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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

Journal XX (XXXX) XXXXXX Author et al

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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


3

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


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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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


6

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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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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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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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

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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

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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

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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

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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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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

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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

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