nanomaterials
Article
Fabrication of Gelatin Methacrylate (GelMA)
Scaffolds with Nano- and Micro-Topographical
and Morphological Features
Ana Agustina Aldana 1 , Laura Malatto 2, Muhammad Atiq Ur Rehman 3,4 ,
Aldo Roberto Boccaccini 3,* and Gustavo Abel Abraham 1
1 Instituto de Investigaciones en Ciencia y Tecnología de Materiales, INTEMA (UNMdP-CONICET),
Av. Juan B. Justo 4302, Mar del Plata B7608FDQ, Buenos Aires, Argentina; aaldana@fi.mdp.edu.ar (A.A.A.);
gabraham@fi.mdp.edu.ar (G.A.A.)
2 Instituto Nacional de Tecnología Industrial, Centro de Micro y Nanoelectrónica del
Bicentenario (INTI-CMNB), Av. Gral. Paz 5445, San Martin B1650KNA, Buenos Aires, Argentina;
laura@inti.gob.ar
3 Institute of Biomaterials, Department of Materials Science and Engineering, University of
Erlangen-Nuremberg, 91058 Erlangen, Germany; muhammad.rehman.ur@fau.de
4 Department of Materials Science and Engineering, Institute of Space Technology Islamabad, 1,
Islamabad Highway, Islamabad 44000, Pakistan
* Correspondence: aldo.boccaccini@ww.uni-erlangen.de; Tel.: +49-9131-85-28601
Received: 12 December 2018; Accepted: 12 January 2019; Published: 18 January 2019


Abstract: The design of biomimetic biomaterials for cell culture has become a great tool to study and
understand cell behavior, tissue degradation, and lesion. Topographical and morphological features
play an important role in modulating cell behavior. In this study, a dual methodology was evaluated
to generate novel gelatin methacrylate (GelMA)-based scaffolds with nano and micro topographical
and morphological features. First, electrospinning parameters and crosslinking processes were
optimized to obtain electrospun nanofibrous scaffolds. GelMA mats were characterized by SEM,
FTIR, DSC, TGA, contact angle, and water uptake. Various nanofibrous GelMA mats with defect-free
fibers and stability in aqueous media were obtained. Then, micropatterned molds produced by
photolithography were used as collectors in the electrospinning process. Thus, biocompatible GelMA
nanofibrous scaffolds with micro-patterns that mimic extracellular matrix were obtained successfully
by combining two micro/nanofabrication techniques, electrospinning, and micromolding. Taking into
account the cell viability results, the methodology used in this study could be considered a valuable
tool to develop patterned GelMA based nanofibrous scaffolds for cell culture and tissue engineering.
Keywords: biomimetic scaffolds; gelatin; electrospinning; micromolding; biomaterials
1. Introduction
The design of biomimetic biomaterials as scaffolds for cell culture is a powerful tool for
studying and understanding fundamental cell behavior, specific tissue environment, degradation,
and reasons for tissue damage [1]. Scaffolding structures should mimic not only biological properties
of extracellular matrix (ECM), but also morphological and topographical features [2–4]. The ECM
directs and modulates cell behavior, is composed of fibrous proteins (mainly collagens and elastin),
glycosaminoglycans (GAGs), proteoglycans, and glycoproteins [5,6].
In order to emulate the natural structure of the ECM, different technologies have been developed.
Current techniques for generating topographical features on polymeric scaffolds for cell culture,
especially those with nano-scale resolution, are typically complex and expensive. Thus, a simple
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and tunable fabrication method for the production of patterned biomimetic scaffold is still pending.
By using electrospinning technology, it is possible to obtain micro- and/or nano-fibrous mats that
mimic ECM [7,8]. The optimization of different processing parameters and the use of post-processing
treatments allow handling dimensions, porosity, morphology, and the spatial arrangement of
nanofibers. A huge variety of natural and/or synthetic polymeric solutions has been electrospun.
Composition and processing techniques determine the scaffold architecture, mechanical performance,
degradation rate, and cell-material interactions. Aligned and randomly oriented electrospun mats have
been also developed to study how morphology affects cell behavior. Gao et al. studied the influence
of aligned and randomly oriented fibrous gelatin/PLLA scaffolds to guide the growth of corneal
stroma cells [9]. The aligned scaffold not only increased cell viability more significantly than that in
a randomly oriented scaffold, but it also provided an external stimulus for the orderly arrangement
of cells. Similar results were observed by Shalumon et al., who prepared aligned and randomly
PLLA/gelatin nanofibrous scaffolds [10]. In these structures, an increase in viability and proliferation
of human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) was observed.
On the other hand, substrates with various micro- and nano-features such as lines, wells, and holes
among others have been explored to introduce significant effects on cell behavior [11]. Most of these
reports relate the topographical features with cell orientation, migration, morphology, proliferation,
cell gene expression, and differentiation [12–14].
The main goal of this work is to design gelatin-based scaffolds with micro and nano-topographical
and morphological features, achieving a high resolution, and performance with low cost. In addition,
gelatin, a biocompatible and biofunctional polymer, and benign solvents (Class 3 according to
ICH guidelines) [15] are used for scaffold fabrication. Gelatin is an inexpensive biomacromolecule
obtained from denatured collagen, and presents integrin cell-binding motifs, such as RGD and matrix
metalloproteinases (MMP) degradable sites [16,17]. Compared to native collagen, gelatin has lower
antigenicity and less batch-to-batch variation due to the denaturation process, in which tertiary protein
structures are removed. Functionalization of amine-containing groups of gelatin with methacrylate
groups was used to provide a photopolymerizable biomaterial named GelMA that has been widely
investigated for cell-based studies and tissue engineering applications [18–22]. Crosslinking of the
methacrylic side groups results in hydrogels with stiffness and density that can be controlled by varying
the polymer dry mass, degree of functionalization, photo-initiator concentration, ultraviolet (UV)
intensity, and exposure time. In this work, electrospinning and photolithography techniques were used
to design 3D scaffolds with novel topographical features in micro- and nanoscale, while UV exposition
time was varied. Moreover, the use of micropatterned molds with different sizes as collectors in the
electrospinning process is proposed to produce electrospun fibrous mats.
2. Materials and Methods
2.1. Materials
Gelatin type A from porcine skin Gel Strength 300, methacrylic anhydride (MAA), and glacial
acetic acid (AA) were purchased from Aldrich (Darmstadt, Germany). The photoinitiator
1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure®2959) was kindly
provided by BASF (Nienburg, Germany). Phosphate-buffered saline (PBS) was freshly prepared
in the laboratory.
2.2. Synthesis of GelMA
The preparation of GelMA has been recently described by the authors [23]. Briefly, gelatin was
dissolved in PBS (pH 7.4) at a concentration of 1% (wt/v) and 50 C. Then, a predetermined amount of
methacrylic anhydride was added, under vigorously magnetic stirring conditions. The mixture was
left to react for one hour at 50 C and was afterwards dialyzed by using a 12–14 kDa cutoff membrane
against distilled water for several days. Finally, functionalized gelatin was frozen and freeze-dried.

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The degree of methacrylation, defined as the percent of amine groups converted to methacrylamide
groups, was determined by Habeeb’s test [24]. Finally, GelMA was characterized by 1H-NMR (Bruker
400 MHz NMR spectrometer, Bruker Biospin, Rheinstetten, Germany) and FTIR spectroscopy (Mattson
Instruments Inc., model Genesis II, Madison, WI, USA).
2.3. Fabrication of Electrospun GelMA Nanofibers
A predetermined amount of GelMA was completely dissolved in acetic acid at a 250 mg/mL
concentration. Then, Irgacure 2959 was added to GelMA solution. The solution was electrospun
through a blunt 18-gauge stainless steel needle onto an aluminum collector plate 10 cm away. A solution
flow rate of 0.2 mL/h and an applied high-voltage of 12 kV were used. All experiments were carried
out at room temperature and a relative humidity of 50%.
2.4. Photocrosslinking of GelMA Nanofibers
The obtained electrospun GelMA scaffolds were crosslinked by UV irradiation. Fibrous meshes
were initially wetted in anhydrous ethanol and then exposed to UV light (UVL-28 lamp, 365 nm),
during different time periods (0, 6, 9 and 12 min) at 2.5 cm (named NG-0UV, NG-6UV, NG-9UV and
NG-12UV, respectively). After crosslinking treatment, samples were removed from aluminum foil and
dried at room temperature in a vacuum oven.
2.5. Fabrication of Micropatterned Molds
Master mold layouts were designed using L-EditTM from Tanner EDA and printed on
polyester-based film using a 3600 DPI printer. Molds were then created on silicon wafers by
spin-coating SU8 2000 negative photoresist (10 s at 500 rpm and 30 s at 3000 rpm, Microchem
Inc., Westborough, MA, USA). Wafers were previously cleaned with the standard piranha solution.
The coated wafers were then soft-baked on a hot plate for 20 min at 65 C, followed by 25 min at 95
C, before exposing the photoresist through the photomask. Near UV (365 nm) was applied with an
exposure dose of 650 mJ/cm2 (EVG 620, EV Group). Post exposure bake conditions were 5 min at
65 C, followed by 10 min at 95 C. Molds were finished by 10 min of immersion on the developer
(MicroChem’s SU-8 Developer) with strong agitation, rinsed with isopropyl alcohol, and dried with
nitrogen stream.
2.6. Fabrication of Micropatterned Nanofibrous GelMA Scaffolds
The electrospinning of micropatterned GelMA scaffolds was carried out by using the same
procedure as described above except that the collector plate consisted in a micropatterned mold
obtained by photolithography. After electrospinning, the meshes were crosslinked by UV irradiation
for 9 min just as it was previously described.
2.7. Morphology Characterization
Surface morphology of the nanofibrous scaffolds before and after crosslinking was examined by
scanning electron microscopy (SEM). Samples were placed on double-sided graphite tape, attached
onto a metal surface, and sputter-coated with gold for 10 s. SEM micrographs were acquired
with different magnifications using a SEM (Jeol USA Inc., model JSM-6460LV, Peabody, MA, USA).
The average fiber diameter and fiber diameter distribution were estimated from SEM images, using
ImagePro-Plus 6.0® software.
2.8. Infrared Analysis
Chemical composition of the gelatin scaffolds was assessed by Fourier transform infrared
spectroscopy with attenuated total reflectance (FTIR-ATR) using a Nicolet 6700 Thermo Scientific
(Waltham, MA, USA) spectrometer equipped with a diamond crystal at a nominal incidence angle of

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45 and ZnSe lens. Spectra were recorded in the range of 600–4000 cm 1 at 32 scans with a resolution
of 4 cm 1.
2.9. Contact Angle Measurements
Water contact angles of the fibrous scaffolds were measured with a Ramé-hart goniometer using
the sessile drop method. The samples were attached to a glass slide and placed in the sample stage.
A droplet of deionized water (10 L) was automatically dispersed onto the sample surface and its
evolution with time was recorded using a CCD video camera attached to the equipment. From the film
frames, the water contact angles along time were automatically calculated by the equipment software.
2.10. Water Uptake Measurements
Electrospun GelMA scaffolds discs (1 cm in diameter) were cut and weighted (Wo). Then, samples
were immersed in PBS at room temperature. After predetermined immersion times, mats were
retrieved and weighted (Wt). The water retention (WR) at time t was calculated according to the
following equation:
WR(%) = (Wt Wo)/Wo 100, (1)
2.11. Tensile Testing
Tensile properties of the electrospun scaffolds were tested by using an Instron 4467 universal
testing machine (Instron, Norwood, MA, USA). Prior to uniaxial tensile testing, the electrospun fibrous
sheets were cut into rectangular shapes (50 mm 10 mm). Samples were secured between opposing
clamps which were approximately 30 mm apart from each other. For tensile testing, samples were
stretched until failure at 10 mm/min.
2.12. Thermal Analysis
Thermal properties were determined by thermogravimetric analysis (TGA) and differential
scanning calorimetry (DSC). TGA were performed on a Shimadzu TGA-50 analyzer from ambient
temperature to 300 C at 10 C/min under nitrogen atmosphere. DSC thermograms were obtained in
a Perkin-Elmer Pyris 1 calorimeter (PerkinElmer Inc., Waltham, MA, USA). Scans were carried out
from 25 to 300 C at a heating rate of 10 C/min under a nitrogen atmosphere.
2.13. X-ray Diffraction
The XRD patterns were obtained using a X-ray diffractometer (PANalytical Model X’pert PRO,
Royston, UK). Film samples with dimensions of 4.0 cm 1.5 cm were cut and fixed in a circular clamp
of the instrument. The analysis was carried out directly and the conditions were as follows: (i) voltage
and current: 40 kV and 40 mA, respectively; (ii) scan range from 3 to 30 ; (iii) step: 0.1 and (iv) speed
1 /min, equipped with a secondary monochromator of graphite beam. The samples were stored at
25 C and 50% Relative humidity (RH) and analyzed in triplicate.
2.14. Roughness Measurements
Roughness was measured by using a laser profilometer (UBM™, ISC-2). A measurement length
of 5–7 mm was used with a scanning velocity of 400 points per second. The roughness was calculated
using the LMT Surface View UBM™ software.
2.15. Cell Viability
For cell culture studies MG-63 osteoblast-like cells (Sigma-Aldrich, Darmstadt, Germany) as an
adequate model for bone cells were used [25]. Culture medium Dulbecco’s modified Eagle’s medium
(DMEM, Gibco, Darmstadt, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS, Merck,
Darmstadt, Germany) and 1% (v/v) penicillin/streptomycin (PS, Merck, Darmstadt, Germany) was

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chosen. The electrospun GelMA samples were cut into pieces at a diameter of 10 mm and then placed
onto the bottom of the culture plates, followed by sterilization under UV light. MG-63 cells were seeded
onto the samples in 24-well plate at a density of 1 104 cells per well, and conserved into an incubator
at 37 C in a humidified atmosphere of 95% air and 5% CO2 for 48 h. After cell culture, the cell viability
was determinate by the enzymatic conversion of tetrazolium salt (WST-8 assay, Sigma-Aldrich) to
formazan. A volume of 1 mL of a solution of 1% WST-8 assay n cell culture medium was added to each
sample, which were incubated for 4 h. The absorbance at 450 nm was measured with a plate reader
(typo Phomo, Anthos Mikrosysteme GmbH, Krefeld, Germany). As a blank value, the cell media
containing 1% WST-8 without contact to a sample, was used and measured after 4 h of incubation.
3. Results
3.1. GelMA Nanofibrous Mats
3.1.1. Synthesis of GelMA
GelMA was synthesized according to previously reported methods, in which methacrylate
functional groups were grafted onto the gelatin backbone through reactions between methacrylic
anhydride and lysine residues [21]. GelMA infrared spectrum showed peaks at 1645, 1526, and
1240 cm 1 related to the C=O stretching (amide I), N–H bending (amide II), and C–N stretching plus
N–H bending (amide III), respectively. Moreover, a N–H stretching (amide A) could be observed at
3284 cm 1. The modification of lysine residues with methacrylate groups was confirmed by a decrease
in the lysine signal at 2.9 ppm, and the appearance of the methacrylate group signal at 5.4 ppm and
5.7 ppm and the methyl group signal at 1.8 ppm. A degree of methacrylation of 71% was calculated
using Habeeb’s test [24].
3.1.2. Fabrication of Electrospun GelMA Matrices
Electrospun GelMA mats were prepared by electrospinning technique. Defect-free nanofibrous
matrices were obtained as shown in Figure 1 (NG-0UV). Then, electrospun mats were successfully
crosslinked by UV irradiation. As it is well-known, photo-chemical crosslinking takes place only in the
presence of a photoinitiator, and thus hydrogel formation is triggered by the external source of UV
light. Irgacure 2959 is usually chosen as photoinitiator due to its low toxicity at the used concentration
as demonstrated in previous studies [23]. To evaluate the UV irradiation process time, GelMA mats
were immersed in ethanol and exposed at different irradiation times. Figure 1 shows SEM micrographs
of GelMA mats with 0, 6, 9 and 12 min of UV irradiation. In a first step of UV irradiation, fibers
increased their diameter as result of their swelling in polar solvents. After UV irradiation, mats still
exhibited their fibrous structures and their diameters were reduced with respect to the diameter of
NG-0UV. Probably, this decrease in fiber size is related to the crosslinking of GelMA. The increase of
diameter in NG-12UV could be ascribed to the increase in temperature produces by UV device.
The mean fiber diameter is summarized in Table 1.
Table 1. Fiber diameter, contact angle, water uptake (WR), and DSC results, melting temperature (Tm),
and the onset of glass transition temperature (Tg), of GelMA mats.
Matrix.
Mean
Diameter ( m)
Contact Angle
( )
WR (%)
Tgonset
( C)
Tmonset ( C)
NG-0UV 1.05 0.17 0 n/d 57.6 74.1
NG-6UV 0.70 0.10 43.2 390 21 52.3 66.5
NG-9UV 0.60 0.12 66.1 405 10 52.9 71.2
NG-12UV 0.80 0.12 56.0 290 15 53.9 68.6

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Figure 1. SEM micrographs of samples at different irradiation times: 0 (NG-OUV), 6 (NG-6UV), 9
(NG-9UV) and 12 min (NG-12UV).
The FTIR spectra of GelMA mats showed typical peaks of gelatin (Figure 2a) as described for
GelMA. On the other hand, TGA thermograms in Figure 2b show the thermal degradation behavior
of GelMA mats. All samples showed a small loss of mass in the range of 50 °C to 120 °C, probably
caused by the loss of water molecules. At temperatures above 150 °C, the degradation of biopolymer
started for all samples. In addition, their onset points of degradation increased slightly with the time
exposed to UV irradiation. Probably, it is due to an increase in the crosslinking degree.
Figur 1. SE micrographs f samples t different irradiatio times: (NG-OUV), (NG-6UV),
(NG-9UV) an 2 mi (NG-12UV).
Th FTI spectr GelM mat showe typica peak gelati (Figu describe fo
GelMA. the hand, thermograms i Figu 2b sho the thermal degradatio behavior
of GelMA mats. All samples showe small of mas th rang t 12 obabl
cause th los f wate molecules. temperatu abov 15 th degradatio biopolyme
starte fo all samples. addition thei onse point degradatio inc ease slightl wit th tim
expose t irradiation. obabl , it is due to an increase in the crosslinking degree.

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Figure 2. Characterization of samples by (a) ATR-FTIR, (b) TGA, (c) DSC, (d) XRD.
Differential scanning calorimetry (DSC) curves of electrospun GelMA mats are shown in Figure
2c. All samples displayed two endothermic peaks at around 50 and 70 °C. The first peak could be
attributed to glass transition of amino acid blocks in the peptide chain relating to the amorphous
regions of gelatin. Uncrosslinked gelatin showed a higher Tg than the observed in crosslinked gelatin
due to peptide chains, could interact physically by intermolecular bonds. In addition, Tg of GelMA
increased with the crosslinking percentage because of the reduced polymeric chain mobility. As
suggested in the literature, the endothermic peak at around 70 °C is attributed to denaturation protein
[26]. The high Tm of uncrosslinked gelatin could be associated to physical crosslinking of peptide
chains. All samples showed endotherm falling at around 97 °C; this is attributed to water loss and
degradation of protein.
In order to correlate thermal behavior with structural organization, XRD patterns of samples
were obtained (Figure 2d). In spite, gelatin has a crystalline structure originated from α-helix and
triple helical; the amorphous structures were observed for all the samples. Probably, the
electrospinning processing hinders the re-crystallization of gelatin and the XRD pattern showed an
amorphous halo. This peak could be associated with the short range order of protein chains. In
addition, the intensity of amorphous peak decreased with the increase in crosslinking degree.
Probably, the crosslinking hindered the peptide chains re-ordering.
The hydrophilicity of the electrospun meshes was evaluated by measuring their water contact
angles (Figure 3a, Table 1). The contact angle of uncrosslinked gelatin mat could not be measured
because it was absorbed instantaneously when the PBS solution was in contact. However, crosslinked
gelatin nanofibers are stable at these conditions. As expected, the CA values of crosslinked gelatin
confirm the hydrophilic character of matrices. UV irradiation time could affect not only crosslinking
extension, but could also modify the structure, as can be seen in SEM images. Thus, CA values are
determined by both chemical and morphological properties.
Figure 2. Characterization of samples by (a) ATR-FTIR, (b) TGA, (c) DSC, (d) XRD.
Differential scanning calorimetry (DSC) curves of electrospun GelMA mats are shown in Figure 2c.
All samples displayed two endothermic peaks at around 50 and 70 C. The first peak could be attributed
to glass transition of amino acid blocks in the peptide chain relating to the amorphous regions of
gelatin. Uncrosslinked gelatin showed a higher Tg than the observed in crosslinked gelatin due to
peptide chains, could interact physically by intermolecular bonds. In addition, Tg of GelMA increased
with the crosslinking percentage because of the reduced polymeric chain mobility. As suggested in the
literature, the endothermic peak at around 70 C is attributed to denaturation protein [26]. The high
Tm of uncrosslinked gelatin could be associated to physical crosslinking of peptide chains. All samples
showed endotherm falling at around 97 C; this is attributed to water loss and degradation of protein.
In order to correlate thermal behavior with structural organization, XRD patterns of samples were
obtained (Figure 2d). In spite, gelatin has a crystalline structure originated from -helix and triple
helical; the amorphous structures were observed for all the samples. Probably, the electrospinning
processing hinders the re-crystallization of gelatin and the XRD pattern showed an amorphous halo.
This peak could be associated with the short range order of protein chains. In addition, the intensity
of amorphous peak decreased with the increase in crosslinking degree. Probably, the crosslinking
hindered the peptide chains re-ordering.
The hydrophilicity of the electrospun meshes was evaluated by measuring their water contact
angles (Figure 3a, Table 1). The contact angle of uncrosslinked gelatin mat could not be measured
because it was absorbed instantaneously when the PBS solution was in contact. However, crosslinked
gelatin nanofibers are stable at these conditions. As expected, the CA values of crosslinked gelatin
confirm the hydrophilic character of matrices. UV irradiation time could affect not only crosslinking
extension, but could also modify the structure, as can be seen in SEM images. Thus, CA values are
determined by both chemical and morphological properties.

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Figure 3. Results of (a) CA, (b) WR, (c) Tensile properties, (d) cell viability of GelMA matrices
Although water uptake measurements were carried out for all samples, only crosslinked gelatin
electrospun mats were stable in the PBS solution (Figure 3b, Table 1). Swelling percentages of the
matrices were around 300%, demonstrating the high hydrophilic character and wettability of
matrices. There were no significant differences between water uptake values of NG-6UV and NG-
9UV samples. However, NG-12UV showed the lowest swelling degree. This fact can be probably
ascribed to the major crosslinking extension of this sample, which clearly affects the water uptake
capability.
In order to confirm the crosslinking, the mechanical properties of NG-9UV and NG-0UV
nanofiber mats were tested (Figure 3c). A uniaxial tensile testing was performed and the Young’s
modulus (YM) was determined for both matrices. YM value of NG-9UV sample was seven times
higher than the measured for NG-0UV (0.717 ± 0.001 MPa and 4.89 ± 0.03 MPa, respectively). Thus,
the crosslinking methodology used in this work for GelMA curing was successful. Moreover, a
relationship between the morphological/chemical properties and UV irradiation time was observed.
Thus, a UV irradiation time of 9 min was chosen for further studies, according to the obtained results.
3.2. Micropatterned Nanofibrous GelMA Scaffolds
3.2.1. Fabrication of Micropatterned Molds
3D structures were created by optical lithography of epoxy-based photoresist on silicon wafers,
obtaining 115 μm thick molds. Designs included patterns of 50 μm, 100 μm, 200 μm, and 400 μm.
Figure 4 shows a microscope image (BX 51, Olympus) of a chip with 200 μm structures.

Figure 3. Results of (a) CA, (b) WR, (c) Tensile properties, (d) cell viability of GelMA matrices.
Although water uptake measurements were carried out for all samples, only crosslinked gelatin
electrospun mats were stable in the PBS solution (Figure 3b, Table 1). Swelling percentages of the
matrices were around 300%, demonstrating the ig hydrophilic character and wettability of matrices.
There were no significant differences between water uptake values of NG-6UV and NG-9UV samples.
However, NG-12UV showed the lowest swelling degree. This fact can be probably ascribed to the
major crosslinking extension of this sample, which clearly affects the water uptake capability.
In order to confirm the crosslinking, the mechanical properties of NG-9UV and NG-0UV nanofiber
mats were tested (Figure 3c). A uniaxial tensile testing was performed and the Young’s modulus (YM)
was determined for both matrices. YM value of NG-9UV sample was seven times higher than the
measured for NG-0UV (0.717 0.001 MPa and 4.89 0.03 MPa, respectively). Thus, the crosslinking
methodology used in this work for GelMA curing was successful. Moreover, a relationship between
the morphological/chemical properties and UV irradiation time was observed. Thus, a UV irradiation
time of 9 min was chosen for further studies, according to the obtained results.
3.2. Micropatterned Nanofibrous GelM Scaffolds
3.2.1. Fabrication of Micropatterne Molds
3D structures were created by optical lithography of epoxy-based photoresist on silicon wafers,
obtaining 115 m thick molds. Designs included patterns of 50 m, 100 m, 200 m, and 400 m.
Figure 4 shows a microscope image (BX 51, Olympus) of a chip with 200 m structures.
Nanomaterials 2019, 9, x FOR PEER REVIEW 8 of 12
Figure 3. Results of (a) CA, (b) WR, (c) Tensile properties, (d) cell viability of GelMA matrices
Although water uptake measurements were carried out for all samples, only crosslinked gelatin
electrospun mats were stable in the PBS solution (Figure 3b, Table 1). Swelling percentages of the
matrices were around 300%, demonstrating the high hydrophilic character and wettability of
matrices. There were no significant differences between water uptake values of NG-6UV and NG-
9UV samples. However, NG-12UV showed the lowest swelling degree. This fact can be probably
ascribed to the major crosslinking extension of this sample, which clearly affects the water uptake
capability.
In order to confirm the crosslinking, th mechanical properties of NG-9UV and NG-0UV
nanofiber mats were tested (Figure 3c). A uniaxial tensile testing was performed and the Young’s
modulus (YM) was determined for both matrices. YM value of NG-9UV sample was seven times
higher than the measured for NG-0UV (0.717 ± 0.001 MPa and 4.89 ± 0.03 MPa, respectively). Thus,
the crosslinking methodology used in this work for GelMA curing was successful. Moreover, a
relationship between the morphological/chemical properties and UV irradiation time was observed.
Thus, a UV irradiation time of 9 min was chosen for further studies, according to the obtained results.
3.2. Micropatterned Nanofibrous GelMA Scaffolds
3.2.1. Fabrication of Micropatterned Molds
3D structures were created by optical lithography of epoxy-based photoresist on silicon wafers,
obtaining 115 μm thick molds. Designs included patterns of 50 μm, 100 μm, 200 μm, and 400 μm.
Figure 4 shows a microscope im 0 μm structures.
Figure 4. Microscope images of micropatterned mold.

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3.2.2. Fabrication of Micropatterned Nanofibrous GelMA Scaffolds
Once molds were fabricated, micropatterned nanofibrous scaffolds were obtained.
The electrospun mats were collected over micropatterned molds and then they were peeled
out carefully. GelMA nanofibrous mats were obtained using five different types of surface
topographies: concentric-circles, dot, parallel-lines, random-lines (maze), and squares. After
crosslinking, samples were examined by SEM. The electrospun mats successfully reproduced the
micropatterned design of the molds, keeping the nanofibrous structure when the size of structures are
200 m or more. Two of the micropatterned designs are shown in Figure 5.
Surface roughness is among the key parameters for biomaterials, which affect the cell attachment
and proliferation. Roughness measurements were carried out on the patterned GelMA electrospun
mats (Table 2). Fibers produced with 400 m of thickness (NG-P400) were rougher than the sample
produced with 200 m of thickness (NG-P200). The depth of the patterns on the mats can be estimated
from the roughness values recorded at Z-axis. The depth of the step on the mat NG-P400 was around
4 m, whereas the depth of the step on the NG-P200 was estimated to be 2.5 m. For all GelMA
fiber mats with different topographies, the measured values of roughness showed an increase of
surface patterning.
Table 2. Roughness values 1 of patterned GelMA electrospun mats.
Sample Ra ( m) Rz ( m) Rmax ( m)
NG-P400 1.5 0.5 4.0 0.2 14 1
NG-P200 0.7 0.05 2.5 0.4 6 1
1 Ra: arithmetical mean deviation of the assessed profile, Rz: arithmetical mean deviation of the assessed profile,
and Rmax: maximum peak-to-valley height.
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Figure 4. Microscope images of micropatterned mold.
3.2.2. Fabrication of Micropatterned Nanofibrous GelMA Scaffolds
Once molds were fabricated, micropatterned nanofibrous scaffolds were obtained. The
electrospun mats were collected over micropatterned molds and then they were peeled out carefully.
GelMA nanofibrous mats were obtained using five different types of surface topographies:
concentric-circles, dot, parallel-lines, random-lines (maze), and squares. After crosslinking, samples
were examined by SEM. The electrospun mats successfully reproduced the micropatterned design of
the molds, keeping the nanofibrous structure when the size of structures are 200 μm or more. Two of
the micropatterned designs are shown in Figure 5.
Surface roughness is among the key parameters for biomaterials, which affect the cell attachment
and proliferation. Roughness measurements were carried out on the patterned GelMA electrospun
mats (Table 2). Fibers produced with 400 μm of thickness (NG-P400) were rougher than the sample
produced with 200 μm of thickness (NG-P200). The depth of the patterns on the mats can be estimated
from the roughness values recorded at Z-axis. The depth of the step on the mat NG-P400 was around
4 μm, whereas the depth of the step on the NG-P200 was estimated to be 2.5 μm. For all GelMA fiber
mats with different topographies, the measured values of roughness showed an increase of surface
patterning.
Table 2. Roughness values 1 of patterned GelMA electrospun mats.
Sample Ra (µm) Rz (µm) Rmax (µm)
NG-P400 1.5 ± 0.5 4.0 ± 0.2 14 ± 1
NG-P200 0.7 ± 0.05 2.5 ± 0.4 6 ± 1
1 Ra: arithmetical mean deviation of the assessed profile, Rz: arithmetical mean deviation of the
assessed profile, and Rmax: maximum peak-to-valley height.

Figure 5. Optic images of 200 μm row and maze patterned molds (a and d, respectively) and SEM
images of 200 μm patterned GelMA electrospun mats (b and c: row patterns; e and f: maze patterns).
3.3. Cell Viability
GelMA electrospun mats were tested in vitro to determine the biocompatible character of the
process (Figure 3d). Cell viability percentages of all samples were above 90%. The high number of
living cells indicates that the crosslinking process and the modified gelatin were compatible with the
used cell-line MG-63. Thus, gelatin based electrospun mat with micro- and nanotopographical
features have a great potential for tissue engineering.
Figure 5. Optic images of 200 m row and maze patterned molds (a and d, respectively) and SEM
images of 200 m patterned GelMA electrospun mats (b and c: row patterns; e and f: maze patterns).
3.3. Cell Viability
GelMA electrospun mats were tested in vitro to determine the biocompatible character of the
process (Figure 3d). Cell viability percentages of all samples were above 90%. The high numb r of
living cells indicates that the crosslinkin process and th modified gelatin were compatible with the
used cell-line MG-63. Thus, gelatin based electrospun mat with micro- and nanotopographical features
hav a great potential for tissue engineering.

Nanomaterials 2019, 9, 120 10 of 12
4. Discussion
The use of gelatin as a biopolymer scaffolding material for tissue engineering applications is
directly related to its high biocompatibility, hydrophilicity, and bioactivity associated with specific
peptide sequences. Electrospinning is a very attractive, complex, and versatile technique to prepare
advanced functional nanofibrous scaffolds for a variety of tissue engineering and cell culture
applications. Current efforts are focused on preparing electrospun scaffolds with controlled multilevel
hierarchical structures. GelMA electrospun scaffolds have been reported using synthetic polymers
and/or hazard, expensive solvents [27–29]. However, a pure gelatin scaffold that mimics the important
features of natural ECM obtained by electrospinning is still pending. In this work, GelMA nanofibers
without defects were obtained using acetic acid, a low toxic potential solvent. Furthermore, in order
to overcome the limitations of the low stability of gelatin in aqueous media as well as the possible
toxicity of chemical crosslinkers, the as-spun scaffolds were crosslinked by UV irradiation at different
exposition times. The same strategy, but while varying the photoinitiator concentration, was reported
by Lai et al. for cultivation of limbal epithelial cells [30].
The GelMA matrix was soluble in PBS before UV irradiation (NG-0UV) while crosslinked GelMA
nanofibers were stable in PBS (NG-6UV, NG-9UV and NG-12UV). The mechanical properties of UV
irradiated mats (NG-9UV) increased 7 times related to mats unexposed to UV (NG-0UV), indicating
the efficiency of crosslinking.
In the last few decades, surface engineering technologies have been used as important tools to
clarify the effects of the microenvironment on cellular behavior [3,11–14]. The fabrication of the surface
topographies with geometrical micro and nanopatterns, like channels, pillars and pits with controlled
dimensions have been possible through the use of various methods as photolithography, electron beam
lithography and microfluidics. These geometric and topographical factors can have an influence in
cell adhesion, migration, differentiation, and the shape of cells. To modulate cell behavior through
surface engineering methods is not only useful to stimuli stem cell differentiation, but also to generate
a favorable response of the implant. In order to control substrate topography, the micropatterns
molding with different geometries and dimensions, were developed to fabricate scaffolds. Molds were
obtained by photolithography and they were used as collector in electrospinning process. Therefore,
GelMA electrospun mats were obtained with nano structure and microroughness. The SEM images
and roughness analysis showed that micropatterns were successfully copied over GelMA electrospun
mats. To the best of our knowledge, this is the first report in literature in which GelMA nanofibrous
scaffolds with micropattern topographical features were designed and prepared.
By combining molding and electrospinning processing, it was possible to obtain scaffolds which
mimic ECM. Thus, this simple and easy technique can be useful for developing sophisticated and
complex materials for tissue engineering and cell culture. In future works, we will study deeply how
different patterns influence the behavior of specific cell lines.
5. Conclusions
In summary, an inexpensive and rapid method for the fabrication of well-defined micro- and
nanotopographic features on uniform bead-free electrospun GelMA fibers were developed in order to
design 3D scaffolds that mimic ECM. The synthesis of GelMA and its processing by electrospinning
was optimized, so therefore, defect-free GelMA nanofibrous mats were obtained. The results showed
that crosslinking took place successfully and it could modulate the final properties. Furthermore,
the use of micropatterned molds obtained by photolithography as a collector in the electrospinning
process allowed controlling the roughness of mats. Taking into account the cell viability results,
the methodology used in this study is a valuable tool to develop patterned GelMA based nanofibrous
scaffolds for cell culture and tissue engineering.
Author Contributions: Ana Agustina Aldana performed the experimental work and wrote the first drafts of
the manuscript and final version, Laura Malatto designed and prepared micropatterned molds, Muhammad

Nanomaterials 2019, 9, 120 11 of 12
Atiq Ur Rehman measured the roughness, Aldo Roberto Boccaccini advised during the project progress and
collaborated in writing the manuscript and Gustavo Abel Abraham contributed to the conception of the project
and collaborated in writing the manuscript.
Funding: This research was funded by CONICET (grant UE73) and MINCYT-DAAD (DA/16/02 Project).
The APC was funded by University of Erlangen-Nuremberg.
Acknowledgments: The authors would like to thank Alina Grünewald and Vanesa Fuchs for technical support in
cell viability and X-ray diffraction studies, respectively.
Conflicts of Interest: The authors declare no conflict of interest.
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