Materials Science and Engineering C 74 (2017) 347–356

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Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Heparin binding chitosan derivatives for production of pro-angiogenic

hydrogels for promoting tissue healing
Muhammad Yar a,⁎, Sohail Shahzad a,b, Lubna Shahzadi a, Sohail Anjum Shahzad c, Nasir Mahmood d,f,
Aqif Anwar Chaudhry a, Ihtesham ur Rehman a,e, Sheila MacNeil e,⁎
a
Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000, Pakistan
b
Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
c
Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
d
Department of Allied Health Sciences and Chemical Pathology, University of Health Sciences, Lahore, Pakistan
e
Materials Science and Engineering, North Campus, University of Sheffield, Broad Lane, Sheffield S3 7HQ, UK
f
Department of Human Genetics and Molecular Biology, University of Health Sciences, Lahore, Pakistan

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

Article history: Our aim was to develop a biocompatible hydrogel that could be soaked in heparin and placed on wound beds to
Received 29 July 2016 improve the vasculature of poorly vascularized wound beds. In the current study, a methodology was developed
Received in revised form 10 November 2016 for the synthesis of a new chitosan derivative (CSD-1). Hydrogels were synthesized by blending CSD-1 for either
Accepted 5 December 2016
4 or 24 h with polyvinyl alcohol (PVA). The physical/chemical interactions and the presence of specific functional
Available online 7 December 2016
groups were confirmed by Fourier transform infrared (FT-IR) spectroscopy and proton nuclear magnetic reso-
Keywords:
nance (1H NMR). The porous nature of the hydrogels was confirmed by scanning electron microscopy (SEM).
Chitosan Thermal gravimetric analysis (TGA) showed that these hydrogels have good thermal stability which was slightly
Hydrogels increased as the blending time was increased. Hydrogels produced with 24 h of blending supported cell attach-
Poly vinyl alcohol ment more and could be loaded with heparin to induce new blood vessel formation in a chick chorionic allantoic
Heparin membrane assay.
Angiogenesis © 2016 Elsevier B.V. All rights reserved.

1. Introduction
Hydrogels are attractive in biological systems because of their three-
dimensional network and their ability to absorb large amounts of fluids
without dissolution [1,2]. They are used in various biomedical applica-
tions such as biosensors [3], membranes [4], artificial organs [5], carriers
for controlled drug delivery [6,7], tissue engineering [8,9] and wound
dressings [10]. Their soft and compliant nature also makes them attrac-
tive in terms of mechanical properties as scaffolds for tissue replace-
ment [11,12].
Biocompatible polymers can be synthesized from many materials
and by many routes [13,14]. Chitosan is one of the most widely available
polysaccharides in nature, usually extracted from marine sources, and
has a high capacity for supporting cell proliferation and differentiation
[15]. Therefore, chitosan has been widely used for drug delivery, gene
therapy and tissue engineering applications due to its biocompatibility,
biodegradation properties [1,16], non-toxicity, and anti-bacterial activi-
ty [1]. Chitosan derivatives have been used as anti-bacterial agents [17,
⁎ Corresponding authors.
E-mail addresses: drmyar@ciitlahore.edu.pk (M. Yar), s.macneil@sheffield.ac.uk
(S. MacNeil).
18], as a BACE1 inhibitor [19], as antifungal agents [20] and as an antiox-
idant agent [21,22]. In recent years, hydrogels made of chitosan and chi-
tosan derivatives have been investigated for skin regeneration [8], drug
release [23] and human osteoblast proliferation [24]. Accordingly, spe-
cial attention has been paid to its chemical modification to obtain deriv-
atives with the improved required properties [22]. Chitosan chemical
modifications can achieve better physiochemical, biochemical and bio-
logical properties such as improved solubility [25,26], better carrier effi-
ciency for gene therapy [27], antioxidant activities [21,28], antibacterial
properties [18,29], better cell adhesion [30,31], adsorption of cationic
dyes [32,33], wound healing applications [34] and controlled drug re-
lease [35]. Similarly, chemical modification of chitosan gives an impor-
tant derivative carboxymethyl chitosan which has distinctive
chemical, physical and biological properties of high viscosity, good bio-
compatibility, a large hydrodynamic volume and the ability to form
films, fibers and hydrogels [36,37]. Thus, it has been widely used as a
moisture-retention agent, bactericide, wound dressing agent, blood an-
ticoagulant and for drug delivery applications [38–40].
The existing methods of chitosan chemical modification include
alkylation [41,42], acylation [43], quaternization [44,45], hydroxyalky
lation [46], carboxyalkylation [47,48], thiolation [49], sulphation [50],
mesylation [51] and graft copolymerization [52].

http://dx.doi.org/10.1016/j.msec.2016.12.021
0928-4931/© 2016 Elsevier B.V. All rights reserved.
348 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356
PVA is a cheap, non-toxic and water soluble synthetic polymer. It has
a film forming ability and provides porous and hydrophilic interfaces
which interact well with human tissues. It is widely used in many bio-
medical applications including contact lenses, artificial muscle, burn
wound dressings and vocal cord reconstruction [53–55]. The compati-
bility of PVA with tissues and blood (plasma proteins) has been widely
reported, it has been used in numerous blood contacting devices and tis-
sues such as drug-delivery systems, wound dressings, dialysis mem-
branes, cardiovascular devices, artificial cartilage, tendon, ligament,
cornea, lens, skin, and intervertebral disc [56–58]. It is also non-carcino-
genic with good biocompatibility and a high degree of swelling in aque-
ous solutions [59–61].
The Mannich reaction is a well-known and commonly used method
for the derivatization of amines. The process is simple and cost-effective
because of easily available inexpensive reagents and it is also robust due
to the formation of iminium ion during this Mannich reaction. This
iminium ion is quite reactive towards a variety of nucleophiles [62]. As
chitosan contains primary amino group these can easily be modified
by the Mannich reaction and this could be an alternative way to prepare
chitosan derivatives and this was explored in this study.
The aim of this research was the synthesis of Mannich base like diba-
sic chitosan derivatives for tissue engineering applications. An efficient
protocol was also developed for the production of porous pro-angiogen-
ic hydrogels from a Mannich base chitosan derivative suitable for bind-
ing heparin and promoting new blood vessel formation in wound beds.
Heparin is a negatively charged protein which has been exploited by
many groups, including our own [63,64], for its pro-angiogenic proper-
ties. There are several methods for loading heparin into materials in-
cluding covalent or ionic interactions. Here we sought to bind heparin
electrostatically.
In summary we report the production of a blend of PVA with a new
chitosan derivative by a Mannich reaction to produce 3D porous hydro-
gel membranes which readily bind heparin and promote new blood
vessel formation.
2. Experimental
2.1. Materials
Chitosan (CS) (degree of deacetylation (DD) 83%, intrinsic viscosity
30.78 ml/g, Mw: 110,019.06 g/mol) was synthesized in our laboratory
using the procedure described in Section 2.2. Polyvinyl alcohol (PVA)
(Mw: 72,000, degree of hydrolysis 98%), hydrochloric acid (HCl), form-
aldehyde, 2-chloro aniline was purchased from Merck (Germany). Gla-
cial acetic acid (CH3COOH) was purchased from AnalaR BDH Laboratory
Supplies (UK). NaOH and MeOH were purchased from Sigma Aldrich.
Heparin was supplied by B. Braun Melsungen AG (Germany). Ethanol
(99.8%) was purchased from Riedel-deHaen (Germany). Phosphate
buffer saline (PBS) (pH 7.4) tablets were acquired from bioPlus Fine Re-
search Chemicals (USA).
2.2. Synthesis of chitosan
CS was synthesized from chitin, extracted from shrimp shells, as pre-
viously described [65,66] with some modifications. For the detailed pro-
cedure of chitin extraction and chitosan synthesis, please see our
previously published papers [67,68].
2.3. Synthesis of chitosan derivatives (CSD-1)
1 g CS was dissolved in 16 ml of mixture of CH3OH/CH3COOH with a
ratio of (3:1), respectively. The solution was stirred for 15 min at 70 °C.
Then 0.48 ml of formaldehyde was added and the solution was stirred
for 5 min at 80 °C. Then the respective amount of 2-chloroaniline was
added and the solution was stirred and refluxed for 17 h at 80 °C.
Then the solution was cooled and filtered. The product was washed
with a minimum amount of methanol. A yellow coloured product was
obtained. The abbreviation CSD-1code was used to represent the CS
derivative.
The schematic representation of the chemical reaction for the prep-
aration of CSD-1 is given in Scheme 1.
2.4. Synthesis of CSD-1/PVA (CSDP) blended hydrogel films
0.3 g of CSD-1 was dissolved and stirred in 10 ml of 1% acetic acid so-
lution at room temperature. Then 0.3 g of PVA was dissolved separately
in a round bottom flask in 10 ml of distilled water at 80 °C. Both solu-
tions were mixed together and stirred for 4 h at room temperature.
After 4 h, half of the solution was taken out and the remaining half
was stirred for a further 20 h at room temperature. Hydrogel films
were prepared by pouring the homogenized mixture into separate
petri dishes. Petri dishes were placed in a vacuum oven at room temper-
ature for 2 h to remove air bubbles and then in a freezer at −80 °C for
24 h. Finally, hydrogels were lyophilized for 24 h at − 56 °C using a
Christ (Alpha 1–2 LD plus) Freeze-Dryer (Christ, Alpha 1–2 LD plus, Ger-
many). This resulted in blended solutions forming hydrogel films. The
codes CSDP-4 and CSDP-24 and CSDP-24-H are used to represent the
CSD-1/PVA hydrogel films with 4 and 24 blending hours and 24 h blend-
ed hydrogel with heparin loading, respectively.
2.5. Characterization of CS derivatives and CSDP blended hydrogel films
2.5.1. 1H NMR analysis
1
H NMR spectra of CSD-1 was recorded in CH3COOHD2O with Bruker
AM 500 spectrometers (Rheinstetten – Forchheim, Germany) operating
at 400 MHz. 1H chemical shifts are reported in δ (ppm).
2.5.2. FTIR analysis
Chemical characterization of the materials (chitosan, CSD-1, PVA,
CSDP-4 and CSDP-24) was carried out using (FTIR) spectroscopy
coupled with photo acoustic sampling cells, which allows the analysis
of neat materials without the need for any sample preparation. Spectra
were recorded within 4000–400 cm−1, averaging 256 numbers of scans
at 8 cm−1 resolution on a Thermo-Nicolet 6700 P FTIR Spectrometer
(USA).
2.5.3. Thermal gravimetric analysis
Thermal Gravimetric Analysis (TGA) and Differential Scanning Calo-
rimetry (DSC) were carried out to evaluate the thermal stability and to
determine decomposition temperature of the CS, CSD-1, PVA, CSDP-4
and CSDP-24 using a Q600 DSC/TGA instrument. DSC/TGA runs were
carried out in a temperature range of 12–500 °C with a constant heating
rate of 10 °C min−1 under nitrogen purging rate of (100 ml min−1) in all
the experiments.
2.5.4. Scanning electron microscopy
The surface morphology of the hydrogel films (CSDP-4 and CSDP-
24) was examined by SEM, JEOL model number JSM 6480. The samples
were gold sputter coated prior to their SEM analysis. The images were
analyzed at different magnifications.
2.6. Solution absorption experiments
The solution absorption behavior of the CSD-1/PVA (CSDP) blended
hydrogel films was evaluated by using 10 mg of each well dried (CSDP)
hydrogel film, which was immersed in 10 ml of PBS solution (pH = 7.4)
in a vial at 37 °C. The sample was withdrawn from the PBS solution at
regular intervals of 10 min and the excess liquid removed by patting
with tissue paper. Then the swollen weight of the sample was measured
and the sample was again immersed in the vial containing PBS solution
until equilibrium swelling of the samples was reached. PBS solution
 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356
Scheme 1. Schematic representation of synthesis of CSD-1 novel derivative from chitosan via the Mannich reaction.
absorption % (S) was measured according to the following equation:
S ¼ ðWs −Wd =Wd Þ  100
where Ws and Wd represent the weight of swollen and dried state sam-
ples, respectively.
2.7. Cell compatibility of CSD-1/PVA blended hydrogel films
The cell viability/cyto-compatibility of blended hydrogel films was
investigated by using the VERO cell line (derived from kidney epithelial
of African green monkey) and using a neutral red dye (NRD) uptake
assay as described by Repetto et al. [69] with some modifications. The
cell line was kindly provided by the Veterinary Research Institute
(VRI) Lahore. Cells were grown under aseptic conditions in GMEM
(Glasgow Minimum Essential Medium, Sigma Aldrich) supplemented
with 10% FBS (fetal bovine serum, Gibco) in a 25 cm3 cell culture flask
with a humidified atmosphere of 5% CO2, 95% air at 37 °C and then sub-
jected to trypsinization by 0.2 ml trypsin-EDTA solution (0.05% trypsin
and 0.5 mM EDTA, pH 8.0) at 80% confluence. For the NRD assay, both
the hydrogel films were cut to fit into the base of 96 well flat bottom
plates. The hydrogel films were sterilized with 70% ethanol for 5 min
followed by washing with distilled autoclaved sterile water to remove
ethanol traces before fitting into the bottom of each well in the culture
plate under strict aseptic conditions. GMEM medium (0.02 ml) contain-
ing a cell suspension at a density of 2 × 105 cells/ml was added to each
well containing hydrogel films. Wells without hydrogel films and con-
taining 0.02 ml GMEM medium with cells suspension were used as
the control. After adding the suspension to each well, the plate was in-
cubated at 37 °C for 72 h with 5% CO2 supply. After the incubation peri-
od, the medium was gently decanted from the wells and each well was
rinsed with 0.02 ml of 1 × PBS solution twice very gently without
disturbing the hydrogel films at the bottom of the wells. 0.02 ml of
GMEM medium containing neutral red dye (4 mg/10 ml of GMEM)
was added into each well and incubated again for 1 h at 37 °C. After
1 h of incubation, the dye containing medium was decanted and each
well was rinsed gently three times with PBS solution to remove the un-
absorbed neutral red dye contained in the wells. 0.02 ml of acidified eth-
anol (50% ethanol and 1% acetic acid) was added to each well and
incubated for 10 min at 37 °C. The acidified ethanol from each well
was pipetted out and poured into sterilized Eppendorf tubes. The absor-
bance of acidified ethanol solution containing extracted neutral red dye
was measured at 540 nm using a spectrophotometer (Perkin Elmer
Lambda 25). We examined the cytotoxicity of the CSDP-24-H derivative
without heparin. We did not perform cytotoxicity experiments for the
heparin loaded hydrogel (CSDP-24-H), as we and many others have
previously shown that heparin is not cytotoxic.
2.8. Loading of heparin
Heparin was loaded onto the CSDP-24 and CS-H hydrogel. To load
heparin, a solution was prepared containing 1 mg/ml of heparin in dis-
tilled water. Then the scaffolds were submerged in the solution over-
night at room temperature. The hydrogels absorbed the majority of
349
this solution. The resulting heparin containing scaffolds were dried at
room temperature for 24 h.
2.9. Assessment of angiogenic activity for heparin loaded scaffolds using the
chorioallantoic membrane (CAM) assay
For the CAM assay, fertilized chicken eggs were incubated at 37 °C
from days 2 to 7 of fertilization. At day 8, a small window was cut
(1 cm2), sterilized and a PBS washed hydrogel sample was placed
onto the CAM. Each egg was implanted with one hydrogel sample. The
egg shell was closed with parafilm and eggs were again incubated
until day 14. At day 14, the eggs were opened and scaffolds were re-
trieved and eggs were sacrificed. Angiogenesis was initially recorded
by taking light microscope pictures of the samples before sacrificing
the eggs. Eight eggs were used for each group and only results from
eggs where embryos were clearly developing well were assessed for
blood vessel formation.
3. Statistical analysis
Results are expressed as mean ± standard deviation. The cell culture
data were analyzed by one way analysis of variance (ANOVA), and then
Dunnett's test was used to analyze multiple differences between the
groups and control by using IBM-SPSS software, version 21. A probabil-
ity of b0.05 was considered to be statistically significant.
4. Results and discussion
The hydrogels CSDP-4 and CSDP-24 were prepared using the same
method except CSDP-4 was blended for 4 h whereas CSDP-24 was
blended for 24 h. The longer blending time made the reaction mixture
more homogenous. This homogeneity clearly affected the TGA/DSC re-
sults, appearance, SEM results and swelling responses of the two pre-
pared hydrogels. Both hydrogels were cell compatible with the 24 h
blended hydrogel promoting cell compatibility slightly more than the
4 h blended version. Also following binding heparin the CSDP-24 hydro-
gel significantly induced angiogenesis in the CAM assay.
4.1. 1H NMR analysis of CSD-1 (Fig. 1a)
1
H NMR (D2O/CF3COOH (few drops), 400 MHz) δ 2.36 (s, CH3
acetamide), 3.4–3.6 (m, CS ring), 4.46 (s, NHCH2NH), 7.03–7.27 (m,
45aromatic CH).
1
H NMR analysis of chitosan (Fig. 1b)
4.2. FTIR analysis
The FTIR spectra of synthesized CS, CSD, pure PVA and CSD-1/PVA
blended (CSDP-4 and CSDP-24) hydrogel films are shown in Fig. 2(a–
e). FTIR spectrum (a) represents CS which showed a broad peak at
3400–3200 cm−1 for O\\H and N\\H stretching vibrations. The peaks
at 2916 cm−1 and 2881 cm−1 are assigned to asymmetric alkyl C\\H
stretching and symmetric alkyl C\\H stretching respectively. The
amide I band or C _O and amide II band or N\\H bending appear at
1658 cm−1 and 1586 cm−1 respectively. The peaks at 1415 cm−1 and
350 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356

Fig. 1. a) 1H NMR spectrum of CSD-1; b) Chitosan [70].

1381 cm− 1 are due to CH2 bending. The amide III band appears at observed at 895 cm− 1. The nature of the peaks, vibration mode and
1322 cm−1. The peaks at 1151 cm−1 and 1088 cm−1 are due to C\\O vibration frequency of the various groups present in CS are shown in
and C\\O\\C stretching frequencies respectively. CS ring stretching is Table 1.
Fig. 2(b) shows spectra of CSD-1 respectively, which indicated clear-
NH absorption in CS 0 ly different values of certain peaks compared to CS. The N\\H and O\\H
Amide I band groups show a broad peak due to involvement of inter and intra molec-
(a)
ular hydrogen bonding. The stretching absorption frequencies of these
C-O-C stretch
groups (O\\H and N\\H) decreased in CSD-1 compared to chitosan,
0.5
(b) due to the formation of amino group bonds. Thus the number of
Alkyl CH stretch
amino groups in the chitosan skeleton is decreased. The derivative
showed a peak at 2973 cm−1 which is due to an aromatic C\\H stretch.
NH bend
1 This peak is absent in the chitosan spectra, providing evidence for the
Transmittance (%)

(c) attachment of the aromatic ring with the chitosan moiety. The peak at
2901 cm−1 is due to alkyl C\\H stretching frequency in the derivative
spectra. The small peaks at 1650 cm−1 and 1635 cm−1 are due to an
Aromatic CH stretch 1.5
amide I band or C _O present in the CS skeleton, which is a part of
(d) the derivative. These peaks have been split due to the formation of

2 Table 1
FTIR values of chitosan.
(e) sr. no Vibration mode Vibration Nature of peak
frequency
2.5
01 O\
\H/N\
\H stretching 3345 Very broad
peak
02 Asymmetric alkyl C\\H stretching 2916 Sharp peak
03 Symmetric alkyl C\\H stretching 2881 Sharp peak
3
CH bend 04 Amide I band or C_ O 1658 Sharp peak
NH/OH stretch
05 Amide II band or N\\H 1586 Sharp peak
deformation
06 CH2 bending and CH3 deformation 1415 Sharp peak
3.5 07 C\
\H bending 1381 Sharp peak
3900 3400 2900 2400 1900 1400 900 400 08 Amide III band 1322 Sharp peak
Wavenumber (cm-1) 09 C\
\O stretching 1151 Sharp peak
10 C\
\O\ \C stretching 1088 Broad peak
11 Ring Stretching 895 Sharp peak
Fig. 2. FTIR spectra of (a) chitosan (b) CSD-1 (c) PVA (d) CSDP-4 (e) CSDP-24.
 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356
Fig. 3. [A] TGA thermograms of CS, CSD-1, PVA, CSDP-4 and CSDP-24. [B] DSC
thermograms of CS, CSD-1, PVA, CSDP-4 and CSDP-24.
hydrogen bonding of C _O group with other groups. The Amide II band
or N\\H bending peak in derivatives shifted at 1550 cm−1 compared to
1586 cm−1 in CS. This shift in peak indicates the change in CS skeleton
and the formation of new derivative. The absorption of this peak has
also been decreased due to the involvement of an amino group in
bond formation. This also provides strong evidence for the attachment
of a chitosan amino group with formaldehyde and aniline to form a de-
rivative. The FTIR spectrum of the derivative also showed a CH2 bending
and CH3 deformation peak at 1406 cm−1. The absorption of this peak
has been increased due to presence of greater number of CH2 groups
in the derivative as compared to chitosan, providing an evidence for
the formation of derivative. The Amide III band at 1322 cm−1 in CS is
slightly shifted to near1304 cm− 1 in the derivative. The peak at
Fig. 4. Camera images of the (a) CSDP-4 and (b) CSDP-24 blended hydrogel films.
351
1255 cm−1 in derivative spectra is due to C\\N stretching vibration.
The C\\O and C\\O\\C stretching peaks at 1151 cm− 1 and
1088 cm− 1 respectively in CS are slightly shifted to 1155 cm− 1 and
1067 cm− 1 in the derivative. The peak at 895 cm− 1 in CS has been
shifted to 891 cm−1 which is due to a modification in the ring skeleton
of chitosan.
Fig. 2(c) represents a PVA spectrum which showed a very broad
peak from 3500 cm− 1 to 3100 cm− 1, assigned to O\\H group
stretching. The sharp peak at 2943 cm−1 is assigned to C\\H stretching
vibrations of methylene group (CH2). The peak observed at 1710 cm−1
is assigned to C _O stretching vibration of acetyl groups present in the
PVA skeleton. The vibrational band observed at 1409 cm−1 refers to CH2
bending vibrations. The bands at 1143 cm− 1 and 1099 cm− 1 are
assigned to C\\O\\C and C\\O stretching vibrations, respectively.
Fig. 2(d) and (e) represent CSDP-4 and CSDP-24 samples. FTIR spec-
tra of both samples confirmed the presence of incorporated compo-
nents. Both samples show a broad peak from 3500 to 3100 cm−1 due
to the presence of O\\H and N\\H stretching vibrations. The C\\H
stretching vibration peaks appeared at 2942 cm− 1 in both samples.
The peaks at 1710 cm−1 in CSDP-4 and CSDP-24 samples are due to
C _O stretching vibration of acetyl groups of PVA skeleton. Both sam-
ples show peaks at 1586 cm−1 and 1409 cm−1 due to N\\H and C\\H
bending vibrations. The vibration band at 1094 cm−1 is due to C\\O\\C
stretching vibration in both samples. The slight shift in frequency values
of both samples as compared to CSD and PVA indicates formation of hy-
drogen bonding interactions.
4.3. TGA/DSC analysis:
The thermograms of CS, CSD-1, PVA, CSDP-4 and CSDP-24 samples
are shown in Fig. 3A. Weight loss of these composite materials occurred
in three steps. The first weight loss occurred between 20 and 240 °C, re-
lated to loss of moisture contents and glass transition temperature of
PVA and CS. The second weight loss occurred between 240 and
340 °C, related to melting of PVA and CS and CSD-1. The third step oc-
curred between 340 and 500 °C, which is due to thermal degradation
of composite materials.
The major weight loss of pure CS occurred from 280 °C to 340 °C
and 55% of its mass was lost by 500 °C (Fig. 3A). In contrast to this
CSD-1 underwent a major weight loss from 220 °C to 360 °C and
77% of its mass was lost by 500 °C (Fig. 3A). This showed that
there is a large difference in resistance to temperature between
CS and its derivative (CSD-1). Similarly pure PVA showed a major
weight loss from 220 °C to 280 °C and 87% of it was decomposed
by 500 °C (Fig. 3A). The CSDP-4 hydrogel film underwent major
weight losses from 240 °C to 300 °C, with 91% decomposition
(Fig. 3A). Similarly the major weight loss of CSDP-24 hydrogel
352 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356

Fig. 5. (A): (A1–A2): SEM images of the CSDP-4 (4 h) blended hydrogel films at different magnifications (500× and 1000×); (B): (B1–B2): SEM images of the CSDP-24 (24 h) blended
hydrogel films at different magnifications (500×, 1000×).

film occurred from 220 °C to 320 °C and 79% of film was lost by
500 °C (Fig. 3A). These TGA results suggest that the increased
blending time slightly enhanced the thermal stability of the hydro-
gel films as the CSDP-24 film lost 79% compared to 91% for the
CSDP-4 film.
the CSDP-24 hydrogel films was pale yellow. In contrast the
CSDP-24 film components appeared well mixed and the film was
soft and elastic like a sponge. This increase in blending time from
4 to 24 h appeared to improve the mixing of both CSD-1 and PVA
to make the hydrogels more homogenous.

4.3.1. DSC analysis

4.5. Morphological analysis
DSC was used to study the purity, crystallinity and the intermo-
lecular interactions present between the CSD-1 and PVA molecules.
SEM images of CSDP-4 and CSDP-24 freeze dried hydrogel films
DSC curves of CSDP-4 and CSDP-24 hydrogel films are shown in Fig.
at different magnifications are shown in Fig. 5A and B, respectively.
3B. DSC results were in good agreement with those obtained by
The average pore size was taken as an average of the vertical and
TGA curves. There is a melting endothermic peak at 300 °C (Fig. 3B)
the horizontal dimensions of 50 pores by using image analysis soft-
for pure CS. Compared to the pure CS, the CSD-1 did not show a
ware (ImageJ launcher). The film CSDP-4 has a smooth and opaque
clear endothermic peak at any temperature (Fig. 3B). The pure PVA
surface with a porous structure with an average pore size of 11 ±
exhibited a sharp endothermic peak at 280 °C (Fig. 3B), which
0.75 μm (Mean ± SEM). The film CSDP-24 was opaque with an
shows its crystalline nature. The CSDP-4 hydrogel film showed an
endothermic peak at 270 °C (Fig. 3B), while the CSDP-24 hydrogel
film peak appeared at the slightly higher temperature of 275 °C
(Fig. 3B). This increase in temperature was due to the greater blend-
ing time. It is proposed that the increased blending time allowed
some extra molecular interactions in the CSDP-24 hydrogel films
compared to CSDP-4 films.
The DSC curves suggested that as the sharpness of pure PVA was de-
creased in the CSDP-4 and CSDP-24 hydrogel films and the peaks were
broad, this confirmed the amorphous nature of the blended hydrogel
films. The curves also suggested that increasing blending time slightly
enhanced the thermal stability of these hydrogel films.

4.4. Appearance and morphology of the films

The appearances of CSDP-4 and CSDP-24 hydrogel films are

shown in Fig. 4(a–b). The color of the CSDP-4 hydrogel film was
white and this was partially transparent. This hydrogel was clearly Fig. 6. Solution absorption (%) of CSD-1/PVA blended CSDP-4 CSDP-24 and CSDP-24-H
nonhomogenous with dispersed particles throughout. The color of hydrogels.
 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356 353

Fig. 7. [A] Cell culture assay of Vero cells on CS-derivative/PVA freeze dried samples. The data are presented as means ± S.D. (N = 3). The bars on columns represent means ± S.D. [B]
Morphology of Vero cells after contact, attachment and proliferation with CS− Derivative/PVA samples at 40× (Scale bar is 100 μm). (a) Control, without any testing material (b)
CSDP-4 (c) CSDP-24.

Fig. 8. SEM images of CSDP-24 before (A1 and A2) and after (B1 and B2) heparin loading (scale bar is 10 μm and 200 μm, respectively).
354 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356

average pore size of 8 ± 0.58 μm (Mean ± SEM) p b 0.05. The PVA
and CSD-1 content appeared to be distributed in a more uniform
manner in the CSDP-24 sample compared to CSDP-4 sample due
to the increased blending time. In addition, the porous structure
in the CSDP-24 sample appeared to have a sponge-like structure.
Thus, the increase in blending time has induced appreciable chang-
es in the surface structure of the films. Overall, porosity of the films
was improved but a decrease in the pore size was observed with
the increase of blending time.
The surface of the films seems to be changed by increased blending
time and this modification had a slight effect on the resulting culture
of VERO fibroblast cells.
4.6. Solution absorption response in PBS solution (pH 7.4)
The solution absorption response of CSDP-4 CSDP-24 and CSDP-
24-H (heparin-loaded CSD-1/PVA membrane with 24 h blending
time) samples was performed in PBS solution (pH = 7.4) as
shown in Fig. 6. All samples absorbed solution to a large extent in
the first 10 min. All samples showed maximum solution absorption
within 40 min and no further solution was then absorbed. CSDP-24
showed a greater degree of solution absorption than both CSDP-4
and CSDP-24-H. A possible reason is that the CSDP-24 sample has
a greater number of slightly smaller pores than the CSDP-4 hydro-
gel so the overall surface area available for solution absorption was
more in CSDP-24 than CSDP-4. In the case of CSDP-24-H the solu-
tion absorption was less than CSDP-24. This might be due to the
coating of the porous structure by heparin which can be seen in
Section 4.7 Fig. 8 – this might effectively have reduced the surface
area.
4.7. Growth of cells on hydrogels
The neutral red dye (NRD) assay was used to measure the ability
of cells to attach and proliferate on these hydrogels. This assay is
based on the uptake of neutral red dye by diffusion into the living
cells through the cell membrane with subsequent accumulation
in the lysosomes. The accumulated dye is then released using acid-
ified ethanol and quantified spectrophotometrically by reading ab-
sorbance at 540 nm. In this study cell viability was measured after
72 h of seeding VERO cells (derived from kidney epithelial cells of
green monkey) on the substrates. The VERO cells not only attached
well to the substrate but remained viable and proliferated with
growth rates only slightly less than that seen for growth of these
cells on tissue culture plastic. (Fig. 7B). Assuming viability of cells
on tissue culture plastic as 100%, then attachment and growth of
cells on CSDP-4 was 76% and on CSDP-24 86% (Fig. 7A). There was
a slight increase (7%) in the growth of cells on the CSDP-24 hydro-
gel compared to the CSDP-4. This was statistically significant
p b 0.043. The reason for slightly more attachment and prolifera-
tion of cells with the CSDP-24 hydrogel film may be explained by
the smaller pore size (average pore size 8 μm) but greater number
of pores (hence surface area) of the sample mixed for 24 h com-
pared to the sample mixed for 4 h (average pore size 11 μm) [71].
The increase in cell viability using the CSDP-24 membrane could
also be due to the greater homogeneity of this hydrogel achieved
with an increase in the blending time. Heparin loaded hydrogel

Fig. 9. [A] CAM assay for control (left), heparin loaded (middle) hydrogels, heparin loaded CS hydrogel (right) [B] The quantification of angiogenesis of scaffold assessed using the
chorioallantoic membrane (CAM) assay. Scaffolds were placed on the CAM for 7 days and then removed. Before removal of hydrogels from eggs, images were taken by light
microscopy (Motic, China). These images were then blindly scored by four assessors (from two experiments with 5 samples in each). Results are means ± SD of 5 viable chicks
surviving from an original group of 8 fertilized eggs per group. The number of blood vessels was counted inside a circle drawn 1 mm away from the each edge of scaffold. The results
shown are mean ± SD and ****p b 0.0001 for control and CSDP-24-H (significant difference), same value of p was observed for CSDP-24-H and CS-H (****p b 0.0001, significant
difference). The difference between control and CS-H was also significant ***p b 0.0004.
 M. Yar et al. / Materials Science and Engineering C 74 (2017) 347–356
(CSDP-24-H was not included in the cytotoxicity tests because in
our other publications on heparin binding to chitosan derivatives
we have shown that heparin bound to chitosan is not toxic to
cells. Also there is extensive literature that heparin is not cytotoxic
[72].
4.8. Loading of heparin into hydrogels
For heparin loading, CSDP-24 was selected because of the apparent
better homogeneity of this hydrogel and slightly better results in pro-
viding a surface for cell attachment and proliferation. Heparin was
also loaded on chitosan hydrogel (CS-H) for comparative purposes in
the CAM assay.
Heparin was loaded on CSDP-24 and CS-H by physical absorption. A
heparin solution of 1 mg/ml of heparin in distilled water was used and
hydrogels were soaked in this overnight at room temperature and
then dried at room temperature. There were clear differences in appear-
ance as seen by SEM images of hydrogels taken before and after heparin
loading (Fig. 8). It can be seen from the images that while the larger pore
structures were retained the fibrous extensions in between pores disap-
peared following heparin loading and the number of pores per unit area
was reduced.
4.9. Assessment of angiogenic activity using the chorioallantoic membrane
(CAM) assay
The embryonic chicken is well-characterized in vivo biological sys-
tems [73]. The Chorioallantoic Membrane (CAM) is a blood vessel rich
membrane system that surrounds the chick embryo. The assay was per-
formed to examine any difference in response to the angiogenesis po-
tential of heparin-loaded and control (without heparin) chitosan
derivative/PVA (CSDP-24) hydrogels and also heparin-loaded chitosan.
It was evident from Fig. 9A that heparin-loaded hydrogels showed
far more angiogenic activity than the control samples. In control sam-
ples, the vasculature around the hydrogels showed no evidence of
being affected by the presence of hydrogels while in the heparin-loaded
hydrogels the blood vessels covered the hydrogels. There was a thick
network of blood vessels around and over the heparin-loaded samples
which confirmed the angiogenic potential of heparin-loaded CS deriva-
tive/PVA hydrogels (CSDP-24). The heparin-loaded CS derivative
hydrogels also exhibited better angiogenic potential than the CS hydro-
gel even after loading with heparin. This might be due to the better
binding of derivative with heparin as compared to CS. The statistics
showed significant difference in number of blood vessels of CS deriva-
tive hydrogel and CS.
5. Conclusion
In summary, we describe the production of new compounds from
chitosan derivative blended with PVA to produce porous membranes
which are able to bind heparin and their subsequent pro-angiogenic ef-
fects were very promising. FTIR analyses confirmed the presence of a
new chemical functional group (\\NH\\CH2\\NH\\) produced as a re-
sult of the reaction between chitosan and aniline. This was further sup-
ported by the 1H NMR spectroscopy. TGA and DSC investigations
showed the thermal stability of the films prepared from newly synthe-
sized chitosan derivative and PVA and thermal stability was increased as
the blending time was increased from 4 h to 24 h. Similarly, SEM results
showed that increasing the blending time resulted in a more porous and
sponge like structure of hydrogel films. Hydrogel films produced as a re-
sult of 24 h blending (CSDP-24) showed slightly greater solution ab-
sorption and slightly better proliferation of VERO cells (p = 0.043)
compared to 4 h blended (CSDP-4) membranes. Finally, CSDP-24 was
loaded with heparin (CSDP-24-H) and its effects in the CAM assay
showed significant angiogenic potential compared to the unloaded
355
control hydrogel. This strongly supports the use of these biomaterials
as promising pro-angiogenic scaffolds for tissue engineering
applications.
Acknowledgment
We acknowledge the Higher Education Commission (PM-IPFP/HRD/
HEC/2012/2733) and Ministry of Science and Technology Pakistan
(MOST- PC1) for financial support.
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