Materials Letters 214 (2018) 224–227

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Materials Letters
journal homepage: www.elsevier.com/locate/mlblue

Biological activity of radiation-induced collagen–polyvinylpyrrolidone–

PEG hydrogels
Maykel González-Torres a,⇑, Gerardo Leyva-Gómez b,1, Margarita Rivera c, Edgar Krötzsch b,
Rogelio Rodríguez-Talavera d, Ana Leonor Rivera a,e, Alejandro Cabrera-Wrooman b,⇑
a
Centro de Ciencias de la Complejidad, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
b
Laboratorio de Tejido Conjuntivo, Instituto Nacional de Rehabilitación ‘‘Luís Guillermo Ibarra Ibarra”, Ciudad de Mexico 14389, Mexico
c
Instituto de Física, Universidad Nacional Autónoma de México, Ciudad de Mexico 04510, Mexico
d
Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Querétaro 76230, Mexico
e
Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: The synthesis of novel degradable hydrogels, with designable structures and good biocompatibility, pro-
Received 13 June 2017 duces attractive materials for use in connective tissue regeneration applications. However, the way in
Received in revised form 21 November 2017 which human fibroblast cells respond to terpolymer hydrogels such as collagen–polyvinylpyrrolidone–
Accepted 3 December 2017
polyethylene glycol (C–PVP–PEG) remains largely unknown. The aim of this study was to synthesize a
Available online 5 December 2017
gamma-radiation-induced C–PVP–PEG hydrogel and to evaluate its structure, morphology and cell viabil-
ity in fibroblasts. Fourier transform infrared spectroscopy showed shifting of amide I to higher frequen-
Keywords:
cies, as evidence of crosslinking produced by the radiation. The morphology of the hydrogel varied with
Hydrogels
Collagen
the ratio of the polymers. We show a proposal for the mechanism of the hydrogel synthesis. The in vitro
Polyvinylpyrrolidone assessment of the hydrogels suggested that at 50 kGy, the presence of collagen decreased the cell viability
Polyethylene glycol for all samples, except for C–PVP. However, when PEG and PVP were added to the collagen, the cell’s via-
Cell viability bility increased with respect to the irradiated collagen sample.
Fibroblasts Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction PVP can be blended via gamma-irradiation with methyl hydrox-

Radiation-induced crosslinking is an environmentally friendly
method used to prepare hydrogels [1]. The main advantages of this
method are that it can be carried out without catalyst or chemical
initiation, it is an effective and fast way to synthesize homoge-
neous three-dimensional networks in one step, and it can be con-
trolled by varying the radiation doses and dose rate. Additive-free
collagen (C) hydrogels have been successfully obtained by this
method. At low doses and high concentrations, the hydrogel was
found to be conducive to the growth of fibroblasts (in vitro) and
showed excellent biodegradability and biocompatibility (in vivo)
[2]. The biological effect of fibrillogenesis on crosslinked collagen
has also enabled it to be utilized as scaffolds for tissue engineering
[3]. Other chemical substrates of interest are polyvinylpyrrolidone
(PVP) and polyethylene glycol (PEG). Previous reports showed that
⇑ Corresponding authors.
E-mail addresses: mikegcu@gmail.com (M. González-Torres), acabrera@inr.gob.
mx (A. Cabrera-Wrooman).
1
Current address: Departamento de Farmacia, Facultad de Química, Universidad
Nacional Autónoma de Mexico, Ciudad de México 04510, Mexico.
yethyl cellulose [4] and carrageenan [5] to produce hydrogels for
use in biomedicine and antimicrobial wound dressing. Besides
reports claiming that PEG is a versatile compound that might be
used to prepare hydrogels under mild conditions [6], other studies
have confirmed collagen mimetic peptide-PEG as a novel class of
polymer for use as a synthetic collagen mimic [7]. Moreover,
gamma-radiation played an interesting role in the development
of tissue-engineered hydrogels to form membranes [8] and valves
with acceptable recellularization and durability [9]. Thus, the
preparation of c-irradiated C-PVP, as well as its physicochemical
and functional characterization [10], and the network structure
of superabsorbent hydrogels have been studied [11]. However, it
is not yet known whether the C–PVP–PEG hydrogels can be gener-
ated by radiation-induced polymerization and what is the nature
of the biological activity of these new biomaterials. To the best of
our knowledge, this terpolymer has not been reported on. The
aim of this work is to evaluate the synthesis of the proposed hydro-
gel, to survey its structure, and to investigate the cell viability in
fibroblasts.

https://doi.org/10.1016/j.matlet.2017.12.006
0167-577X/Ó 2017 Elsevier B.V. All rights reserved.
 M. González-Torres et al. / Materials Letters 214 (2018) 224–227
2. Experimental section
2.1. Materials
The pepsinized porcine (type I) collagen (C) was dialyzed before
use (5 mM, acetic acid, DMS Branch Pentapharm, Aesh, Switzer-
land). Polyvinylpyrrolidone (PVP, ISP Investments, Inc., Wilming-
ton, DE, U.S.A.) and polyethylene glycol (PEG, Sigma-Aldrich, St.
Louis, MO, U.S.A.) of 8000 and 400 Da respectively were used.
The polymers were mixed in a phosphate buffer solution (PBS,
0.1 M, pH = 7.4, Sigma-Aldrich, St. Louis, MO, U.S.A.). Standard
autosampler vials with cap and septa, 12  32 mm, 8–425 thread
(Sigma-Aldrich) were employed to prepare the solutions.
2.2. C–PVP–PEG hydrogel preparation
Several solutions in the PBS were prepared; the first one
(labeled M1) was roughly 0.35 mg/mL of collagen; the second
one (M2) was a mixture of C + PEG (0.35 mg/ml; 50 mg/ml); the
third sample (M3) contained C + PVP (0.35 mg/ml; 25 mg/ml),
while the samples M4, M5 and M6 consisted of C–PVP–PEG mix-
tures with increasing concentrations of PVP/PEG polymers keeping
the collagen concentration constant for all samples: M4 (0.35 mg/
ml; 50 mg/ml, 25 mg/ml), M5 (0.35 mg/ml; 125 mg/ml, 50 mg/ml),
and M6 (0.35 mg/ml; 150 mg/ml, 75 mg/ml). The synthesis of the
hydrogel was achieved via the simultaneous irradiation method,
where the mixture of polymers is subjected to the source of
60
Co-c-radiation in air (Gamma Beam 651PT, Nordion Interna-
tional), at a dose rate of approximately 3.6 kGy/h and a dose of
50 kGy (measured with a Fricke dosimeter). Argon flushing was
carried out to assist in degassing the samples before irradiation.
2.3. Hydrogel characterization
Fourier transform infrared (FTIR) spectra were recorded on a
Spectrum TwoTM spectrophotometer (Perkin Elmer, U.S.A.) in order
to estimate the functional groups presented in the wet and dry
hydrogels. We prepared previously the hydrogels for scanning
electron microscope (SEM) study. The polymers were dried in ace-
tone solution by critical point drying (Critical Point Dryer CPD,
K850, Quorum). The morphological aspects of the sputter-coated
(carbon) dehydrated hydrogels were studied by SEM (JEOL, JSM-
5600 LV).
2.4. Cytotoxicity study
For this experiment, human fibroblast (BJ1) cell line (generously
donated by PhD. Iván Velasco UNAM, México) with different
hydrogels was cultivated in 96 well plates. 2x104 cells were plated
in each well with 200 ml of Dulbecco’s Modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum and 5% of
antibiotic for 24 h at 5% CO2 and 37 °C. The hydrogels were
removed and added to the medium of 10 ml of 5 mg/ml 3-(4,5-dime
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and
incubated for 3 h [12]. The supernatant was removed, and each
well was washed with saline solution at room temperature. Subse-
quently, the formazan precipitate was dissolved with 200 ml of
DMSO/isopropanol 1:1, and then measured by colorimetry at
570 nm with an iMARKTM microplate Absorbance Reader (Bio-Rad
Laboratories, Inc). Microscopy images were obtained using an
Axion Observer Z1, Carl Zeiss inverted light microscope with a 1
0  N.A.
225
3. Results and discussion
Fig. 1 shows the FTIR of the PVP, PEG, and M1–M6 samples. As
can be seen in Fig. 1a, the signals at 3000–2800 cm 1, 1651 cm 1,
1436 cm 1, and 1282 cm 1 correspond to C-H stretching, carbonyl
absorption peak, characteristic C-H stretching vibrations (1493–
1423 cm 1) and C-N stretching of PVP, respectively. The figure also
shows the hydroxyl signal (3445 cm 1) and the skeletal vibration
of PEG (1100 cm 1) [13]. It is of note that collagen displays a broad
band, centered at 3290 cm 1 and assigned to amide A (Fig. 1a and
b). The stretching vibrations at 3074 cm 1 and 2941 cm 1 are asso-
ciated with the amide B. The signal at 1628 cm 1 (amide I) is par-
ticularly useful for assessing conformational changes. Two other
bands of interest are observed at 1528 cm 1 and 1229 cm 1, which
are attributed to amide II (bending) and amide III (stretching)
vibrations. The shifting towards higher wavenumbers, for instance,
from 1628 cm 1 to 1641 cm 1, reveals an evidence of crosslinking
of C + PEG (M2; Fig. 1a). The most striking feature of the M3 sample
is the decrease of the bending vibration (C-N) and also the shifting
of amide I (1628–1658 cm 1, Fig. 1a; Fig. S1, supplementary data)
[11]. The M4–M6 spectra correspond to the prepared terpolymer
(C–PVP–PEG). As shown, they follow the same trend as the M2
and M3 samples, which indicates that the polymers are cross-
linked. In addition, a decrease can be observed in the relative
intensity of the amide II and III and the broadening of amide A
and B. This result suggests that PVP and PEG are forming inter-
molecular interactions through hydrogen bonds with collagen,
and also that the collagen backbone has chemically reacted with
these polymers [14].
The morphology of the samples was characterized by SEM
(Fig. 2a–f). As seen, the hydrogels have a porous structure induced
by the critical point dryer. The size of the pores decreased for sam-
ple M3 (Fig. 2c). The increase of the PVP/PEG ratio caused the
absence of pores in samples M5 and M6 (Fig. 2e and f) [15].
Here, we show the proposal for the mechanism of synthesis of
the C–PVP–PEG (Fig. S2, supplementary data). First, the presented
species are formed by the radiolysis of water. Second, the PEG,
PVP and collagen radical are produced by hydrogen abstraction.
Third, crosslinked species and macro-radicals can be obtained by
radical coupling and propagation reactions. Finally, the macro-
radicals of PVP and PEG react with collagen macro-radical to yield
complex structures as shown at the end of the reaction. We repre-
sent the collagen as Gly-X-Y, where Gly stands for glycine, while X
and Y symbolize proline and hydroxyproline.
Measurement of the cell viability in fibroblasts showed a
decrease of 78% in the cellular viability value for irradiated colla-
gen (Fig. 3, M1). These results are corroborated with the phase con-
trast images (Fig. 4b). A compact collagen cylinder was formed in
the solution (see graphical abstract). It is suggested that the
high-dose radiation affected irreversibly the collagen structure
[10]. The addition of PEG produced an increase in cell viability with
respect to collagen (Fig. 4c, M2). Previous reports indicate that PEG
can be used to protect against the toxic effect on the cells at high
radiation doses [16]. Interestingly, the combination of C–PVP
(M3) showed a better cell viability (Fig. 4d). In an earlier work, it
was demonstrated that radiation-induced C–PVP hydrogel can be
obtained and structurally characterized [10]. Furthermore, the
addition of different proportions of PEG + PVP to collagen (M4–
M6) showed a reduction of cell viability to levels similar to that
of C–PEG. The samples did not show significant differences in the
absorbance values for cell viability. The new polymer configuration
seems to be adapting better to the cell line than irradiated collagen
(Fig 3, M1). However, with a greater increase in the amount of
PVP–PEG there is a marked alteration in the cell morphology
(Fig. 4f and g).
226 M. González-Torres et al. / Materials Letters 214 (2018) 224–227

Fig. 1. FTIR spectra of: (a) the samples PVP, PEG, and M1-M6; and (b) sample M1.

(a) (b) (c)

(d) (e) (f)

Fig. 2. SEM images of the samples: M1 (a), M2 (b), M3 (c), M4 (d), M5 (e) and M6 (f).

0.12

0.10
Absorbance

0.08

0.06

0.04

0.02

0.00
0

6
M

Fig. 3. Cell viability assay of samples M0 (control) and M1–M6 in fibroblasts.

 M. González-Torres et al. / Materials Letters 214 (2018) 224–227
(a) (b)
(d) (e)
Fig. 4. Phase contrast images of: basal (a), M1 (b), M2 (c), M3 (d), M4 (e), M5 (f) and M6 (g) samples.
4. Summary
A C–PVP–PEG hydrogel was prepared by gamma-radiation-
induced polymerization. The terpolymer was structurally charac-
terized by FTIR and SEM. We also studied the cell viability in
fibroblasts. The spectroscopic analysis verified the synthesis of
the hydrogel by changes in the amide I signal of collagen, which
is conformation dependent. The evaluation of the morphology
revealed that the porosity of the samples decreased with the
increasing PEG/PVP concentration. The proposed reaction mecha-
nism highlights the great complexity of this reaction. Moreover,
collagen hydrogel showed major damage at 50 kGy, leading to
low viability of the fibroblasts. This effect was countered with
the addition of PEG/PVP. The novel material showed some
improvement in cell viability at lower PEG/PVP concentrations
than at higher ratios, probably due to the increase of the crosslink-
ing density and degradation. These results provide a viable
approach to prepare tridimensional terpolymer scaffolds for cell
attachment.
Disclosure Summary
The authors have no conflicts to disclose.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.jcis.2017.12.006.
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