Three-Dimensional Microstructured Azobenzene-Containing Gelatin

Dec 20, 2017 - Light-triggered expansion of gelatin microstructures induced an in-plane nuclear deformation of physically confined NIH-3T3 cells. The ...
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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Three-Dimensional Microstructured Azobenzene-Containing Gelatin as a Photoactuable Cell Confining System Fabrizio A. Pennacchio,†,‡ Chiara Fedele,†,‡,§ Selene De Martino,†,‡ Silvia Cavalli,*,† Raffaele Vecchione,*,†,‡ and Paolo A. Netti*,†,‡ †

Center for Advanced Biomaterials for Healthcare, IIT@CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci, 53, 80125 Napoli, Italy ‡ Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industriale, DICMAPI, Università degli Studi di Napoli Federico II, Piazzale Tecchio, 80, 80125 Napoli, Italy S Supporting Information *

ABSTRACT: In materials science, there is a considerable interest in the fabrication of highly engineered biomaterials that can interact with cells and control their shape. In particular, from the literature, the role played by physical cell confinement in cellular structural organization and thus in the regulation of its functions has been well-established. In this context, the addition of a dynamic feature to physically confining platforms aiming at reproducing in vitro the well-known dynamic interaction between the cells and their microenvironment would be highly desirable. To this aim, we have developed an advanced gelatinbased hydrogel that can be finely micropatterned by two-photon polymerization and stimulated in a controlled way by light irradiation thanks to the presence of an azobenzene cross-linker. Light-triggered expansion of gelatin microstructures induced an in-plane nuclear deformation of physically confined NIH-3T3 cells. The microfabricated photoactuable gelatin shown in this work paves the way to new “dynamic” caging culture systems that can find applications, for example, as “engineered stem cell niches”. KEYWORDS: azobenzene, photoactuation, two-photon lithography, hydrogel, cell confinement

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INTRODUCTION Engineered biomaterials that can confine cells represent a relevant tool to control cell functions and guide their behavior in a precise way. In particular, the scientific literature reports examples of cell caging platforms where the physical confinement is used, for instance, to regulate the embryonic stem cell spherogenenesis or to get a deeper comprehension over the mechanisms involved in cellular mechanotransduction pathways.1,2 However, up to now, the use of biomaterials whose properties cannot be changed in time has been mostly explored providing just “static” interactions between the cells and the material itself, thus limiting the possibility to reproduce in vitro the well-known dynamic interaction between the cells and their microenvironment.3 Therefore, it would be highly desirable to develop engineered “smart” materials that can generate in vitro spatio-temporal complex cell signaling events and regulate “on-demand” cell−material dynamic interplay.4,5 In the literature, there are examples of stimuli-responsive materials that can change their properties in response to several external stimuli, such as pH, temperature,6 electrical and magnetic fields,7,8 enzymes,9 and light,10,11 and offer the possibility to introduce a temporal control over cell © XXXX American Chemical Society

signaling. In particular, among all these stimuli, light represents the ideal source for biological investigations because it can be precisely localized over a substrate and is finely spatiotemporally tunable and remotely addressable, without altering the environmental cell culture conditions.12−14 For these reasons, there is accumulating evidence that in the near future, light-responsive materials, in particular those containing azobenezenes, will drive forward the research in the biological field.15 For example, azopolymer thin-film photopatterning has been recently used for controlling real-time cell behavior,16 whereas azobenzene-containing poly(ethylene glycol) (PEG)based hydrogels, whose elastic modulus was affected by azobenzene photoinduced isomerization, were found biocompatible and promising for valvular interstitial cell differentiation into myofibroblasts.17 Recently, photoresponsive liquid crystalline elastomers have been elegantly exploited as dynamic cell culture supports.18−20 Received: August 31, 2017 Accepted: December 20, 2017 Published: December 20, 2017 A

DOI: 10.1021/acsami.7b13176 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces In this scenario, to the best of our knowledge, no examples of light-responsive materials used for the dynamic stimulation of physically confined cells have been reported so far. Motivated by all of these reasons, we have modified and fabricated gelatin to produce a novel light-responsive photoactuable hydrogel platform to confine and deliver a local mechanical stimulation at a single-cell level. Here, cell positioning was obtained by microstructuring the material as an array of interconnected microchannels and combining this topographical guidance with the material adhesive properties. Gelatin is an attractive candidate as a starting material for preparing hydrogels for cell culture as it presents important biochemical cues of the extracellular matrix (ECM), enhancing cell−material interaction. However, the use of native gelatin under physiological condition is limited by a reversible sol−gel transition at 37 °C, a drawback that we overpassed by modifying the material with acrylic groups, which allowed photopolymerization by means of a two-photon polymerization (2PP) process.21,22 Additionally, adding an azobenzene cross-linker molecule in the mixture, besides increasing microscale-patterning resolution, allowed the custom-made gelatin photoresist to perform as a photodeformable gelatinbased microstructured platform. Indeed, once irradiated with an adequate laser source, these structures underwent deformation, which can stimulate living cells. To demonstrate the feasibility of our approach, we used immortalized fibroblasts (NIH-3T3 cells) and we assessed aspects of biocompatibility of the substrate, including cell viability/ cytotoxicity, cell adhesion, and morphological compatibility. Photostimulation by means of the multiphoton laser of a confocal microscope allowed a real-time observation of cell response to the biomechanical cue in a cell-friendly environment.

Our photoresist mixture was formulated including Irgacure 369 as a photoinitiator (PI), acrylamide-modified gelatin, and finally an azobenzene-based bisacrylamide cross-linker (Scheme S1 in the Supporting Information). The presence of the azobenzene moiety gave the possibility to deform the polymerized and microstructured material with the use of light. The synthesized molecule was characterized in solution by means of ultraviolet−visible (UV−vis) spectroscopy, showing a reversible isomerization behavior under specific illumination conditions (see Figure S3 in the Supporting Information). The photodeformation process of gelatin microstructures was studied employing the multiphoton laser of a confocal microscope. Gelatin parallelepipeds (30 × 30 × 10 μm3) were fabricated with the Nanoscribe 2PP system on a glass substrate and used as a simple test structure to study the photomechanical response of the microstructured material. The structures were designed to be on a dimensional scale interesting for the localization and manipulation of single cells. Additionally, gelatin blocks were used as simple structures for the investigation of the photomechanical effect to work as modular elements for cell stimulation. The employment of the microscope laser was not only a finely tunable stimulus for the photoactuation but also represented a powerful tool to observe, record, and evaluate in real-time the material microdeformations with spatio-temporal accuracy, even in the presence of living cells. Furthermore, with the use of the multiphoton laser, we were able to precisely localize the UV absorption to set the experimental conditions for the biological studies. In fact, because UV light is known to be harmful for cells, this method minimized cytotoxic effects, while maximizing the light penetration depth. Exposing the structures to a 700 nm laser (a voxel wavelength of 350 nm) at 13 ± 0.5 mW output power and at a fixed z position for 10 min, an in-plane expansion of the structures of about 10% was registered, as shown in Figure 1. All the stimulation experiments were conducted under water immersion of the samples. Using a laser power higher than 15 ± 0.5 mW, the material was instead damaged.

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RESULTS AND DISCUSSION Gelatin Fabrication and Photostimulation. Gelatin is a natural hydrogel extensively used in biological applications and obtained from the partial hydrolysis and denaturation of collagen. As such, it is inherently biocompatible and biodegradable and promotes cell adhesion through collagen motifs.23 Conversely, the fabrication of well-defined gelatin microstructures that resemble the extracellular microenvironment structural features results challenging, and as a consequence, the application of gelatin in the production of advanced cell instructive platforms is limited.24,25 In this work, the combination of the cell-adhesive characteristics of gelatin with light responsiveness in precisely fabricated structures is proposed. More in details, through the design of an acrylamide-modified gelatin containing azobenzene-based cross-linkers, a smart photoactuable platform aimed at stimulating cells on demand was fabricated by means of a three-dimensional (3D) lithography system. When illuminated with a specific wavelength, the azobenzene molecule undergoes isomerization from the more stable trans isomer to the less stable cis isomer, the former having a near planar, whereas the latter a bent conformation. From the materials science perspective, this isomerization leads to interesting photomechanical effects when azobenzenes are incorporated into polymer networks, such as cross-linked hydrogel matrices.26 The light-induced isomerization can change different material properties such as the mesh size and/or its swelling behavior, and under specific conditions, it can also trigger notable shape deformations.10

Figure 1. Deformation of the gelatin structures upon MP illumination at 700 nm for 10 min. (A) Before illumination and (B) after illumination. As shown in figure, aided by the grid, the illuminated structure increased its x and y dimensions (10%). The scale bars are 10 μm.

As a negative control, the same structure was exposed to a laser tuned at 780 nm (a voxel wavelength of 390 nm), a wavelength at which the azobenzene molecule presents an adsorption valley (see Figure S3 in the Supporting Information) and does not isomerize. In these conditions, (780 nm and 13.5 ± 0.5 mW) no shape change was observed, B

DOI: 10.1021/acsami.7b13176 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

fibroblasts has been used because it is a well-recognized approach to gain general information on cellular behavior.16,18,31 Combining the ability to microstructure and photoactuate our gelatin with its cell adhesive properties, we have built up a remotely controllable platform for the mechanical stimulation of cells. In our strategy, cells are physically confined between the adjacent gelatin blocks, whose photoexpansion compressed them at the nuclear site (Figure 3A).

confirming that the deformation was related to the azobenzene isomerization (Figure S4 in the Supporting Information). To characterize possible photostimulation-related variations in the mechanical properties of the material, we measured the Young modulus of the microstructures before and immediately after the photoactuation by an atomic force microscopy (AFM) force spectroscopy analysis. From the collected data, a decrease of the Young modulus from 6.5 to 3.89 kPa was observed (Figure S5 in the Supporting Information). All of the works present in the literature reporting hydrogel azobenzenebased photoactuation describe a variation of the material mechanical properties. However, each study observed different material behaviors upon photostimulation probably because of the differences in the molecular architecture of each material, as, for example, shrinking accompanied by an increase in the storage modulus in an azobenzene-containing poly(amide acid)27 or a decrease in the elastic modulus in branched PEG− azobenzene.17 In hydrogel matrices, it is worth noticing that material stiffness plays a fundamental role in mediating cell behavior and function.28 Local variations in hydrogel stiffness may cause, for example, a preferential cell migration on hydrogel substrates. Therefore, the capability of our smart gelatin-based material to change its mechanical properties upon light exposure could be further exploited for the development of stiffness-tunable platforms, usable to precisely control cell fate and study cell behavior in response to stiffnessrelated dynamic mechanical stimuli.11,29 Besides conferring photoactuability to the gelatin, the azobenzene moiety was opportunely designed to act also as a cross-linker participating to the material structuration and compatible with the working specifications of our 3D lithography system.30 Presenting in fact an adsorption valley at 390 nm, the two-photon wavelength at which the Nanoscribe works, this molecule does not interfere with the adsorbed radiation and the consequent excitation of the PI, a primary condition for the material 2PP fabrication process.30 As it is evident from the images (Figure 2), the azo cross-linker

Figure 3. (A) Graphical representation of the gelatin platform. (B) CAD structures of the entire platform. (C) Z-stack maximum intensity projection of a fixed cell cultured on gelatin structures (nuclei were stained in blue with Hoechst, whereas the cytoskeleton is colored in red with rhodamine phalloidin). The scale bar is 10 μm. (D) Lower magnification image of cells confined between gelatin microchannels. The scale bar is 30 μm.

Here, thanks to the adhesive motives of gelatin, we did not need to selectively functionalize the microstructured platform to obtain cell confinement as done in a previous work.32 The distance between the structures was chosen following the preliminary results (data not shown), which show that the 10 μm channel width was the best distance for cell confinement. Moreover, the well-known phenomenon of contact guidance was exploited, for which nano- or micropatterns together with cell adhesive motives influence cell orientation and morphology.33−37 More in details, a network of interconnected 10 μm wide channels was produced with the Nanoscribe system, fabricating an array of acrylate gelatin parallelepipeds (30 × 30 × 10 μm3) directly attached to a glass substrate. Here, taking advantage of the unique 3D fabrication ability of a 2PP equipment such as the Nanoscribe system, we included a linear topography (3 μm pitch) on the lateral surface of each gelatin parallelepiped (Figure 3A,B). Introducing such a pattern on the channel walls, an enhancement of the cell confinement was observed (Figure S6 in the Supporting Information). This result is in line with the literature data that have highlighted the capability of micrometric linear topographies to align fibroblasts.38 Confined cells showed a mature cytoskeletal organization (Figure 3C,D), an essential condition for stress transmission to the nucleus, activating molecular pathways that regulate important cell functions such as gene expression, migration, proliferation, and differentiation.39−41 Material biocompatibility was tested with a LIVE/DEAD assay that showed no

Figure 2. Bright field images of micrometric structures obtained with the same writing parameters (A) with the azo cross-linker in the photoresist mixture and (B) without it. The scale bars are 10 μm.

improved the computer-assisted design (CAD) reproducibility and the structural stability of the fabricated microstructures, which are the two critical issues in the photopatterning of gelatin by means of a direct laser writing (DLW)-2PP technique. This result considerably improved the material potentialities in the reproduction of ECM microfeatures and gives them the possibility to evaluate their effect over the cellular behavior down to the single cell level.25 Biological Investigation. For our biological investigations, an established cell line such as immortalized NIH-3T3 C

DOI: 10.1021/acsami.7b13176 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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in Figure 5B. Our results match with the data presented in the literature, which reported, in fact, that a higher polarization of the nucleus is associated with a lower cellular compliance.42 Potentially, the capability to finely modulate the nucleus deformation controlling its polarization degree can give the possibility to study with high accuracy cell response in a broad range of conditions. To this end, next developments will include the obtainment of a higher control on the nucleus polarization, therefore increasing the reproducibility of the results. Chemical, mechanical, and topographical properties of the cell microenvironment are able to determine cell shape, and thus, its fate.43 Consequently, the ability to locally trigger a cellular mechanotrandsuctive pathway through nuclear deformation is fundamental to understand the role of molecular forces in the modulation of cell functions. In the call for new experimental platforms that allow the smart manipulation of force-responsive structures in cell-friendly conditions, engineered hydrogels, such as our gelatin photoresist, emerge as an extremely advantageous tool for the realistic evaluation of cellular behavior. In this context, the use of azobenzene-based polymers as a smart cell-instructive platform designed to dynamically interact with cells has allowed in the last years to expand scientific knowledge about mechanisms behind the regulation of cell functions.16−18,44 However, following this strategy, despite notable steps done toward the comprehension of the cellular dynamic behavior, up to now, the majority of the conducted studies has been based on the employment of twodimensional interfaces, which, as well-known, do not faithfully replicate the physiological environment. To overcome this limitation, in our proposed approach, we combined the advantages of the azobenzene-based materials with those related to the use of cell-instructive hydrogel microstructures. More in details, by means of a 3D lithography system, we have given a first example of a microstructured photoactuable hydrogel platform that could represent a valuable tool and a starting point for an evaluation of cell reactions to dynamic modulation of cell shape in a more realistic environment. This approach could be appealing, for instance, for closely mimicking the stem cell niche, which orchestrates stem cell phenotype, proliferation, and differentiation through the modulation of key elements of ECM such as composition and architecture.45,46 This strategy would aim in perspective at designing a sophisticated 3D engineered stem cell niche, whose properties could be tuned through an external light trigger on demand, regulating interplay between cells and their associated ECM and analyze this cross talk effect on stem cell selfrenewal, differentiation, quiescence, and apoptosis.47 For this reason, we are currently investigating the behavior of the human mesenchymal stem cell upon in situ photostimulation of the proposed hydrogel-based platform. Therefore, we foresee the use of such photoactuating systems as a novel paradigm in stem cell research.

cytotoxic effects after 24 h (see Figure S7 in the Supporting Information). Moreover, from other preliminary results, we observed that the material did not compromise cellular viability even after longer periods, that is, up to 25 days of cell culture (data not shown), thus showing the potential use of this platform also for investigations requiring longer culture timing. Once confined, living cells were mechanically and selectively stimulated by photoactuating their surrounding structures. Laser scanning was precisely restricted using the region-ofinterest (ROI) editor of the microscope software, thus selecting the structures to be stimulated and avoiding cell exposure to UV radiation. To observe the cell body and nucleus during the stimulation, cells were stained with the vital CellTracker deep red and Hoechst, respectively. The photoinduced expansion of the structures provoked the deformation of the nuclear area (Figure 4), a factor that could deeply

Figure 4. MP images of NIH-3T3 living cells. (A,C) Before light irradiation. (B,D) After 10 min of MP stimulation. Cells are stained with the vital CellTracker deep red for the cell body, whereas Hoechst is used for nucleus staining. The scale bars are 10 μm.

influence cellular behavior and function. After stimulation, living cells were followed every hour, up to 4 h, to evaluate possible cell-noxious effects eventually related to the stimulation process. Indeed, from the collected data (Figure S8, Supporting Information), we did not observe cytotoxic effects related to the UV light exposure in the two-photon excitation event. Comparing cell nuclei before and after stimulation (Figure 5A), a decrease of the nuclear area was observed and calculated as described in the Experimental Section. From our investigation, a range of deformations between 3 and 20% was registered. The variability of these results was related to the nuclear polarization before the stimulation. The variation in the nuclear area was quantified by comparing the fluorescent signal of the nucleus before and after the stimulation, and the initial polarization degree of the nucleus was calculated as the ratio between its two main axes (Imax/Imin). By fitting experimental points with a power law function, a correlation between nuclear area contraction and Imax/Imin was evidenced, as shown

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CONCLUSIONS In this work, we have exploited the photoactuation of azobenzene-containing hydrogel microstructures opportunely designed to alter on demand the shape of NIH-3T3 fibroblasts, a cell line commonly used as a model for mechanotransduction studies. To this end, we have added an azobenzene molecule as a cross-linker to a photopolymerizable gelatin that was structured by means of a 2PP DLW technique. Here, gelatin was the material of choice thanks to its strong and well-known D

DOI: 10.1021/acsami.7b13176 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 5. (A) Cell nuclei before, immediately after light stimulation, and an overlay of the two images. Cell nuclei are stained with Hoechst. (B) Correlation between the nucleus polarization and its area variation (12 cells). became clear, 4 wt % azo cross-linker (for its synthesis and characterization see the Supporting Information, Scheme S1 and Figure S2)48,49 and 3 wt % Irgacure 369 were added. DLW-2PP Process. DLW-2PP was performed on a Nanoscribe Photonic Professional GT system (Nanoscribe GmbH). This system uses a 780 nm Ti−sapphire laser emitting ∼100 fs pulses at 80 MHz with a maximum power of 150 mW and is equipped with a 63×, 1.4 NA oil immersion objective. The substrate was placed in a holder that fits into a piezoelectric x/y/z stage. A galvo scanner determines the laser trajectories. Gelatin photoresist was first heated at 40 °C and then poured on a circular glass coverslip (30 mm diameter and 0.17 mm thickness) previously washed with 2-propanol and dried with nitrogen. To minimize solvent evaporation from photoresist, gelatin was dropped in a closable homemade poly(dimethylsiloxane) reservoir carefully placed on the glass surface. Gelatin was fabricated at room temperature (solid state) with an output power of 24 mW and a writing speed of 7500 μm/s. To minimize the optical aberrations related to the already polymerized gelatin, structures were written in a “top-down” sequence (the first layer was the farthest from the substrate). Thanks to the solid state of the resist during fabrication, this writing sequence could be used without recurring to supporting structures. After exposure, gelatin was developed in water at 45 °C for 20 min. After development, the sample was stored in water at room temperature to prevent solvent evaporation. Cell Culture and Staining. NIH-3T3 fibroblasts (ATCC CRL1658) were cultured in a high glucose Dulbecco’s modified Eagle’s medium and incubated at 37 °C in a humidified atmosphere at 5% CO2. Prior to cell seeding, substrates were sterilized in a solution of penicillin−streptomycin in PBS (phosphate-buffered saline) (1:2, v/ v) for 4 h. On each sample, 7000 cells/cm2 were seeded and left adhering overnight in the incubator. Live cell staining was performed for nuclei by using Hoechst (150 μL cell culture medium of a solution 1:10 000 v/v in PBS) and for cell body with a treatment in a vital CellTracker deep red solution (1:1000 in a cell culture medium without fetal bovine serum) for 30 min in the incubator. Fixed cell staining was performed as follows: cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 3 min. Actin filaments were stained with rhodamine phalloidin. Samples were incubated for 30 min at room temperature in the phalloidin solution (dilution 1:200), and finally, cells were

capability to promote cell adhesion. Our gelatin-based photoresist was found highly efficient for 2PP lithography. The fabrication results highlighted the role played by the azobenzene cross-linker that has substantially improved the CAD reproducibility and the structural stability of the material on the micrometric scale, which are the two main issues in gelatin fabrication. Moreover, the results shown in this work demonstrated for the first time the possibility to photoactuate gelatin, which can be used as a “dynamic” microstructured hydrogel for cell culture. Thus, combining the light sensitivity of the material with the possibility to microfabricate it, we have built up an array of interconnected microchannels for the confinement and mechanical stimulation of living cells upon material expansion as a consequence of the illumination. Choosing the multiphoton laser of a confocal microscope as a light source, we were able to selectively deform the single cell with a high spatio-temporal accuracy in a biocompatible environment. Our results evidenced that the photostimulation was efficiently transferred from the structures to the cell nuclei, provoking a decrease of the cell nuclear area. Moreover, from preliminary AFM nanoindentation analysis, the material showed a variation in stiffness in response to the light stimulus. Taken altogether, these findings highlighted the applicability of our proposed platform as a versatile, smart hydrogel microscaffold for studying and guiding the cell mechanotransduction process in real time. Overall, our results encourage the microfabrication of more complex 3D light-responsive hydrogel-based structures for the development of “dynamic” engineered platforms, going from static to dynamic intelligent materials for cell culture applications.

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EXPERIMENTAL SECTION

Gelatin Photoresist Preparation. Acrylamide-modified gelatin B (20 w/v %) (for its synthesis and characterization see the Supporting Information, Figure S1)22 was dissolved in a citrate buffer (pH 3.1) at 40 °C overnight by gently stirring. When the solution E

DOI: 10.1021/acsami.7b13176 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces incubated for 15 min at 37 °C in Hoechst solution (dilution 1:1000) to stain cell nuclei. Photostimulation. A multiphoton (MP) confocal microscope (TCS SP5 MP, Leica Microsystems, Germany) was used to photoactuate the hydrogel 3D structures. The azobenzene isomerization and the consequent structure deformation were activated by using a MP laser, tuned at 700 nm wavelength (two-photon absorption wavelength at 350 nm, 13 ± 0.5 mW output power). The structures were immersed in water-based media and photostimulated by controlling the laser beam focusing and position. The exposed area was determined through the ROI editor of the microscope software. The deformation of the structures was live monitored and recorded via software, by time-lapse imaging. Quantification of Cell Deformation. Cell nucleus polarization and area were evaluated from stained cells analyzed with the ImageJ MomentMacroJ version 1.3 script (hopkinsmedicine.org/fae/mmacro. htm). On the basis of a fluorescence threshold, this macro evaluates the nuclear area and its principal moments of inertia (i.e., maximum and minimum). The anisotropy index was calculated as the ratio of the principal moments (Imax/Imin), where the higher Imax/Imin, the higher the nucleus polarization.

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valuable contribution in recording NMR data. Pietro Melone is instead acknowledged for the fruitful discussion regarding chemical synthesis and Valentina La Tilla for her irreplaceable aid in the production of the TOC figure.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b13176. 1 H NMR spectrum of modified gelatin B, synthesis of azo cross-linker, 1H NMR spectrum of azo cross-linker, UV−vis absorption spectra of azo cross-linker, negative control of photostimulation, AFM analysis of mechanical properties, topographical cell confinement, LIVE/DEAD assay, and cell viability after gelatin photostimulation (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.C.). *E-mail: raff[email protected] (R.V.). *E-mail: [email protected] (P.A.N.). ORCID

Silvia Cavalli: 0000-0002-8435-1785 Raffaele Vecchione: 0000-0002-8831-7891 Present Address §

Laboratory of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, Tampere FI 33101, Finland.

Author Contributions

F.A.P. and C.F. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

Financial support was provided by IIT (Istituto Italiano di Tecnologia). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Valeria Panzetta is acknowledged for her important guidance during AFM nanoindentation measurements and Lucia Rossano for her appreciated help in some biological experiments reported in the Supporting Information. We thank Dr. Fabio Formiggini for his precious support during photostimulation experiments and Dr. Luca Raiola for his F

DOI: 10.1021/acsami.7b13176 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.7b13176 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX