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This is a repository copy of Hybrid hydrogels based on
polysaccharide gum karaya, poly(vinyl alcohol) and silk
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paper:http://eprints.whiterose.ac.uk/142199/
Version: Accepted Version
Article:
Postulkova, H., Nedomova, E., Hearnden, V.
orcid.org/0000-0003-0838-7783 et al. (2 more authors) (2019) Hybrid
hydrogels based on polysaccharide gum karaya, poly(vinyl alcohol)
and silk fibroin. Materials Research Express, 6 (3). 035304. ISSN
2053-1591
https://doi.org/10.1088/2053-1591/aaf45d
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IOP Publishing Journal Title
Journal XX (XXXX) XXXXXX https://doi.org/XXXX/XXXX
xxxx-xxxx/xx/xxxxxx 1 © xxxx IOP Publishing Ltd
Hybrid hydrogels based on 1
polysaccharide gum karaya, 2
poly(vinyl alcohol) and silk fibroin 3
4 Received xxxxxx 5 Accepted for publication xxxxxx 6 Published
xxxxxx 7
Abstract 8
This work focuses on preparation of a hybrid hydrogel consisting
of both natural and 9 synthetic polymers including the
polysaccharide gum karaya which is both inexpensive and 10
abundant, the protein silk fibroin which exhibits remarkable
mechanical properties and 11 poly(vinyl alcohol). These polymers
were primarily selected due to their biocompatibility, but 12 also
through their ability to be combined together in an aqueous,
non-toxic route, thus 13 facilitating their potential future use as
burn dressings. A range of structural, mechanical and 14 practical
techniques were employed to characterise the hydrogels including,
FTIR, UV/VIS, 15 phase contrast microscopy, XRD, DMA, swelling and
hydrolytic stability. Finally, looking 16 towards application as a
dressing, these materials demonstrated low cell adhesion through a
17 keratinocyte cell culture assay. The results support both the
potential application of these 18 hydrogels and provide insight
into the role of each component polymer in the material. 19
Therefore, we propose hybrid hydrogels such as these offer a unique
combination of 20 performance, ease of processing and low cost that
can serve as inspiration for the next wave 21 of bespoke medical
products. 22 23
Keywords: Biocompatible polymers; Hydrogel; Gum Karaya;
Poly(vinyl alcohol); Silk fibroin 24
25
1. Introduction 26
Gels are cross-linked macromolecular networks swollen 27 in a
liquid [1–3] and if done so in water are termed hydrogels 28 [4].
Their three-dimensional networks are capable of 29 retaining large
volumes of water or biological fluids (up to 30 thousands of times
their dry weight) [5]. As such hydrogels 31 are widely studied in
biomedical applications because their 32 physical properties are
similar to human tissues and they 33 possess excellent
biocompatibility [6] making them highly 34 desirable for burns
treatment. 35
Specifically for burns treatment, hydrogels provide a 36 moist
environment which is has been shown to be an 37 important factor in
accelerating the wound healing process 38 [7] as well as providing
a cooling effect, a barrier against 39 infection and can be easily
removed without pain [3,7–10]. 40 Yet despite several solutions
currently on the market there is 41 still plenty of room for
improvement and designing 42
hydrogels and skin wound coverings that satisfy a range of 43
technical requirements, at an affordable price, is a big 44
challenge [11–14]. However, previous work has suggested a 45
potential solution; a hybrid hydrogel material based on a 46
mixture of natural and synthetic biopolymers which can meet 47
these complex requirements for successful wound healing 48 [15].
49
Addressing this challenge, this work focuses on the 50
combination of four types of biomaterials in order to design 51
hydrogels for potential future use in burns treatment. These 52
hydrogels are based on a natural polysaccharide, gum karaya, 53 a
natural protein, silk fibroin, a synthetic biopolymer 54 poly(vinyl
alcohol) and finally glycerol. 55
Gum karaya (Sterculia urens, GK) is a natural gum which 56
consists of a complex, branched and partially acetylated, 57
hydrophilic, anionic polysaccharide containing く-D-58 galactose,
L-rhamnose, く-D-glucuronic acid and D-59 galacturonic acid. It is
commonly available and is considered 60
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Journal XX (XXXX) XXXXXX Author et al
2
a relatively cheap, biodegradable and biocompatible material 1
[9,16]. GK has garnered widspread interest as it has unique 2
material features such as a high viscosity and capacity for 3
swelling and water retention, it is both gel and film forming 4 and
has adhesive properties [16]. From a chemcial 5 perspective GK is
resistant to hydrolysis by mild acid and it 6 is partly resistant
to bacterial and enzymatic degradation 7 [16,17]. In recent years,
GK and its combination with other 8 polymers has been explored when
developing hydrogels for 9 drug delivery systems (i.e. with PVA
[3,8,9,18], acrylic acid 10 [19,20] and others [2,17,20,21]).
However its potential has 11 been somewhat limited due to its water
solubility, although 12 this can be altered through alkali
treatment [22]. 13
Poly(vinyl alcohol) (PVA) is a biocompatible, hydrophilic 14
water soluble polymer [8,18,23] which is not biodegradable 15 in
most physiological conditions [24]. It is widely used in 16
biomedical and tissue engineering applications because of its 17
good processability, ability to form films, mechanical 18
properties (e.g. sufficient strength) and temperature stability 19
[8,25]. Hydrogels based on PVA are mainly prepared by 20
crosslinking (e.g. glutaraldehyde) [26] or radiation and 21
repeated freezing/thawing methods [24,27] 22
PVA has been previously combined with different types 23 of
biopolymers to obtain hydrogels for tissue engineering, 24 for
example with chitosan [25], starch [28], cellulose [6], 25 alginate
[29], dextran, glucan [30], gelatine [31], PVP [32], 26 silk
fibroin [33] etc. Hydrogels based on PVA and 27 polysaccharides
have been found to be suitable for producing 28 transparent,
flexible, mechanically strong, biocompatible, 29 effective and
economical hydrogel dressings [3,20]. PVA is 30 also known as a
anti-biofouling material i.e. it is a non-31 favourable substrate
for cell adhesion, proliferation and 32 exhibits minimal adsorption
of proteins [23]. Such a 33 combination of desirable properties
makes PVA an excellent 34 candidate for the use in burn dressings
because of the need 35 for frequent dressing changes on wounds
without destroying 36 newly grown tissue underneath. 37
Over the past decade silk fibroin (SF) has rapidly become 38 a
biomaterial of choice for a range of applications due to 39 a
combination of excellent intrinsic mechanical properties, 40
biocompatibility, biodegradability [34,35] and extrinsic 41
properties achieved through aqueous processing such as film 42
formation, oxygen permeability and ease of sterilisation 43
[34,36–38]. Extending its capability, silk has been used in 44
mixtures with other biomaterials to create films or hydrogels 45
for biomedical applications including alginate [39], 46 hyaluronic
acid [40], chitosan [41], PVA [42–44], PEG [45], 47 polyacrylamide
[46] and polyurethane [47]. 48
Finally glycerol is a non-toxic, low molecular weight 49
compound which is also often used in biomedical 50 applications as
a plasticizer [38]. This is best evidenced in 51 the case of PVA/SF
hydrogel blends where glycerol has been 52 used to improve
mechanical properties for over a decade [48] 53
and has been shown to reduce the degree of phase separation, 54
acting as a compatibiliser and resulting in increased breaking 55
strength and elongation of films [38]. 56
Therefore, under the premise that a combination of the 57 above
materials can be determined that results in a hybrid 58 hydrogel
whose properties exceed that of any individual 59 materials
contribution. This work reports the preparation of 60 hydrogels
based on natural polysaccharide gum karaya, 61 synthetic biopolymer
poly(vinyl alcohol) and protein silk 62 fibroin and subject them to
characterisation by FTIR, 63 UV/VIS, phase contrast microscopy,
XRD, DMA, swelling 64 and stability studies and cell culture
assays. 65
2. Materials and methods 66
2.1 Chemicals 67
Gum karaya was purchased from Sigma-Aldrich (Mw of 68 approx. 9
500 000 g·mol-1), sodium hydroxide and 69 hydrochloric acid were
purchased from Lach-Ner, s.r.o., 70 Czech Republic, ethanol (96%)
was obtained from Moravian 71 distillery of Kojetín, Czech
Republic, ultrapure water (Type 72 I, resistivity 18.2 Mっ:cm) was
prepared by a MilliQ Plus 73 185 machine and distilled water (Type
II, resistivity 15 74 Mっ:cm) was prepared by a Bibby Merit 4000
still. Lithium 75 bromide, sodium carbonate, poly(vinyl alcohol) 76
(Mw 130 000, 99+% hydrolysed) were purchased from 77 Sigma-Aldrich
and glycerol (99 wt. %) from 78 Fisher Scientific. Silkworm cocoons
(commercial grade) 79 were spun in-house from a stock of B. mori
silkworms. 80 Dulbecco’s medium, fetal calf serum, L-Glutamine, 81
penicillin, MTT solution and resazurin solution was 82 purchased
from (Sigma Aldrich, Dorset, UK). 83
2.2 Chemicals Solubilisation and purification of 84 raw gum
karaya 85
Raw gum karaya powder was combined with ultrapure 86 water and
magnetically stirred at 300 rpm in a beaker for 24 87 hours at room
temperature to obtain a visually homogenous 88 dispersion.
Solubilisation was carried out following a 89 previously described
deacetylation method [49]. Briefly, a 90 dispersion of GK was
solubilized by sodium hydroxide 91 (1 mol/l). Three volumes of a GK
dispersion were mixed 92 with one volume of hydroxide solution and
stirred for 93 5 minutes at room temperature. Diluted hydrochloric
acid 94 was used to neutralize any excess hydroxide after GK 95
solubilisation. The solubilized sample of GK was filtered 96
through polypropylene filters (pore size of 42 たm) and 97
centrifuged for 40 minutes at 40 °C, 15 000 rpm to remove 98
impurities. Afterwards the samples were filtered again 99 through a
paper filter (pore size 4 札 7 たm). The sample was 100 then
precipitated with ethanol in a ratio 2:1 and air-dried for 101
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Journal XX (XXXX) XXXXXX Author et al
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24 hours. Finally the dry sample was powdered and stored 1 in a
glass vial. 2
3
2.3 Degumming process of silk fibroin (SF) 4
Commercial quality B. mori silkworm cocoons were cut 5 into
small pieces (~4 mm2) and washed with distilled water 6 in a food
processor at its highest speed for 15 minutes three 7 times. They
were washed again with sodium carbonate 8 solution having 0.05
mol/l concentration (70 °C) using the 9 food processor for 20
minutes four times and finally washed 10 with distilled water.
Fibres were dried in an oven at 50 °C 11 overnight. Finally, dry
fibres were blended in the food 12 processor for 5 minutes to
become ‘fluffy’ to assist with the 13 following dissolution. 14
2.4 Dissolution of silk 15
Dissolution of silk fibres was carried out with 16 9.3 mol/l
lithium bromide at 70 °C in a water bath for 17 80 minutes. The
resulting solution was dialysed in a dialysis 18 bag (molecular
weight cut-off 12-14 000 g·mol-1) against 19 ultrapure Type I water
for 2 days at 4 °C and then stored in 20 the fridge until required.
The concentration of silk solution 21 was determined by gravimetry
and then diluted to 1 wt. % 22 solution with ultrapure water.
23
2.5 Preparation of hydrogels based on blend of 24 gum karaya,
poly (vinyl alcohol), silk fibroin and 25
glycerol 26
GK and PVA were dissolved together in ultrapure Type I 27 water
to prepare 0.3 wt. % and 3 wt. % solutions, 28 respectively. GK/PVA
solution was prepared by dissolving 29 raw powders of GK and PVA
together to produce final 30 concentration of GK 0.3 wt. % and PVA
3 wt. % in a given 31 volume. The solution was made by dissolving
polymers 32 overnight on hot plate stirrer at 90 °C. The solution
was then 33 dialysed against ultrapure Type I water for 2 days at 4
°C 34 (molecular weight cut-off 12-14 000 g·mol-1) and then 35
filtered through filter paper. GK/PVA solution was mixed 36 in 2 ml
Eppendorf tubes with different ratios of 1 wt.% silk 37 solution
and glycerol (G). Ratios of solutions used for 38 hydrogel mixtures
are described in Table 1. Solutions were 39 mixed overnight at room
temperature and then cast 40 onto round 35 mm Petri dishes and
dried on an orbital shaker 41 in a fume hood. The resulting dry
Xerogels (in a film form) 42 were peeled off the next day and
stored in plastic bags. 43
2.6 Characterisation 44
2.6.1 Attenuated Total Reflectance - Fourier 45 Transform
Infrared Spectroscopy 46
Infrared spectra were recorded with a NICOLET 380 47 FTIR
spectrometer (Thermo Scientific) purged with dry air 48 between 4
000 and 800 cm-1 averaging 32 scans and a 49
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Journal XX (XXXX) XXXXXX Author et al
4
resolution of 4 cm-1 The samples were analysed in xerogel 1 form
using an attenuated total reflection (ATR) accessory 2 with a
diamond crystal (Golden Gate, Specac, UK). 3
2.6.2 UltravioletにVisible Spectroscopy 4
UV/VIS analysis was carried out with UV2 UV/VIS 5 Spectrometer
(UNICAM). Spectra were recorded between 6 200 and 800 nm with lamp
change was at 340 nm applying 7 240 nm.min-1 speed with 2 nm data
point intervals. 8
2.6.3 Phase Contrast Microscopy 9
Phase contrast microscopy was carried out using an 10 inverted
Nikon Diaphot microscope (Nikon systems, Japan) 11 and phase
contrast optics with 10, 20 and 40x objectives and 12 imaged using
a Motic Moticam 5MP digital camera (Motic 13 Systems, Spain).
14
2.6.4 X-ray diffraction (XRD) 15
X-ray diffraction analysis was carried out on a benchtop 16
X-ray diffractometer Rigaku MiniFlex 600 using Cu anode, 17 40 kV
tube voltage and 15 mA tube current. Xerogel films 18 (2x2 cm) were
analysed in scanning range from 2 to 60° (2し). 19
2.6.5 Dynamical Mechanical Analysis (DMA) 20
DMA measurements were performed using a 21 TA Instruments DMA
Q800 Dynamic Mechanical Analyser 22 (TA Instruments, Delaware, USA)
equipped with the 23 film/fibre tension accessory. Xerogel films
were heated 24 under a nitrogen atmosphere from -100 to 220 °C at a
heating 25 rate of 3 °C/min with frequency of 1 Hz, 0.01% strain
and 26 1N preload. 27
2.6.6 Swelling behaviour 28
Hydrogel swelling was carried out in Type I water using 29 the
gravimetric method. Xerogel films were cut into small 30 pieces of
the same weight (approximately 1x1 cm) 31 put onto Petri's dishes
and immersed in an excess of 32 ultrapure Type 1 water. At set time
points (1, 3, 5, 7, 10, 20, 33 30, 40, 60 and 120 minutes), the
excess water in the Petri 34 dish was removed by paper tissue and
the hydrogel was 35 immediately weighed. The swelling ratio was
calculated as 36
37 where Ws is weight of swollen hydrogel and Wd is weight
38
of xerogel. 39
2.6.7 Hydrogel stability 40
After swelling, a stability test was carried out. Samples were
41 placed into vials and immersed in ultrapure Type I and kept 42
in an incubator at 37 °C. Resulting hydrogel stability was 43
measured on day 3, 10, 20 and 60. To do so, samples were 44
removed from their vials and weighed to determine the 45 weight
loss. Hydrogel stability was calculated using the 46 formula (2),
where wt is the weight of sample at a specific 47 timepoint and w0
is the dry weight of initial mass of sample. 48
49
2.6.8 Adhesion test and MTT proliferation assay 50
A HaCaT skin keratinocyte cell line was used and cells 51 grown
in cell culture media consisting of Dulbecco’s 52 Modified Eagle’s
medium (DMEM) supplemented with 10% 53 (v/v) fetal calf serum,
100i.u./ml penicillin and 100たg/ml 54 streptomycin and 2mmol/l
L-Glutamine (Sigma Aldrich) and 55 cultured in a humidified cell
culture incubator at 37°C with 56 5% CO2. Xerogels were sterilised
using UV light (emission 57 253.7 nm) in Esco Labculture Class II
Biological Safety 58 Cabinet for 40 min. 59
The adhesion assay was performed with HaCaT 60 keratinocyte cell
line cultured in 6 well plates. A confluent 61 layer of HaCaT cells
(cell number ~400 000) was seeded 62 onto the tissue culture
plastic surface of the 6 well plate to 63 produce a layer of
epithelium. Sterilised hydrogel discs (1 cm 64 in diameter) were
then added into wells and left to swell in 65 cell culture media
for 30 min. After 30 minutes, hydrogel 66 discs were weighed down
by light metal grid to ensure 67 contact between the hydrogel and
the cell layer. After 24 68 hours of direct contact, hydrogels were
peeled off the cell 69 layer and both the hydrogel surface and cell
layer were 70 examined with using phase contrast light microscopy
for 71 signs of cell adhesion. 72
An MTT (3-(4,5- dimethylthiazol-2-yl)-2, 5-73
diphenyltetrazolium bromide) assay was used to measure cell 74
adhesion and survival on the tissue culture plastic (TCP) 75
surface and on hydrogel surfaces. 2 ml of 0.5 mg/ml MTT 76 solution
(Sigma Aldrich) in PBS was added to cells or 77 hydrogels and
incubated for 40 min. After 40 minutes, the 78 unreacted MTT
solution was removed and the purple 79 intracellular formazan salt
(produced by dehydrogenase 80 reduction of MTT) was solubilised and
released from cells 81 using acidified isopropanol (125たl of 10
mol/l HCl in 100ml 82 isopropanol). The eluted dye was transferred
to a 96 well 83 plate. The optical density of the solution was
measured at 84 540nm with a reference at 630nm, using a
spectrophotometer 85 (BioTek ELx800). A positive control was
conducted which 86 represents results from cells which were not in
contact with a 87 hydrogel and the negative control represents
measurements 88 from wells without any cells present. 89
3. Results and discussion 90
Transparent and flexible hydrogels were prepared by 91 physical
crosslinking based on strong intra and 92 intermolecular hydrogen
bonds in PVA with a high degree of 93
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Journal XX (XXXX) XXXXXX Author et al
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hydrolysis [23]. Not using a chemical crosslinker was 1
desirable as this lowers the possibility of negative effects of 2
unreacted crosslinker on cell viability and thus the overall 3
healing process, simplifies regulatory approval as well as 4
reducing the overall cost of production. We estimate the 5 price of
1 cm2 of prepared hydrogel in this study to be 6 ~£0.02/$0.03
(based on material costs for 10cm2 of a ~40 µm 7 thick hydrogel).
In comparison, complex dermal treatment 8 applications as Integra
(silicone layer on top of a porous 9 matrix comprising a chemically
cross-linked coprecipitate of 10 bovine collagen and shark-derived
chondroitin-6-sulfate) 11 costs about $15–30 per cm2 [12].
Subsequently the prepared 12 xerogels were characterized by various
techniques to 13 understand their properties, structure and mutual
interaction 14 of the materials when combined together. 15
3.1 Effect of composition on chemical structure 16
FTIR spectroscopy was used to characterize specific 17 chemical
groups in the individual materials which can then 18 be used to
inform of their presence or absence in subsequent 19 blends.
Individually, spectra of GK, PVA, SF and final 20 blended xerogel
films are depicted in 21
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Journal XX (XXXX) XXXXXX Author et al
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1
2 3
Figure 1A. The ATR-FTIR spectra of GK shows a diagnostic 4 broad
peak of hydroxyl stretching at 3650-3000 cm-1 [9], 5 stretching of
aliphatic C-H bonds at 2920 cm-1 [9], vibrations 6 of carboxylate
salt group (-COO-) at 1605 and 1418 cm-1 7 [50] and C-O stretching
and group vibration of sugar rings at 8 1180–940 cm-1 [51]. For PVA
spectra the band for CH2 9 groups at 1470-1410 cm-1 [52], resonance
of CH-OH at 10 1320 cm-1 [53] and broad band representing C-O bonds
at 11 1150-1085 [52] are indicative of its presence. Significant 12
bands in SF spectra represent the OH and NH stretching at 13
3600-3100 cm-1, amide I, II and III at 1640, 1510 and 1230 14 cm-1,
respectively. The peak at 1050 cm-1 belongs to 15 vibration of
serine [54]. 16 When investigating subsequent blends to confirm all
17 introduced materials are present, spectra of a GK/PVA 18 mixture
clearly shows a combination of characteristic bands 19 for PVA and
GK. However in these spectra bands assigned 20 to GK were less
distinctive which we believe is due to its 21 lower ratio in the
mixture. Hence bands are not only a useful 22 indicator of
presence, but band intensity also informs of a 23 materials’
relative proportion in the xero/hydrogels. 24 Spectra of the
xerogel films most suited towards potential 25 application and
explored via cell culture later, contain 26 primarily bands of PVA
and SF which are the major 27 components of these samples. As can
be seen from the 28 spectra in Figure 1, GK/PVA/SF- and
GK/PVA/SF-/G 29 spectra show a decreased intensity in bands related
to amide I 30 and II due to lower SF ratio compared to GK/PVA/SF+
and 31 GK/PVA/SF+/G. 32 Finally the addition of glycerol increased
the intensity of the 33 broad peak representing hydroxyl stretching
(3650-3000 cm-34 1) for samples GK/PVA/G, GK/PVA/SF+/G and 35
GK/PVA/SF-/G due to presence of hydroxyl groups in 36 glycerol
structure [48]. 37
38 The UV/VIS absorption spectra of prepared xerogel films
39
are shown in 40
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Journal XX (XXXX) XXXXXX Author et al
7
1
2 3
Figure 1B. Absorption spectra of samples containing silk 4
fibroin displayed a wide peak in region of 250-300 nm. The 5 main
chromophores absorbing in this region are aromatic 6 amino acids
such as tyrosine and tryptophan which are 7 known to be present in
silk [55]. The absorbance observed in 8 these samples is further
confirmed to be attributed to silk as 9 the intensity of UV
absorption for tyrosine peak decreases 10 with decreasing silk
content of the samples 11 (SF>GK/PVA/SF+>GK/PVA/SF-). 12 A
minor increase in absorbance was observed for GK 13 samples in the
same region as for silk whereas samples 14 where PVA was major
component (PVA and GK/PVA 15 samples) did not show any UV
absorbance. This increase in 16 absorption at the beginning of the
spectra is therefore most 17 probably caused by water adsorbing to
the dried xerogel 18 films [56], which speak to the ability of GK
to become 19 hydrated easily. All samples had a very low absorbance
20 in the rest of the spectra towards higher wavelengths and 21
notably the addition of glycerol did not affect the UV/VIS 22
spectra of any of the films. These results are important as it 23
clearly demonstrates a high degree of 24 transmission/transparency
in the visible range of light. This 25 is particularly useful for
wound dressings as it would enable 26 the underlying tissues to be
inspected by healthcare 27 professionals without the need to remove
the dressing. 28
3.2 Morphology of xerogel film surfaces 29
Phase contrast microscopy was carried out to characterize the 30
morphology of xerogel film surfaces and study any potential 31
macroscale phase separation of the materials in the films 32
(Figure 2). GK and GK/G films were smooth with only small 33
aggregates or bubbles (Figure 2A, B). Films from PVA and 34 GK/PVA
(Figure 2C, E) were also smooth, lacking any 35 significant surface
morphology. However SF films displayed 36
both surface roughness and inhomogeneities (Figure 2G). 37
Looking towards the blends, the GK/PVA/SF+ sample 38 (Figure 2I)
had a similar structure to SF. This structure is not 39 homogenous
and indicative of a phase separation which 40 appears to happen
spontaneously when SF and PVA solution 41 are mixed together and
cast into films [57]. GK/PVA/SF- 42 (Figure 2K) also showed phase
separation but in this case, a 43 finer dispersion was observed
with smaller particles due to 44 the lower SF content. Previosuly
it has been shown that 45 particle size in PVA/SF system with phase
separation can be 46 tailored by sonication [57] and this may be a
useful strategy 47 to adopt in future studies. Finally it was seen
that addition of 48 glycerol to the films did not have any
noticeable effect 49 on hydrogel morphology and phase distribution.
50
3.3 Xerogel film crystallinity 51
XRD measurements were used to evaluate the crystallinity of 52
the separate raw materials and prepared blended xerogel 53 films.
GK and all SF samples clearly showed an amorphous 54 structure.
This is not particularly surprising as GK is a 55 branched
polysaccharide with non-repetitive structures and 56 thus an
amorphous nature was expected. In contrast, SF has 57 ability to
form ordered structures including く-sheets [36], 58 however the
ability of SF molecules to create these more 59 crystalline
structures can be lost due to the detrimental 60 effects of the
preparation/reconstitution process [58]. 61 PVA also has
crystalline and regular regions in its structure, 62 but their
extent is determined by the level of PVA hydrolysis 63 [26]. In our
measurements, pellets of PVA showed a 64 characteristic peak for
PVA at 20° (Figure 3) which is the 65 main crystal peak,
corresponding to a (101) reflection of the 66 monoclinic crystal
[59]. Subsequent lower peak intensities 67 and therefore less
developed crystalline structures (i.e. minor 68 peaks not present)
was observed for the PVA film and all 69 samples with PVA content.
This could be a result of the 70 preparation method, as solutions
were dried whilst being 71 gently mixed. This drying method could
disturb the 72 development of the crystalline structure of a PVA
xerogel 73 film compared to that observed in PVA pellets. Formation
of 74 PVA crystalline and regular regions could be also affected by
75 the presence of other types of polymers in system whose 76
chains could restrict the ability of the PVA polymer chains to 77
crystalise. Following on from this, an effect of SF content 78
towards hydrogel crystallinity was observed. Samples 79 containing
a higher ratio of SF (GK/PVA/SF+ and 80 GK/PVA/SF+/G) showed lower
crystallinity; this could be 81 caused generally by a lower ratio
of PVA in system but also 82 by the higher SF content which
contributes more towards 83 restriction of PVA crystalline regions
formation. 84 Interestingly, the addition of glycerol clearly
affected xerogel 85 film crystallinity. Samples with glycerol
tended to show a 86 higher intensity peak on XRD. This could
suggest that as 87 glycerol acts as a plasticiser is essentially
increases free 88
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Journal XX (XXXX) XXXXXX Author et al
8
volume and supports movement/reptation of polymer chains, 1
promoting higher sample crystallinity. However, this 2 influence
was not significant for PVA/G and SF samples. 3
3.4 The effect of structure on the xerogel film 4
viscoelasticity 5
Dynamical mechanical analysis (DMA) was conducted to 6 measure
changes in viscoelasticity of xerogel films in dry 7 state as a
result of structural relaxation changes. 8
Xerogels based only on GK or SF could not be tested 9 because of
their brittle nature. All samples containing PVA 10 showed the same
general trend during testing (exemplar data 11 depicted in (Figure
4A). From these DMA traces the storage 12 modulus is related to a
material’s ability to store energy and 13 its stiffness. A steady
decrease in the storage modulus is 14 observed from the beginning
of the test, as temperature 15 increased from -100 to 20 °C and is
related to the softening 16 of the material as a result of gamma
and beta transitions (i.e. 17 the beginning of localized bond
movements and bending; 18 stretching and side chain movements). The
broad band for 19 the loss modulus at the beginning of the test
shows energy 20 dissipation. 21
Following, the xerogel films show a significant decrease 22 in
storage modulus (approx. at 50 °C) and concurrent 23 maximum in
loss modulus which is related to the glass 24 transition (Tg) of
major component (PVA). This Tg is more 25 easily denoted by
investigating the tan delta signal for a peak 26 (Figure 4B) which
is reached slightly after maximum of loss 27 modulus (which is
indicative of a second-order phase 28 transition). It is also worth
noting that the Tg for the sample 29 GK/PVA/SF-/G is slightly
reduced by presence of glycerol 30 because of its plasticising
character. 31
Loss modulus showed another softening band in the 90-32 110 °C
region which could be potentially related to structure 33
reordering (possibly reordering amorphous PVA structures 34 into
crystalline ones). This transition is significantly affected 35 by
glycerol which reduces the temperature of this effect, 36 lowering
its intensity and somehow merging this transition 37 together with
band for the Tg (Figure 4A). Interestingly, a 38 slight increase of
storage modulus (intepretted as a hardening 39 of the material) has
been also observed in the region (above 40 100 °C) for both
samples. 41 The aforementioned effect of glycerol on xerogel film
42 properties is also apparent from tan delta plot (Figure 4B). 43
Glycerol serves to merge peaks together, broadening them 44 and
suggesting its positive effect on mixing and blending the 45
polymeric components present, thus also acting as a 46
compatibilser. Here glycerol probably promotes and 47 increases
interactions between different types of polymer 48 chains due to
hydrogen bonding of hydroxyl groups of GK, 49 PVA and glycerol and
amide groups of SF [48,60]. 50 The glass transition temperature of
SF is not apparent in loss 51 modulus data due to its low content
in the film. However, the 52
ordered structures Tg of SF can be observed in the tan delta 53
signal as it usually appears at ~210 °C for B. mori silk [61] 54
Figure 4B. This transition is clearly present for samples with 55
higher SF content but is barely visible from samples with a 56 low
SF content. Interestingly, the temperature for SF’s Tgin 57 the
GK/PVA/SF+/G blend is significantly affected by the 58 presence of
glycerol, reducing the transition temperature to 59 around 160 °C
which would be indicative of promoting more 60 disordered
structures in the film. 61
3.5 Swelling behaviour 62
The effect of various materials and their ratios on hydrogel 63
water absorption (swelling) were carried out. Pure GK and 64 SF
samples were immediately water soluble, thus their 65 swelling
properties were not studied. 66 The highest swelling ratio (above
25x) was observed in 67 samples with GK (GK/PVA and GK/PVA/G).
Despite the 68 low amount of GK present in the samples, the
hydrogel 69 showed the highest swelling potential (Figure 5A).
However, 70 any significant effect of glycerol on hydrogel swelling
was 71 not observed. The glycerol physical crosslinking clearly had
72 a greater influence on the hydrolytic stability of hydrogels as
73 discussed below. 74 The remaining samples showed similar and
relatively stable 75 swelling profiles (around 15x) which remained
consistent 76 through the 2 hours of the test. At the end of the
test all 77 samples had stabilised their swelling ratio apart to 78
GK/PVA/SF+ which showed a lower swelling ratio 79 throughout the
whole test. These observations are in 80 agreement with work
studying PVA/SF hydrogels and their 81 water uptake [62]. No
significant difference was observed in 82 the swelling regardless
amount of added silk. This supports a 83 hypothesis that the higher
swelling in samples GK/PVA and 84 GK/PVA/G is caused by the
presence of GK. 85
3.6 Hydrogel stability 86
A study focused on hydrogel stability was carried out to 87
evaluate stability in ultrapure Type I water at 37 °C over a 60 88
day period. Hydrogel stability is depicted in Figure 5B. the 89
results indicate that hydrogel stability is largely based on the 90
ability of PVA to form a physically crosslinked structure 91
connected by hydrogen bonds [63] without any chemical 92
crosslinking and the structure present is stable over a long 93
time period. 94 Samples with different SF ratios (although
otherwise with 95 the same composition) have similar stabilities,
suggesting 96 that SF content did not have any significant effect
towards 97 hydrogel stability. Improvements in stability of PVA/SF
98 cryo-hydrogels has been previously reported whereby a 99
freeze-thaw regime for cryogels fabrication which ensured 100
better stability in PBS at 37 °C[62]. 101 The presenence of
glycerol improved hydrogel stability was 102 observed for all
samples. This is most likely due to the 103
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9
compatbilising action of glycerol as previously discussed, for 1
our results are in good agreement with observations from 2 DMA and
XRD testing (see above) and previous 3 observations on stability in
SF hydrogels [38]. 4
3.7 Adhesion test and MTT proliferation assay 5
A specific assay was developed to study adhesion of 6 prepared
hydrogels to a keratinocyte cell layer in order to to 7 simulate a
real-world scenario where a hydrogel dressing 8 would be placed
onto the skin surface. In this assay 9 hydrogels were in contact
with a confluent layer of 10 keratinocytes for 24 hours prior to
being removed and cell 11 attachment measured. 12 As a broad
observation, all hydrogels did not show any 13 adhesion to the cell
layer after weight removal and they were 14 freely floating in the
culture medium. Furthermore, using 15 microscopy, no cells were
observed on hydrogel surfaces of 16 PVA, PVA/G, GK/PVA and GK/PVA/G
(Figure 6 A-D) 17 which suggests a low preference of keratinocytes
to adhere to 18 the hydrogel. However, the presence of
keratinocytes on 19 samples containing SF (Figure 6 E-H) could not
be 20 determined by imaging alone because the inherent 21
microstructure of these materials when imaged using phase 22
contrast microscopy gave an uneven appearance. 23 Therefore, moving
past a qualitative visual analysis, in order 24 to quantify cell
attachment to the hydrogels an MTT assay 25 was conducted. In
addition, an MTT was performed on the 26 confluent layer of cells
on the tissue culture plastic to 27 determine if contact with the
hydrogel resulted in cell 28 detachment or a reduction in cell
viability (either through 29 direct contact with the material or
through contact with gel 30 components eluted during swelling and
incubation). 31
The MTT assay was unable to detect any metabolic 32 activity
from cells on the hydrogel surface, demonstrating no 33 viable
cells adhered to the material. This low adherence of 34 cells on
the hydrogel surface is most likely caused by the 35 high content
of PVA which is recognised as a non-favourable 36 substrate for
cell adhesion and proliferation [23]. However 37 this is ideal for
these films’ potential application, as low 38 adhesion of a
hydrogel towards cells is essential for a wound 39 dressing to
avoid removal of any regenerating epithelium 40 when the dressing
is applied and subsequently replaced. 41
Figure 7 shows the metabolic activity of HaCaT cells 42
following contact with each type of hydrogel. The positive 43
control (cells without any hydrogel contact) demonstrated the 44
highest cell activity while all wells with hydrogel contact 45
displayed a slightly reduced cell metabolic activity compared 46 to
the positive control. This is likely as a result of the 47
mechanical disruption as a result of the direct contact assay 48
performed. The highest viability was observed in the samples 49
containing SF. This observation is in agreement with [34,64] 50
where SF has been described as supporting and promoting 51
keratinocyte cells. 52
This data demonstrates that contact with hydrogels largely 53
maintains cell viability. There is no evidence that the 54
hydrogels are able to promote cell proliferation in this short 55
term, two-dimensional cell culture assay. Here further studies 56
are required to determine if the hydrogels are able to promote 57
reepithelialisation in a wound healing model and to fully 58
examine the effect of the material on skin cell viability and 59
integrity. 60
4. Conclusion 61
Novel hydrogels based on a natural polysaccharide gum 62 karaya,
the synthetic biopolymer poly (vinyl alcohol) and the 63 protein
silk fibroin, were designed to address the challenge of 64
developing suitable wound coverings . A range of hydrogels 65 were
produced and studied using different techniques such as 66 FTIR,
UV/VIS, phase contrast microscopy, XRD, DMA, 67 swelling and
stability studies as well as cell culture assays. 68 The results
have helped us to better to understand the 69 structure,
interactions and function of the constituent 70 materials and their
contributions towards the final extrinsic 71 properties of
hydrogels. From the results we propose that 72 hydrogel stability
in water is based on PVA’s ability to 73 create a partly
crystalline structure which acts as physical 74 crosslinking.
Furthermore, DMA and stability studies 75 showed a significant
positive effect of glycerol towards 76 improving hydrogel
properties. Finally, cell culture showed 77 that the hydrogels
produced were non-toxic towards 78 keratinocytes and they exhibited
a low adhesion to them. 79 Low cell adhesion is an essential
feature for hydrogels to be 80 successfully used for burnt skin
regeneration to prevent 81 destroying newly grown tissue as the
covering is replaced. 82 We conclude that the presented method of
hydrogel 83 preparation is straightforward, non-expensive and does
not 84 use any toxic chemicals. Therefore this study seeks to 85
increase the potential of these materials to further develop 86 new
types of affordable and widely available biomedical 87 materials;
hybrid hydrogels for skin burn treatment. 88 89
Acknowledgements 90
91 92
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2
3
Figure 1: A) FTIR ATR spectra of prepared xerogel films 4
(GK = gum karaya, PVA = poly(vinyl alcohol), 5
SF = silk fibroin, G = glycerol, + represents higher silk ratio,
6
- represents lower silk ratio), B) UV/VIS spectra of prepared
7
xerogel films with the small amount of noise at 340 nm being
8
caused by the deuterium to halogen lamp change. 9
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Figure 2: Phase contrast microscopy pictures of prepared 14
xerogel films: A) GK, B) GK/G, C) PVA, D) PVA/G, E) 15
GK/PVA, F) GK/PVA/G, G) SF, H) SF/G, I) GK/PVA/SF+, 16
J) GK/PVA/SF+/G, K) GK/PVA/SF- and L) GK/PVA/SF-/G 17
(scale bar for all pictures is 100 µm). 18
19
20
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1
2
Figure 3: XRD spectra of PVA pellets, PVA, GK/PVA, SF, 3
GK/PVA/SF+, GK/PVA/SF+/G, GK/PVA/SF- and 4
GK/PVA/SF-/G (samples tested in xerogel film form apart 5
from PVA pellets). 6
7
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9
10
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Figure 4: A) Storage and loss modulus for samples and 15
GK/PVA/SF-/G, B) Tan delta for samples GK/PVA/SF+, 16
GK/PVA/SF+/G, GK/PVA/SF- and GK/PVA/SF-/G. 17
18
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1
2
3
Figure 5: A) Swelling ratio of prepared hydrogels depending
4
on time, B) Hydrogel stability on day 3, 10, 20 and 60 (Type
5
I water at 37°C). 6
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10
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12
13
14
15
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Figure 6: Microscope picture of hydrogel surfaces after 18
adhesion assay: A) PVA, B) PVA/G, C) GK/PVA, D) 19
GK/PVA/G, E) GK/PVA/SF+, F) GK/PVA/SF+/G, G) 20
GK/PVA/SF-, H) GK/PVA/SF-/G (magnification 7.5). 21
22
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6
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Figure 7: MTT assay: Normalised UV absorbance 8
corresponding to activity of keratinocytes layer on TCP after
9
hydrogel removal. 10
11