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Engineering enriched microenvironments with gradients of platelet lysate in hydrogel fibers Vitor E. Santo, Pedro S. Babo, Miguel Amador, Cláudia Correia, Bárbara Cunha, Daniela Fernandes Coutinho, Nuno M. Neves, João F. Mano, Rui L. Reis, and Manuela E. Gomes Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00150 • Publication Date (Web): 20 May 2016 Downloaded from http://pubs.acs.org on May 21, 2016

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Engineering enriched microenvironments with gradients of platelet lysate in hydrogel fibers Vítor E. Santo*,¥,†, Pedro Babo¥, Miguel Amador¥, Cláudia Correia§, Bárbara Cunha§, Daniela F. Coutinho¥, Nuno M. Neves¥, João F. Mano¥, Rui L. Reis¥, Manuela E. Gomes*¥

¥

3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho,

Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal. ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal §

iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras,

Portugal

KEYWORDS Tissue engineering, vascularization, photocrosslinkable hydrogels, smart biomaterials, gellan gum.

ACS Paragon Plus Environment

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ABSTRACT

Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute towards morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photocrosslinkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers towards the PL-enriched sections in comparison with the non-loaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions.


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1. INTRODUCTION In vivo tissue microenvironment involves the interaction between different components, such as (i) Extracellular Matrix (ECM); (ii) biochemical factors including cell-cell and cell-matrix interactions, localized soluble factors and gradients of bioactive molecules; (iii) physicochemical parameters, namely pH, temperature, oxygen availability and (iv) mechanical stimuli, comprising shear and tension stresses 1. ECM provides the physical and biochemical cues to guide cellular behavior, such as cell proliferation, differentiation, migration and apoptosis 2. These cues are mostly in the format of spatiotemporally regulated gradients, which play an important role in tissue development, homeostasis and disease progression 3. ECM gradients have been shown to modulate the directional migration and overall behavior of cells during numerous physiological and pathological situations 4. The ability to engineer the microenvironment is crucial for the identification of critical factors involved in these cellular processes 5. Cell adhesion, spreading, migration, proliferation and differentiation can be modulated and recapitulated with degradable and biocompatible hydrogels, designed for therapy approaches or as in vitro tissue constructs. Temporal tuning of biochemical or biophysical cues such as growth factor distribution and hydrogel stiffness induce significant changes in the overall properties of the artificial ECM, causing protein rearrangements 6. Therefore, scaffolds that are able to mimic biochemical cues while offering a topographical guidance present a valuable opportunity in tissue engineering 7. Gradient biomaterials have been used to rapidly screen cell-biomaterial interactions and to study cellular processes such as migration8, in vitro angiogenesis9 and axonal growth10. Gellan Gum (GG) is a linear anionic polysaccharide composed of a tetrasaccharide, with repeating units of two β-D-glucose, one β-D-glucuronic acid, and one α-L-rhamnose. By


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physical crosslinking, in the presence of divalent ions, GG gives rise to a hydrogel

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. GG has

been receiving particular attention for tissue engineering applications, namely for cartilage regeneration

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and intervertebral disk regeneration

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. However, physically crosslinked GG

hydrogels exhibit weak mechanical properties and tend to lose their stability in vivo after implantation

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. This limitation can be overcome by chemical modification of GG with

methacrylate groups followed by photocrosslinking. Photocrosslinkable MeGG hydrogels have been previously proposed by Coutinho et al11 as a novel biomaterial for tissue engineering. The easy and fast production and ability to tailor mechanical and degradation properties by adjusting the chemical crosslinking of these materials provide an additional control element that was not available when using hydrogels based on the original GG polymer

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. MeGG hydrogels were

found to be stable in vitro and in vivo, supporting cell encapsulation and viability, being welltolerated and non-toxic in vivo

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. Nevertheless, MeGG hydrogels present an important

limitation, namely lacking cell-specific adhesion sites, which prevents cell attachment and spread throughout these matrices. Therefore MeGG emerges as a strong material template candidate for further functionalization to study the incorporation of bioactive agents in their bulk structure and to evaluate the impact of a more cell-friendly 3D microenvironment on cell behavior. Platelet Lysate (PL) was selected as the source of bioactive agents to incorporate in the bulk of these materials. The release of growth factors and adhesion proteins stored in platelets can be triggered through different activation mechanisms

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; in this study physical disruption of the platelets was achieved by thermal shock

to extract the lysate

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. Undoubtedly, the use of PL in tissue engineering applications holds an

enormous potential as an alternative source of growth factors, as these proteins are clinically safe and can be obtained using simple and cost-effective procedures

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. Moreover, PL constitutes a


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natural and potentially autologous source of growth factors including Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), among others, which are involved in essential stages of wound healing and cell differentiation 19. PL-enriched hydrogels have been proposed in the past for tissue engineering applications 18b, 18c, 20

with promising results regarding cell adhesion, proliferation and differentiation. Nevertheless,

to our knowledge, no study has described the functionalization of hydrogels with gradients of PL. The goal was to produce these tridimensional enriched hydrogels with microfluidic tools, which have been successfully developed to offer alternatives to other 2D and 3D constructs and to induce the formation of more physiologically relevant in vitro biomaterials displaying spatial patterning of biochemical signals

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. The spatial arrangement of biomolecules within tissues

conveys specific functions that are not achieved by the homogeneous presentation of the basic components

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. One of the emerging features of these microfluidic systems is the ability to

generate shape-controlled hydrogels (microfibers, microparticles, and hydrogel building blocks) for biological applications, such as the design of cell-laden artificial hydrogels Microfabrication techniques, such as photolithography

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and microcontact printing

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.

are well-

suited to create structures with defined shapes and positions on the micrometer scale that can be used to control cell shape and function and to create highly functionalized 3D microenvironments that can work as templates for tissue regeneration 25. Therefore, the rationale behind our approach to design new advanced PL gradient biomaterial consisted in the combination of the advantageous features of PL (such as cell adhesion capacity and growth factor-enriched environment for cell culture) with the high stability provided by MeGG hydrogels. We hypothesized that the combination of a photolabile hydrogel matrices


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based on MeGG with PLs provides appropriate mechanical support and biological cues to create a microenvironment more prone to cell colonization. Moreover, we also hypothesized the ability of using this MeGG:PL combination to generate novel microfibers with gradients of bioactive factors derived from PL towards its application as a bioactive substrate for tissue engineering applications, but also as a platform to study the effect of bioactive factor gradients on the migration, proliferation and differentiation of specific cells throughout the material. As depicted in figure 1, the first section of this manuscript focuses on the development of new 3D PLs-enriched MeGG hydrogels and its effect on the adhesion and viability of human adipose derived stem cells (hASCs). The second part relies on the application of microfluidic tools for the development of PL-enriched MeGG fibers comprising a gradient of bioactive factors and follow-up biological assessment with Human Umbilical Vein Endothelial Cells (HUVECs).

Figure 1. Graphical scheme representing the experimental rationale for the preparation of the Platelet Lysate-enriched MeGG hydrogels. Gellan gum polysaccharide was firstly methacrylated to enable the development of photo-crosslinkable hydrogels. Tailoring the combination of platelet lysate (PL) with the functionalized polysaccharide provides the opportunity to produce hydrogels with different biomechanical and growth factor-releasing properties, improving overall cellular response. The final step of this study was to develop PL-enriched MeGG fibers


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constituting a gradient of cell-anchorage sites and growth factors along the length of the fiber. These PL-gradient fibers can then be used both for regenerative medicine application as well as in vitro platforms to study cell-biomaterial interactions and the effect of specific bioactive factors on cellular behavior.

2. MATERIALS AND METHODS 2.1. Material Low-acyl gellan gum (GG) (Gelzan® (Gelrite®), Mw = 1,000 kg/mol) and Methacrylic anhydride (MA) were purchased from Sigma-Aldrich, USA.

2.2. Synthesis of Methacrylated Gellan Gum Methacrylated Gellan Gum (MeGG) was synthesized by reacting the GG with MA using a previously described chemistry method

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, as depicted in figure 2A. Briefly, 2 g of GG were

dissolved in 200 mL of deionized water at 50 ºC for 20-30 min until complete dissolution. Then, 8 mL of MA were added to this solution at 50 ºC. The reaction was continued under constant stirring for 6 h. After the first correction to pH 8.5

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with 5.0 M NaOH solution (continuing

adjustment during one hour), the pH was re-adjusted to pH 8.5 every 30 min in the first 4 hours and every 45 minutes in the last 2 hours of the reaction. The modified MeGG solution was precipitated at -20 ºC overnight with, at least, an excess of 3 times in volume of cold acetone. The acetone, as well as other unreacted reagents and reaction by-products, were removed by centrifugation at 7500 rpm, 10 min at 21 ºC. The MeGG pellets were resuspended in at least 20


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mL of deionized water and kept at 37 ºC until complete dissolution. The modified MeGG solution was then purified by dialysis (dialysis membrane cut-off 11-14 kDa; Fisher Scientific, USA) for 3 days against distilled water to remove the excess of MA. Water was completely removed and exchanged at least 3 times per day. Due to the high amount of particles in suspension, the purified MeGG was filtered either with cellulose filters or with a sieve. All the batches were lyophilized and stored in a dry place protected from light.

2.3. Characterization of Methacrylate Gellan Gum The chemistry of the modified material was analyzed by Fourier transform infrared spectroscopy (IR-Prestige-21, Shimadzu), in which the samples were prepared with potassium bromide (FTIRKBr) and processed into pellets. The spectra were obtained in the range of 400 to 4000 cm-1 at a 2 cm-1 resolution with 32 scans. Furthermore, the chemical modification to GG was assessed by proton nuclear magnetic resonance (H1-NMR) spectroscopy. H1-NMR spectra were recorded with a Varian Inova 500 NMR equipped with a variable temperature system. Briefly, lyophilized materials were dissolved in D2O (Cambridge Isotope Laboratories, USA) at a concentration of 5 mg/mL and at a temperature of 70 ºC. Chemical shifts were referred to the methyl group of rhamnose as an internal standard, which is at δ 1.45 ppm.27 The degree of substitution (Ds), defined as the percentage of methacryloyl groups per reacting hydroxyl group in the tetramer, was calculated according to Eq. (1), as previously described 11, were IDB refers to the relative peak integration of double bound protons of the methacrylate group (peaks at ~6.20, ~5.77), ICH3rham refers to the relative integration of the internal standard methyl protons of the rhamnose peak (~1.98 ppm).


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nHDB, nHCH3rahm, and nOH correspond to the number of protons in the double bound of the methacrylate group, the number of protons in the internal standard methyl protons of the rhamnose and the number of reactive hydroxyl moieties in the GG structure respectively.

Equation (1)

2.4. Preparation of MeGG:PL hydrogels PL was obtained from different platelet collections performed at Instituto Português do Sangue (IPS, Porto, Portugal), and prepared as previously described 17b, 28. All the platelet products were biologically qualified according to the Portuguese legislation (Decreto-Lei n.º 100/2011). The platelet count was performed at the IPS, and the sample concentration was adjusted to 1x106 platelet/µL. Briefly, PL samples from three different donors were mixed, subject to three repeated temperature cycles (frozen with liquid nitrogen at -196 ºC and heated at 37 ºC) and frozen at -20 ºC until further use. The lyophilized MeGG was dissolved overnight at 1% (w/v) in deionized water, under constant stirring at 21 ºC, protected from light. To guarantee a homogeneous solution of MeGG and PLs, a solution of MeGG:PL was also prepared by dissolving MeGG in PLs solution, also at 1% (w/v) concentration. 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959, Sigma-Aldrich) was used as the photoinitiator (PI) in MeGG and MeGG:PL solutions. Table 1


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describes the different formulations developed for the production of these hydrogels. Three different concentrations of photoinitiator: 0.15% (w/v), 0.25% (w/v) and 0.5% (w/v) and three volumetric ratios of MeGG:PL: 1:0, 1:1, 2:1 (v:v) were evaluated for the production of 3D photocrosslinked hydrogels. Final volume of mixtures for the preparation of the constructs was always the same. Table 1. Summary of the conditions tested for the preparation of MeGG:PL hydrogels. [Photoinitiator] (% w/v)

Condition

Ratio MeGG:PL (v:v) 1:0

MeGG 0.15 % MeGG:PL 0.15% 1:1

0.15

1:1

MeGG:PL 0.15% 2:1

2:1

MeGG 0.25%

1:0

MeGG:PL 0.25% 1:1

0.25

1:1

MeGG:PL 0.25% 2:1

2:1

MeGG 0.5%

1:0

MeGG:PL 0.5% 1:1

0.5

MeGG:PL 0.5% 2:1

1:1 2:1

Due to the low solubility of the photoinitiator Irgacure 2959, three cycles of vortex followed by incubation at 37 ºC for 30 min were performed until complete dissolution. Then, MeGG solutions were mixed with PL in a ratio MeGG:PL of 1:1 and 2:1 (v/v). MeGG solutions without PL were used as controls. The resulting solutions were casted in 5 mm diameter, cylindrical PDMS molds (60 µL/mold) placed on a microscope slide and exposed to UV light (320-500 nm, 7 mW/cm2, EXFO OmniCureS2000, Lumen Dynamics, Canada) for 60 s, in order to obtain chemically crosslinked hydrogels.


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2.5. Design of MeGG:PL Gradients A cross-gradient of PLs through the MeGG fibers was prepared by reverse flow in a microfluidic channel through the creation of chemical gradients, as previously reported

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. A solution of 1%

MeGG dissolved in PL (MeGG:PL) was mixed with a solution of 1% MeGG in H2O (MeGGH2O), both containing 0.25% (w/v) of Irgacure 2959, within a microfluidic channel (1.2 mm diameter x 30 mm length) in a PDMS mold, as schematized in figure 1. The MeGG solutions with or without PL were injected into the microfluidic channels using a peristaltic pump (ISMATEC REGLO E120, ISMATEC, Switzerland). The flow rate used for the creation of gradients was 0.6 mL/min. The photocrosslinking of the fibers was triggered by exposing to UV light (320-500 nm, 8.00 mW/cm2, 7.5 cm height from the light beam) for 90 s. After 3 min of exposure, hydrogel fibers were extracted from the mold by positive pressure, through the inlet, using a syringe loaded with Phosphate Buffered Saline (PBS, Sigma-Aldrich). Fibers were further washed in PBS (pH 7.4) to remove all non-crosslinked material. Figure 1 comprises a graphical representation of the workflow to produce gradient fibers. A vertical layout was chosen due to its ability to produce homogeneous gradient fibers. The reverse-flow technique was performed by filling first MeGG solution through the channel inlet. Afterward, a tube filled with MeGG:PL solution was added in the outlet and MeGG solution was pumped back and forward seven times through the inlet, originating a flow in and out of MeGG:PL solution. During the process MeGG:PL solution mixed with the MeGG solution already in the channel by diffusion, generating a concentration gradient of MeGG:PL in the channel, with decreasing concentration from the outlet to the inlet.


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2.6. Physico-chemical Characterization of MeGG:PL hydrogels 2.6.1. In Vitro Release studies MeGG:PL gels were suspended in 5 mL of PBS solution and stirred at 60 rpm at 37 ºC. Aliquots of 1 mL were withdrawn at predetermined time intervals and the same volume of fresh PBS was added to the suspension. The total amount of protein released was quantified by microBCA protein quantification kit and optical density was measured at 562 nm on a multiwell microplate reader (Synergy HT, Bio-Tek Instruments, USA).

2.6.2. Dynamic Mechanical Analysis (DMA) The mechanical properties of the hydrogels were evaluated by DMA. The viscoelastic measurements were performed using a TRITEC8000B DMA from Triton Technology (UK), equipped with the tensile mode. The experiments were performed at 37 ºC in a wet state to simulate the physiologic conditions. In brief, freshly prepared samples were immersed in a PBS solution until equilibrium was reached. Then, the geometry of the samples was measured and the samples were clamped in the DMA apparatus and immersed in a PBS bath. After equilibration at 37 ºC, the DMA spectra were obtained for a frequency scan between 0.1 and 10 Hz. The experiments were performed under constant strain amplitude (3% of hydrogel height). A static pre-load of 1 N was applied during the tests to keep the sample tight.

2.6.3. Scanning Electron Microscopy (SEM) The morphological structure of the cross-surface of hydrogels was characterized by SEM. The constructs were dehydrated using a graded series of ethanol for 20 min, twice (30, 50, 70, 90,


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100%) and were dried at 21 ºC. Afterward, the constructs were sputter coated with gold (JEOL JFC-1100) and analyzed using a Leica Cambridge S360 scanning electron microscope.

2.6.3. Qualitative characterization of the protein gradient FITC-BSA (Sigma-Aldrich, Germany) was mixed with MeGG:PL solution and MeGG solution was mixed with TRITC (Sigma-Aldrich, Germany), both at 0.003% (w/v) concentration. Samples of hydrogel fibers were observed in a UV transilluminator (BioSpectrum AC Chemi HR 410, UVP) shortly after fiber preparation. The gradient of protein was quantified by measuring the gray values of UV transilluminator images along the fiber sections for each fluorescence marker, using ImageJ software.

2.6.4. Protein content Total protein accumulated in each section of the MeGG and MeGG:PL fibers were quantified. Protein content was then measured by microBCA protein quantification kit, as previously described. Each fiber was evenly sliced in three sections and further sonicated (Ultrasonic processor, VCX 130 PB – VCX 130 FSJ, USA) until the complete disintegration of the matrix.

2.7. In vitro biological studies 2.7.1. Human Adipose Derived Stem Cells Isolation Human subcutaneous adipose tissue samples were obtained from lipoaspiration procedures performed on patients with ages between 35 and 50 years old under a protocol previously established with the Department of Plastic Surgery of Hospital da Prelada in Porto, Portugal. Informed signed consent was obtained from all the donors. All the samples were processed


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within 24 h after the lipoaspiration procedure. Human ASCs were enzymatically isolated from the subcutaneous adipose tissue as previously described 30. The lipoaspirate samples were firstly washed with a solution of PBS and 10% (v/v) penicillin/streptavidin. Liposuction tissue was digested with 0.2% Collagenase Type II solution for 90 min with intermittent shaking, at 37 °C. The digested tissue was filtered using a 100 µm filter mesh (Sigma-Aldrich, Germany). The floating adipocytes were separated from the precipitation stromal fraction by centrifugation at 1250 rpm for 10 min at 21 ºC. The cell pellet was resuspended in lysis buffer for 10 min to disrupt the erythrocytes. After centrifugation at 800 rpm for 10 min, cells were again resuspended and placed in culture flasks with Minimum Essential alpha Medium supplemented with sodium bicarbonate, 1% (v/v) penicillin/streptavidin and 10% (v/v) of Fetal Bovine Serum (FBS) (basal medium). Cells were cultured until sufficient number was reached for the experiment in a humidified atmosphere of 95% air, 5% CO2, at 37 °C, and the medium was exchanged every two days. In our lab, human ASCs have been routinely isolated by enzymatic digestion, and characterized for stemness potential by flow cytometry and RT-PCR for CD44, STRO-1, CD105 and CD90 markers, whose procedure is described in more detail elsewhere 30b.

2.7.2. Encapsulation of hASCs Briefly, a hASCs suspension of 5x106 cell/mL was mixed with the volume of PLs (filtered through a 0.22 µm filter) required to achieve a final hydrogel MeGG:PL ratio of 1:1 or 2:1 (v/v). This mixture was then incorporated into the MeGG 0.25% (w/v) solution prepared as described in section 2.3. Afterward, the cells/PL/polymer mixture was casted into molds (5 mm in diameter


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and 2 mm height) and exposed to UV light (7 mW/cm2) for 60 s. Cells encapsulated in the hydrogel formulations were cultured in 1 mL of basal medium in non-adherent 48-well culture plates up to 7 days, and the medium was exchanged at each 2-3 days.

2.7.3. Seeding of hASCs Human ASCs were seeded in the hydrogels as follows: a drop of 50 µL containing 20.000 cells was placed on each hydrogel (5 mm in diameter and 2 mm height). After 2 h in a humidified atmosphere at 37 ºC, containing 5% CO2, 1mL of fresh basal culture medium was added per well. Cell-hydrogel constructs were statically cultured for 7 days under the culture conditions previously described.

2.7.4. Encapsulation of human umbilical vein endothelial cells (hUVECs) Briefly, HUVECs (Thermo Fisher Scientific, C-003-5C) in passage 4 were seeded in culture flasks previously coated with gelatin and cultivated in medium M199 (Sigma-Aldrich, USA) supplemented with heparin (heparin sodium salt from porcine intestinal mucosa, Sigma-Aldrich, USA) and Endothelial Cell Growth Supplement (ECGS, BD Biosciences), until reaching confluency. Then, cells were trypsinized and a hUVECs suspension of 4x106 cell/mL in 1% MeGG-H2O solution was prepared. A cross-gradient of PLs and MeGG was engineered by mixing a solution of MeGG:PL with the solution of MeGG-H2O, within a microfluidic channel in a PDMS mold, using a vertical layout, as previously described. After crosslinking of the fibers by exposing to UV light (320-500 nm, 8.00 mW/cm2, 7.5 cm height from the light beam) for 90s,


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fibers containing inverse gradients of hUVECs and PL. Also, fibers containing only hUVECs, without gradient, were produced, as controls. Afterward, the fibers were distributed for wells of 12 wells tissue culture plates, and cultured in endothelial medium at 37 ºC, 5% CO2.

2.8. Evaluation of biological response to the hydrogels 2.8.1. Live/Dead Assay Cell viability in the hydrogels was assessed throughout the culture period using the live/dead assay (Calcein AM / Propidium iodide (PI) staining). Briefly, the constructs were incubated for 15 min with 150 µL of calcein AM (C3099, Invitrogen, USA) solution (2 µL calcein/mL DMEM without phenol red) and 150 µL of PI (P1304MP, Invitrogen, USA) working solution (2 µL PI stock solution (1 mg/mL in distilled water) and 20 µL RNAse (78020Y, USB Corporation, USA) stock solution (1 mg/mL in distilled water) in 2 mL PBS), protected from light. The constructs were washed with PBS to remove residual fluorescent staining and observed under fluorescence microscopy (Zeiss HAL 100/HBO 100).

2.8.2. Determination of metabolic activity by alamarBlue Assay Metabolic activity of the cells was assessed at days 1, 3 and 7 using the alamarBlue assay (AbDseroTec), according to the manufacture’s recommendation. Briefly, hASCs-constructs were incubated 4 h with fresh medium containing 10% (v/v) alamarBlue. Fluorescence was measured in 96-well plates using a multi-well microplate reader using the filter parameters: 560 nm of excitation, and 590 nm of emission.


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2.8.3. Determination of cell proliferation by DNA quantification Cell proliferation was evaluated as a function of the total amount of double-stranded DNA present in each cell-construct (cylindrical and fiber hydrogels) at different culture times (days 1, 3 and 7). The Quant-iT™ PicoGreen® dsDNA Assay Kit (Molecular Probes, Invitrogen) was used and manufacturer’s instructions were followed. Briefly, samples were washed twice with PBS and then were preserved in 1 mL of ultrapure water at -80 °C for further analysis. Before the analysis, samples were thawed and sonicated for 15 min. The fluorescence was measured at an excitation wavelength of 485/20 nm and at an emission wavelength of 528/20 nm, on a multiwell microplate reader. The DNA concentration for each sample was calculated using a standard calibration curve.

2.8.4. Phalloidin / DAPI staining The morphology of hASCs seeded on the hydrogels was analyzed using a fluorescent light microscope (Zeiss HAL 100/HBO 100). After 1, 5 and 7 days, cells were fixed with formalin 4% (v/v) and immuno-stained with phalloidin (10 mg/mL, Sigma-Aldrich, USA) for 45 min and with DAPI (Invitrogen, USA) for 2-3 min. Then the samples were thoroughly rinsed with PBS solution and representative images were taken using fluorescence microscopy (Zeiss HAL 100/HBO 100).


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2.9. Statistical Analysis All the experiments were performed with at least three replicates. Data sets were analyzed statistically using GraphPad Prism 6 and tested for normality by ShapiroWilks test. Comparisons between multiple groups were done using a one-way ANOVA with Tukey post-hoc test, with significance levels of p

Engineering Enriched Microenvironments with ... - ACS Publications

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