverted Nikon Eclipse TE2000 microscope in a glass bottom dish (MatTek Corporation) and observed through a 40x water immersion or 40x oil objective respectively. Ca2 transients in cardiomyocytes were studied by field stimulating the cells at 1鈥塇z to induce rhythmic depolarisation, and line scans were recording. A custom made MATLAB code was used to calculate the normalized amplitude as f/f0, time to peak (Tp), times to 50% (T50) and 90% declines (T90) in the transients28.Statistical analysisAll the described experiments were repeated at least three times. With regards to topography, nonparametric analysis was carried out among samples to statistically evaluate the significant difference between ridges and grooves of the mask and the gel, respectively. A Mann-Whitney test was also performed to compare the two groups, and the significance was fixed at 1%.For statistical analysis of cell elongation, morphology and orientation, three different images were acquired per each sample to analyse at least 80 cells for statistical analysis.Mann-Whitney test was employed for evaluating cell elongation, circularity, and area whereas nuclei alignment and calcium transient were performed using an unpaired t-test. Cell area and circularity are presented as box plots, and each box is composed of whiskers indicating the lower extreme and upper extreme of the data, while the top part and the bottom part of the box are the first and third quartiles. The line inside the box is the median. In the plots, *indicates p鈥?lt;鈥?.05 and **indicates p鈥?lt;鈥?.01. The statistical analysis and graphic presentation were performed using OriginPro v.8 SR2 software and Prism 7 software (GraphPad Software Inc.). All data are expressed as a mean鈥壜扁€塻tandard error of the mean.Data AvailabilityThe data that support the findings of this study are available from the University of Southampton institutional repository at: https://doi.org/10.5258/SOTON/D0605.Results and DiscussionHydrogel patterning and characterisationThe anisotropic pattern on PAm was obtained using capillary-based approach17 thanks to capillarity of the pre-polymer solution in hydrophilic vertical channels created in Parylene C masks. Parylene C after plasma oxygen restores its hydrophilicity slower than PDMS29. The feature height on the hydrogel strongly depends on the contact angle, the hydraulic radius of the capillary and the viscosity of the pre-polymeric solution17. We have monitored the contact angle of flat Parylene C and PDMS with the same thickness (~7鈥壜祄) using ICP and reactive ionic etching (RIE). ICP treatment was used to hydrophilize Parylene C for 1鈥塵inute and 40鈥塻econds and RIE was used to treat PDMS with oxygen for 15鈥塻ec. The time of exposure and the kind of treatment were decided based on the fabrication process that has been employed for the two materials. Figure聽2a,b show 10鈥壜祃 water drop on Parylene C and PDMS immediately after plasma treatment. The contact angle of Parylene C and PDMS before plasma oxygen are 84.2鈥壜扁€?.6掳 is 117.4鈥壜扁€?.8掳 respectively. Parylene C restore its hydrophobicity slower than PDMS29,30, and we found that after 6 days the contact angle for Parylene C and PDMS was 36.07鈥壜扁€?.66掳 and 64.97鈥壜扁€?.72掳 respectively. In Fig.聽2c the measurements of the contact angle for six days are shown.Figure 2Contact angle measurements. In (a) and (b), images of 10鈥壜祃 DI water droplets on Parylene C and PDMS at day 0, immediately after the plasma treatment. In (c) the graph reports contact angle measurements as a function of days.Full size imageThe surface of flat polyacrylamide was obtained avoiding surface aberration, and the native hydrate state of the gel was preserved31. In this work, we have used PAm fixed on glass coverslip with a defined formulation to fix the structure and the elasticity of the material5. PAm SEM images generally present pores and voids on the surface because of the failure of the structures during preparation for imaging (e.g., cryo-freeze-dry) and the volume fraction and the distribution of polymeric chains can change accordingly32. The hydrogels were fixed on glass coverslips obtaining a uniform film of almost 300鈥壜祄 of thickness (Fig.聽3a.1). The topography of flat PAm prepared with T鈥?鈥?.48% (w/v), and C鈥?鈥?.66% (w/w) was checked using SEM as illustrated in Fig.聽3a.2, and it results more wrinkled and jagged without evident porosity since the gel was checked immediately after removing it from water and it was almost hydrated.Figure 3Surface VP SEM photograph of flat hydrogel surface and an optical micrograph of the patterned hydrogel. The image (a.1), shows a hydrogel fixed on the glass coverslip and (a.2) is a representative SEM image of flat polyacrylamide. In (b) is shown a micrograph (50x objective) of grooved PAm characterised by pitches (p), ridges (r) and grooves (g) after removing the Parylene C mask. (Red scale bar: 1鈥塩m. White scale bar: 20鈥壜祄).Full size imageThe comparison between the topography of the mask and the hydrogel after patterning was also evaluated extending the results already reported in our previous work19. Grooves (g), ridges (r) and pitches (p) of the hydrogels in the hydrated state are illustrated in Fig.聽3b. The average values of g, r, and p of the masks were here analysed using the profiler, and they were 6.88鈥壜扁€?.6鈥壜祄, 6.32鈥壜扁€?.1鈥壜祄 and 20.62鈥壜扁€?.1鈥壜祄 respectively. The average height of the grooves of the mask was found to be 0.99鈥壜扁€?.04鈥壜祄.The averaged values of the width of g, r and p of the hydrogels were found to be10.1鈥壜扁€?.05鈥壜祄, 7.8鈥壜扁€?.24鈥壜祄 and 19.7鈥壜扁€?.28鈥壜祄 respectively. The comparison between the ridges of the mask and the grooves of the gels was not significant with p鈥?gt;鈥?.01. The average height of the grooves of the hydrated hydrogels was evaluated using the profiler (low force of ~1.96鈥塵N), and it was almost 1鈥壜祄, close to the height of the grooves of the mask.Elastic modulus valuesNanoindentation tests were performed on flat PAm samples as illustrated in Fig.聽4a. These tests were necessary to quantify Young鈥檚 modulus of the substrates, important parameters for cell elongation and function5. They cannot be carried out on the microstructured ones since the radius of the AFM tip was comparable with the width of the grooves. As already mentioned sharper tips would induce plastic deformation on such soft materials. In any case, the contact area between an AFM tip and a steep ridge can never be defined due to the geometry of the system.Figure 4Elastic modulus evaluation. The draw of AFM indentation in liquid (a) and force-indentation curves (b,c) obtained from flat PAm samples underwater (a) and PBS (b).Full size imageTypical load-displacement curves for PAm show a region of the negative load in the unloading portion due to the adhesion between tip and sample (Fig.聽4b,c). Measurements under water and PBS present negligible adhesion force values. Both curves do not present jump to contact and show a linear elastic behaviour. Thus in both cases, the Hertzian model can be applied. The E value was found to be 37.1鈥塳Pa for PAm measured in water, and 22.9鈥塳Pa for PAm measured in PBS. The difference might be due to local changes in the gelification process. However, the two values fall in the desired range for our application.Cell morphology and alignmentIn this work, we have applied a method previously reported to functionalize polyacrylamide hydrogel with collagen IV using Parylene C masks19. Polyacrylamide without collagen treatment was cytotoxic for NRVMs as reported in Fig.聽S1 of Supplementary Information. As expected, cells on substrates without adhesion protein display an apoptotic behavior because of non-adhesiveness properties of PAm hydrogel15. Therefore, adhesion protein is necessary for improving cell adhesion. With our proposed technique we have previously demonstrated that protein is completely transferred from selectively hydrophilized Parylene C mask onto the hydrogel19. In this way, we can obtain a more biocompatible polyacrylamide based scaffolds using a simple and non-toxic technique. In this paper, we supplement our previous findings with the application of this technique towards NRVM culturing.PAm hydrogels are widely used as a scaffold in cell biology especially for cardiomyocytes since it has mechanical properties close to the extracellular matrix of the heart. However, most of the techniques used to pattern this hydrogel are toxic and not easy to carry out16,33. For this reason, we considered the application of this technique for cardiac tissue engineering creating biomimetic platforms that can be applied in synthetic biology. The comparison between isotropic and anisotropic substrates is made to demonstrate the effects of these platforms on cell morphology, orientation, and activity. For each cell, a best-fitted ellipse was outlined with a minor and major axis. A certain orientation (胃) of the major axis concerning the reference axis was considered as shown in Fig.聽5a.Figure 5Cell morphometric analysis. Sketch of cell morphology and orientation along the collagen stripe (a). Dmax and Dmin are the long and short axis, 胃 the angle of orientation of the cells. Cell elongation is quantitatively evaluated through the elongation factor (b). A significant elongation between the two types of samples is evident in (b). Box plots of cell areas (c) and scatter box plots of circularity distributions (d) are shown in the bottom panel. A significant difference was found in cell circularity between cells seeded on FPC and GPC. Notes: *p鈥?lt;鈥?.005, each box has its ends at the quartiles, and the band within the boxes is the median of the distribution ((c), (d)).Full size imageCellular alignment plays an important role in functional and physical characteristics of many tissues, especially for the heart. To demonstrate the application of our substrates on cell elongation quantitative data were extracted. The elongation factor is commonly used to quantify cellular alignment in tissue engineering. Generally, this parameter assumes 0 value for a perfect circle and 1 for an ellipse with an axis ratio of 1:234. The results shown in Fig.聽5b demonstrate the efficiency of GPC scaffolds in promoting the elongation of NRVMs. The elongation factor is significantly higher for cells seeded on GPC with an averaged value of 3.75鈥壜扁€?.49 in comparison to cells seeded on FPC that have a lower value (0.70鈥壜扁€?.67). These results prove that collagen-patterns have a significant influence on NRVMs behaviour.Cell area and circularity quantified cell morphology. Figure聽5c shows the distribution of cell areas on FPC and GPC. Cell areas on FPC are more positively skewed with more dispersed values. The average area of cells on FPC was found to be 558.04鈥壜扁€?28.33鈥壜祄2 in comparison to the area of cells seeded on GPC (average area: 480.04鈥壜扁€?60.01鈥壜祄2). The evaluation of the cell circularity is reported in Fig.聽5d. Cells on FPC were more circular as presented in a whisker plot where data are negatively skewed with an average value of 0.85鈥壜扁€?.14. Cells on GPC present a less circular morphology, and data dispersion is narrower and slightly positively skewed with an average value of 0.50鈥壜扁€?.12. The distributions of circularity denote a significant decrease when cells are seeded on patterned substrates (p鈥?lt;鈥?.005).Cellular orientation alongside the direction of the anisotropic pattern was evident from optical images. We confirmed and quantified the orientation of cells using polar plots to show the difference in orientation of NRVMs on the two type of samples. In the polar plots, the azimuth ranges from 鈭?0掳 to 90掳, and the 0掳 corresponds to the elongation direction of the pattern. A perfectly circular cell would be at the origin, and long and thin cells are far from the origin. Cells on FPC showed a random distribution (Fig.聽6a) with more dispersed values close to the origin of the graph as reported in the graph of Fig.聽6b, whereas cells on GPC have the preferential orientation alongside the pattern (Fig.聽6c). In the polar plot of Fig.聽6d, there are not points close to the origin, confirming that cells on GPC are more elongate than cells cultured on flat uniformly collagen-patterned scaffolds. The percentage of cells with angle values 15掳鈥?lt;鈥壩糕€?lt;鈥?5掳 on FPC is 8.64% less than on GPC where it was found to be 77.64%. Also, cells with random distribution in uniform samples are 50.6掳鈥壜扁€?3.4掳. On collagen-patterned surfaces, the values of angles are 11.69.2掳鈥壜扁€?.9掳.Figure 6Immunofluorescent staining images, analysis of cell and nuclei alignment on FPC and GPC. Representative immunofluorescence staining of sarcomeric 伪-actinin (red) and nuclei stained with DAPI (blue) on FPC (a) and GPC (c). Relative polar plots of the orientation angle of cells on FPC (c) and GPC (d) are also reported. In this polar plot the distance from the origin, R, is the elongation factor, each point is a single cell (81 cells for FPC and 85 cells for GPC) and the angle to the horizontal, 胃, is the angle the long axis of the cell makes with the reference axis. In (e) is shown the modulus of the angle of nuclei alignment on uniformly covered and collagen-patterned polyacrylamide with **p鈥?lt;鈥?.01.Full size imageThe modulus of the angle of nuclei was also measured using DAPI staining. Results reveal that the modulus of the angle of nuclei from the mean axis is smaller for cells seeded on GPC (31.70掳鈥壜扁€?.16掳) than cells seeded on FPC (44.75掳鈥壜扁€?.81掳) with a considerable alignment to the direction of the pattern as shown by the histogram in Fig.聽6e.The bright field image in Fig.聽7a shows the direction of the lines (red arrow) and also the considerable tendency of the cells to follow the pattern. With these results, we also demonstrate that cells alignment is strongly aimed by cell-ECM interaction so effectively that cells follow the ridges functionalized with collagen (Fig.聽7b) or the bridge across the ridges (Fig.聽7c) to create focal adhesions. Immunostaining for connexin43 (Cx43) showed that gap junctions in sarcomeric 伪-actinin were aligned along the perimeter of the elongated cardiomyocytes cultured on GPC, resulting in spatial distribution of Cx43 primarily oriented parallel to ridges (Fig.聽7d), versus more random distribution in glass control (Fig.聽7e). The cx43 expression on cell aligned is indicative of well-formed intracellular contact35,36. In our case, Cx43 also shows random membrane distribution typical of neonatal cardiomyocytes36.Figure 7Cell tendency to bridge across the anisotropic pattern. Bright field image of NRVMs aligned on GPC (a) and a sketch of cells aligned alongside the features (yellow circle, (b)) and bridges across the lines (green circle, (c)). Immunofluorescence images of cells aligned on GPC and glass control with an expression of connexin43 (green) are shown in (d) and (e) respectively. (White scale bar: 50鈥壜祄).Full size imageCell electrophysiologyDifferent patterning techniques have been employed to improve Ca2+ cycling in NRVMs11,24. It has been shown that microgrooved substrates can increase the diastolic and systolic Ca2+ dynamic properties37. Many studies have also revealed that the substrate stiffness can influence the electrophysiology activity of the cardiomyocytes38. In this work Ca2+ cycling properties of NRVMs was studied fixing the stiffness of the scaffolds and comparing two groups: unpatterned (FPC) and patterned (GPC) constructs. No significant differences were found in the amplitude (f/f0), time to peak (Tp) and time to 50% transient decay (T50) between cells cultured on flat and grooved substrates (Fig.聽8a鈥揷), but there is only a significant increase for the 90% time decay (T90) with p鈥?lt;鈥?.05 (Fig.聽8d). We have shown that fixing the stiffness and improving cell alignment Ca2+ amplitude remains the same (1.03鈥壜扁€?.02 in FPC and 1.04鈥壜扁€?.02 in GPC), but Ca2+ diastolic decay slightly increases on GPC constructs. Since the amplitude in NRVM is the same, there are no significant changes in the sarcoplasmic reticulum (SR) function37 and the calcium releasing through ryanodine receptors (RyR)39. The amplitude and the time to peak can also be attributed to the peak force generated by the cells on the substrates8. Since the stiffness of the substrates has been fixed, cells generate the same force on the substrates with Tp equal to 45鈥壜扁€?5.5鈥塵s for FPC and 45.12鈥壜扁€?.9鈥塵s for GPC. The significant decrease of T90 for cells plated on GPC samples (317.98鈥壜扁€?1鈥塵s) instead suggests that there is an increase in diastolic Ca2+ levels40. Slower calcium transients can augment the contraction as myofilaments are exposed to Ca2+ for a longer period41.Figure 8Ca2+ transient amplitude (f/f0), time to peak (Tp), 50% time to decay (T50) and 90% time to decay (T90) of NRVM cultured on unpatterned (FPC) and patterned (GPC) substrates. In (a) Ca2+ signal is shown as f/f0 with fluorescence intensity, f, normalised to the minimal intensity, f0, measured between 1鈥塇z contractions. In (b) time to peak of Ca2+ release in FPC and GPC. In (c) and (d) transient decay times (T50 and T90) are shown, *p鈥?lt;鈥?.05.Full size imageConclusionsMimicking the native extracellular matrix of the cardiac cells is one of the most important steps to design in vitro models. The goal of this research was to demonstrate the possible application of new polyacrylamide patterned scaffolds via Parylene C masks. We have used a versatile and simple method to fabricate flat and grooved constructs transferring collagen directly from hydrophilic Parylene C to PAm. We have demonstrated that Parylene C is a better mask than the classic PDMS and it can be used for better patterning PAm hydrogel. Flat and grooved surfaces were also characterized showing how the pattern is faithfully transferred onto the hydrogel. NRVMs behavior on isotropic and patterned substrates was quantified. 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ProdromakisNational Heart and Lung Institute, Imperial College London, London, UKE. J. Humphrey聽 聽C. M. TerraccianoConsiglio Nazionale delle Ricerche (CNR), INO UOS 鈥楢. Gozzini鈥?Area della Ricerca di Pisa - S. Cataldo, via Moruzzi 1, I-56124, Pisa, ItalyF. DinelliAuthorsI. SanzariView author publicationsYou can also search for this author in PubMed聽Google ScholarE. J. HumphreyView author publicationsYou can also search for this author in PubMed聽Google ScholarF. DinelliView author publicationsYou can also search for this author in PubMed聽Google ScholarC. M. TerraccianoView author publicationsYou can also search for this author in PubMed聽Google ScholarT. ProdromakisView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsI.S. performed the material preparation, microfabrication, characterisation of the scaffolds. I.S. and F.D. conceived the mechanical tests using AFM and data analysis. E.J.H. performed cell culturing, immunostaining, imaging and data analysis with imput from C.M.T. I.S. performed the data analysis of fluorescence images of cells. I.S. wrote the main manuscript text under the supervision of T.P. All authors contributed to improving the manuscript writing.Corresponding authorCorrespondence to I. Sanzari.Ethics declarations Competing Interests The authors declare no competing interests. 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If material is not included in the article鈥檚 Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleSanzari, I., Humphrey, E.J., Dinelli, F. et al. Effect of patterned polyacrylamide hydrogel on morphology and orientation of cultured NRVMs. Sci Rep 8, 11991 (2018). https://doi.org/10.1038/s41598-018-30360-6Download citationReceived: 05 February 2018Accepted: 26 July 2018Published: 10 August 2018DOI: https://doi.org/10.1038/s41598-018-30360-6 CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. 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