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Dual crosslinked keratin-alginate fibers formed via ionic complexation of amide networks with improved toughness for assembling into braids
Polymer Testing 81 (2020) 106286
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Dual crosslinked keratin-alginate fibers formed via ionic complexation of amide networks with improved toughness for assembling into braids Ashmita Mukherjee, Yogesh H. Kabutare, Paulomi Ghosh * CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032, West Bengal, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Dual crosslinking Sustainable fibers Green process Textile Braids
With the dwindling of petroleum resources worldwide, there is an immediate need for a renewable, environment friendly, cost effective and sustainable bio-resource in the textile industry. Here, we report a dual crosslinked fiber (DCF) derived from renewable biopolymers. In this study, keratin was extracted from bio-waste of chicken feathers with a thiol content of 0.172 mM. The extracted keratin was used to prepare dope with alginate at different ratios and N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride via amide linkages. The formation of covalently crosslinked dope was evidenced from FTIR and ninhydrin assay. The dope was then extruded in calcium bath to produce fibers with uniform diameter wherein the calcium ions were used to ion ically crosslink the covalently crosslinked dope. The resulting dual crosslinked fibers were characterized in terms of chemical composition, surface morphology, mechanical properties, thermal degradation, and swelling. The strength, modulus and toughness of the dual crosslinked fibers were substantially improved by 27%, 20%, and 33% respectively than that of control alginate fiber. The gravimetric toughness of the optimised dual crosslinked fiber (724 J g 1) was much higher than the values reported for Kevlar (78 J g 1). We further assembled the dual crosslinked fibers into complex braided architectures using the textile techniques, demonstrating the flexibility of the fibers. We believe that this preliminary work of sustainable fiber production could open new insights into eco-friendly organic textile manufacturing and for tissue engineering applications.
1. Introduction Textile industry depends mostly on the synthetic polymers besides few natural polymers such as cotton, silk and jute. Synthetic and manmade fibers are however not sustainable because of its detrimental ef fect on environment coupled with global depletion of petroleum re serves and rising oil cost [1,2]. Therefore, it is necessary to consider producing fibers from alternative bio-resources with large availability [3,4]. In recent years, sustainable fibers derived from eco-friendly nat ural materials such as cucumber vine, lotus, and coconut coir have received increasing attention [5–7]. Chicken feathers that are otherwise disposed in landfills could be a prospective resource for keratin to pro duce alternative natural polymer based fibers for industrial and medical applications [8–10]. Keratins fabricated into different forms such as sponges, films, nanoparticles, membranes either alone or blended are highly attractive for industrial and biomedical use due to their intrinsic biocompatibility, and physical properties [11–17]. However, drawing of keratin fibers is still not actively pursued due to the inherent poor mechanical properties
of pure keratin. Therefore, many researchers have attempted to increase the processability of keratin by combining it with synthetic or natural polymers such as poly(ethylene oxide) (PEO), poly-(vinyl alcohol) (PVA), silk and cellulose for fiber formation [18–22]. For instance, Katoh et al. [22] used PVA to increase the viscosity of the aqueous keratin solutions to improve upon the fiber drawing capabilities for potential applications as absorbents for toxic substances. However, these fibers were predominantly petroleum-dependent because of high amount of PVA incorporated, and thus were non-sustainable, non-re newable, and had limited industrial potential. Wrzesniewska-Tosik et al. [23] also employed fabrication techniques to prepare keratin-cellulose fibers for possible application as hygienic fabrics. However, introduc tion of keratin into cellulose fibers deteriorated the overall mechanical strength of the fibrous materials. Recently, Xu and Yang [24] reported the preparation of keratin fibers of high tensile strength but other associated parameters such as elastic modulus, toughness, and swelling were not detailed. Thus, there is still a gap to produce sustainable ker atin based fibers with sufficient mechanical properties. This motivates us to use the crosslinking mechanism to produce mechanically robust
* Corresponding author. E-mail address: [email protected] (P. Ghosh). https://doi.org/10.1016/j.polymertesting.2019.106286 Received 6 October 2019; Received in revised form 21 November 2019; Accepted 7 December 2019 Available online 9 December 2019 0142-9418/© 2019 Elsevier Ltd. All rights reserved.
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keratin based fibers. Sodium alginate is a naturally occurring biocompatible polymer composed of blocks of (1–4)-linked β-D-mannuronic acid (M) and α-Lguluronic acid (G) monomers and known for having many biomedical applications including wound healing, drug delivery and tissue engi neering [25–27]. Alginate can be easily modified by enzymatic and chemical means to alter the composition and structure to enhance the functional properties for specific applications [28]. Alginate readily crosslink with divalent cations, usually calcium ions forming ionic linkages with carboxylic acid groups on the polymer backbone. Samorezov et al. [29] employed ionic- and photo-crosslinking to create alginate hydrogels with spatially controlled physical and chemical properties. According to the authors, these dual crosslinked alginate hydrogels, with tunable mechanical properties, has potential to be used in regenerative medicine. In recent years, various other polymeric de rivatives have been developed with dual crosslinking mechanism [30–32]. Nevertheless, to the best of our knowledge, covalently cross linked keratin-alginate forming fibers via ionic complexation and resulting in a dual crosslinked network are not yet documented in literature. Herein, we have extracted keratin from Gallus gallus domesticus (chicken) feathers and characterized it. Thereafter, we have chemically crosslinked keratin with alginate via N-(3-Dimethylaminopropyl)-N0 ethylcarbodiimide hydrochloride (EDC) to form dope. The dope pre pared from varying the amount of keratin, alginate and concentration of crosslinkers was used to prepare uniform diameter fibers via ionic crosslinking in a calcium bath. An elaborate comparative study was done to evaluate the effect of the dual crosslinking on tensile strength, elastic modulus, toughness, swelling, and thermal properties of the fi bers. We further demonstrated the flexibility of the dual crosslinked fi bers by assembling them into complex braided architectures using the regular textile techniques.
subsequently lyophilised to obtain the powdered form of keratin and was stocked in storage containers with lid at room temperature until further use. 2.3. Characterization of keratin The molecular weight of the extracted keratin was estimated by SDS PAGE according to a previously published report [34] and is provided in the supplementary information. Separation was performed at 80 V to run the sample from the stacking gel to the separating end and 120 V was maintained for 2 h to run the protein sample over the gel. Thereafter the gel was stained in the staining solution overnight, de-stained and then was placed in the UV light to observe the separation of protein strands in the gel. The FTIR of freeze-dried chicken feathers and extracted sample from the feathers was determined on a PerkinElmer system (Spectrum-100). The samples were powdered, mixed with KBr to form pellet and analysis was performed between a range of 4000 and 400 cm 1. The thiol content of the feather keratin was determined by Ellman’s assay following a previously published report [34]. Briefly, 0.1 M PBS-EDTA buffer of pH 8.0 was freshly prepared containing 50 μL of 4 mg/mL DTNB. Then, 250 μL of 1% w/v extracted keratin was pipetted to the 2.5 mL PBS-EDTA buffer and mixed well. The sample was then subjected to incubation at 25 � C for 15 min, and the absorbance was taken at 412 nm by UV Spectrophotometer (UV-10, Thermo). Standards were prepared using cysteine to make a standard curve for determina tion of the thiol content. 2.4. Preparation of dope For production of fibers, dope was prepared by carbodiimide cross linking of keratin with alginate. The crosslinking was carried out by varying the concentrations of crosslinker and proportion of the bio polymers (Table 1) according to a previously published report [35]. Briefly, keratin solution% (w/v) was added into the alginate–EDC mixture and stirred at room temperature for 24 h to obtain a clear viscous dope. Alginate dope, 1% (w/v) was also prepared and used as the control.
2. Experimental section 2.1. Materials Chicken feathers of Gallus gallus domesticus were collected from local market (Jadavpur, Kolkata, India). Ethanol (Himedia), urea (Merck), sodium-bisulfite (Lobachemie, India) sodium dodecyl sulphate (SDS, Qualigens), Whatman filter paper (GE Healthcare UK Limited) were used for the process of keratin extraction. 5, 50 -dithio-bis-(2-nitro benzoic acid) [DTNB i.e. Ellman’s Reagent] (Sigma, USA), L-cysteine hydrochloride (Sigma, USA), EDTA (Qualigens), sodium dihydrogen phosphate (Merck) were used for thiol content determination. For per forming SDS PAGE, phosphate buffered saline (PBS, Himedia), glycerol and TRIS HCl (Merck), TEMED (sigma), and the protein marker of the range 245–5 kDa (Puragen, Genetix) were used. Alginate (Himedia) and EDC (Sigma, USA) were used for dope and fiber preparation. Ninhydrin reagent (Sigma) was used for determining crosslinking density.
2.5. Characterization of dope The IR spectrum of lyophilised keratin-alginate crosslinked dope was determined by FTIR using potassium bromide pellet method. The crosslinking index was evaluated quantitatively by determining the amount of free amine using ninhydrin assay as per a previously pub lished report [35]. Briefly, 1 mL of 0.4% w/v ninhydrin aqueous solution was added to 10 mg of minced, pulverized sample (n ¼ 10), mixed and kept in a hot water bath (OVFU) at 90 � C for 20 min. The mixture was subsequently centrifuged after cooling down to room temperature. The mixture’s supernatant was used for determining the absorbance at 570 nm in a microplate reader MULTISKAN GO (Thermo Scientific). The degree of crosslinking was calculated using the formula below:
2.2. Extraction of feather keratin Chicken feathers were washed with warm water, dried at 50 � C in an incubator (RRB Scientific & Surgicals, India) for 24 h and chopped into tiny pieces with a scissor. Thereafter, the feathers were sterilized in ethanol for 15 min and were air dried. The feathers were then delipi dified and keratin was extracted via reduction process as per a previ ously published report [33]. Briefly, 2 g defatted feathers were added in 50 mL aqueous solution consisting of 0.5 M sodium bisulfite, 8 M urea and 0.08 M SDS. The mixture was kept at 50 � C in an incubator under shaking condition for 2 h. After that, the resulting mixture was subjected to centrifugation at 9000�g (REMI, model 8C, India) for 15 min to discard off the insoluble chicken feathers. The supernatant was filtered through a funnel containing filter paper and the filtrate was dialysed using cellulose membrane dialysis tube. The dialysed sample was
Table 1 Amounts of keratin, alginate and EDC for dope preparation. Dope code
K: A
Keratin (w/v) %
Alginate (w/v) %
EDC conc. (mM)
Dope-1:10 Dope-1:4 Dope-1:2 Dope-1:1 Dope-1-1:10 Dope20–1:10 Dope-Alg
1:10 1: 4 1: 2 1: 1 1:10 1:10
0.1 0.25 0.5 1 0.1 0.1
1 1 1 1 1 1
10 10 10 10 1 20
N/A
N/A
1
N/A
K: A, weight ratio of keratin to alginate; Dope-Alg, dope of 1% w/v alginate; N/ A, not applicable. 2
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Crosslinking index ð%Þ ¼
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TGA instrument in the temperature range of 50–800 � C, using aluminum crucibles, under dynamic nitrogen atmosphere (50 mL/min) with a heating rate of 10 � C/min following a published report [38].
ðNHo– NHtÞ � 100 NHo
Where, NHo and NHt are the absorbance of the lyophilised keratin before and after crosslinking, respectively.
2.8. Textile structures
2.6. Fiber fabrication
To determine flexibility for textile manufacture, the dual crosslinked fibers were twisted, knotted, and manually intertwined to form simple and complex braided structures. The textile structures were imaged by FE-SEM.
Fibers were prepared by wet spinning following a previously pub lished report with slight modifications [36]. The alginate and cross linked alginate-keratin dope were degassed by centrifuging at 5500 rpm for 10 min and then the solutions were transferred in the 20 mL syringe. The spinning was performed in a custom-made wet-spinning device at room temperature consisting of a syringe pump (Cole-Parmer, US), a polypropylene coagulation bath chamber, a fiber collection shaft and motor (Elmee, Kolkata, India) for controlling the fiber collection rate. The syringe containing dope of alginate (1%) or crosslinked keratin-alginate was loaded into the syringe pump system operated at a rate of 0.05 mL/min. The tip of the syringe was attached with 30 cm long silicon tubing (2 mm inner diameter X 4 mm outer diameter) fitted to a blunt tip metallic needle of 15 mm, 22 gauge (Supelco, Merck, SGE Luer Lock Needles) submerged into the coagulation bath containing 5% w/v CaCl2. The fabricated fibers were collected at a speed of 60 rpm in a rotating shaft fitted with a motor. Calcium ions induced an immediate coagulation of the alginate concurrently forming continuous micron sized fibers. Finally, the fibers were dried at room temperature.
2.9. Statistical analysis The data obtained in this study were presented as mean � standard deviation and statistically evaluated using Graph-pad prism software. There were significant differences between the experimental groups when the p value was less than 0.05. 3. Results and discussion 3.1. Extraction and characterization of keratin Keratin was extracted by the reduction method from bio-waste of chicken feathers that are abundantly and easily available. The extraction was performed using sodium bi-sulphite, a cheap reducing reagent; urea, a protein denaturant and SDS to prevent agglomeration of the protein chains. The yield of keratin from chicken feather was 74.2% by mass. The molecular weight of the extracted keratin determined by SDS PAGE was found to be around 10 KDa (Fig. 1a). This is very close to the result obtained in a previously published report [34]. The FTIR of the freeze-dried extracted sample from chicken feathers is shown in Fig. 1b. The positions of the bands in the sample indicated the conformations of the protein material: 1659 cm 1 for amide I, 1535 cm 1 for amide II, and 1238 cm 1 for amide III. The peak at 3309 cm 1 could be attributed to the stretching vibrations of O–H and N–H (Amide A), the bands at 2960 cm 1 and 2926 cm 1 could be ascribed to the CH3 stretching vibration (Amide B). The peak observed at 1453 cm 1 is due to bending vibrations of CH2 & CH3. The peaks at 1061 cm 1 and 1025 cm 1 could be due to asymmetric and symmetric S–O stretching vibra tions contributed by cysteine-S-sulphonated residues formed during extraction process. This is in agreement with a previous study [39]. The peaks at 985 cm 1 and 584 cm 1 were due to the characteristic ab sorptions of the C–S and S–S bonds. The IR spectrum of extracted keratin is similar to that of the chicken feather (Fig. S1). The thiol content in 1% w/v keratin sample as obtained from the Ellman’s assay was 0.172 � 0.09 mM. This is similar to a published report where 0.16 mM of thiol was estimated to be present in the extracted feather keratin [34].
2.7. Fiber characterization 2.7.1. Morphological analysis The surface morphology evaluation of the fibers was performed under Field Emission Scanning Microscope (FE-SEM, JEOL, JSM7500F). The fibers were mounted on a copper stub using carbon adhe sive tape and processed for Pd coating. EDX of the samples were also done for determining the elemental composition. 2.7.2. Mechanical characterization Alginate and keratin-alginate crosslinked fibers were evaluated for mechanical tests using 5 cm long fibers with pre-determined diameters measured in a brightfield microscope. Tensile tests of the fibers (n ¼ 10) were performed using a Universal testing machine (Instron 5500R) with a 100 N load cell at a strain rate of 10 mm/min at room temperature according to a published report [37]. The tensile strength, Young’s modulus, and elongation at break were obtained from at least five resultant stress–strain curves. Further, the area under stress-strain curve was used to calculate the toughness of the dual crosslinked and alginate fibers. The density of the fibers (~0.8 g) was measured in a gas pyc nometer (Micromeritics AccuPyc II 1340) using helium gas at a tem perature of 25 � C. Gravimetric toughness was determined by dividing the toughness value obtained by the density of the fibers.
3.2. Preparation of dope and characterization Though keratin is abundantly available, drawing of keratin fibers is rather difficult because of the inherent weak mechanical properties of the biopolymer. Therefore, keratin is covalently crosslinked with algi nate in this study to form a dope for continuous fiber fabrication in a suitable coagulating bath. FT-IR of the crosslinked dope was done to determine the observable changes in the functional groups after the crosslinking process (Fig. 1b). The FTIR spectrum of sodium alginate shows the peaks around 3431, 1603,1416, and 1030 cm 1 that could be attributed to the stretching of O–H,–COO (asymmetric),–COO (sym metric), and C–O–C, respectively. This is in agreement with a previous report [38]. The spectrum of crosslinked keratin-alginate dope shows the characteristic absorption bands of alginate. In the crosslinked dope, the band for Amide A and Amide B was still evidenced similar to the keratin sample. The peaks at 1622 cm 1 and 1417 cm 1 could be – O) asymmetric and symmetric stretching of ascribed to the carbonyl (C–
2.7.3. Water absorption ratio of fibers The water intake efficiency of the fibers (n ¼ 3) was evaluated by soaking dry fibers in PBS for a pre-determined time at 25 � C. After that excess PBS was drawn out. The weight of the fibers before and after the immersion in PBS was determined by weighing balance. The water ab sorption ratio (AR %) was calculated by the equation: AR% ¼
ðWw WdÞ � 100 Wd
Here, Wd and Ww are the weights of the fibers before and after im mersion in PBS [38]. 2.7.4. Thermogravimetric analysis Thermal degradation behavior of keratin, alginate and crosslinked keratin-alginate fibers were evaluated in NETZSCH, STA 449 F1 Jupiter 3
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Fig. 1. (a) SDS PAGE image for molecular weight determination of keratin, (b) FTIR graph of extracted keratin and dope and (c) Crosslinking density of dope prepared from various ratios of Keratin and alginate (Dope-K:A) and 10 mM EDC. Significant differences between groups at **p < 0.01, ***p < 0.001 and ****p < 0.0001. M, marker; S, sample.
Fig. 2. Schematic illustration showing chemistry of dope formation by covalent crosslinking of the biopolymers (keratin with alginate) and then ionic complexation by calcium ions to produce dual crosslinked fibers. 4
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alginate respectively. The peak at 1030 cm 1 could be assigned to C–O–C stretching. This agrees with previous reports [10,40]. The disappearance of the NH2 associated band at 1534 cm 1 and appearance of a new band of amide at 1615 cm 1 suggested successful crosslinking of the biopolymers. Mechanistically, crosslinking of carboxylic group of alginate with amine group of keratin using the crosslinker EDC leads to amide linkages [35]. In the dope, reduced intensities of the peaks be tween the regions. 1190–974/cm and 1480–1370/cm were evidenced, indicating a reduction of the mannuronate and guluronate groups, respectively, compared to that of sodium alginate [35]. The characteristic peak of amide III at 1208 cm 1 from keratin was evidenced in the dope spec trum, which confirmed the incorporation of keratin in dope. The scheme of crosslinking reaction is presented in Fig. 2. The crosslinking index was evaluated from the ninhydrin assay wherein the sample containing higher amount of unbound amine moi eties will result in a higher optical absorbance reading and will have low crosslinking index. Accordingly, a sample that has few free amine functional groups will demonstrate a lower absorbance and subse quently have high crosslinking index. The keratin: alginate ratio
significantly influenced the crosslinking index of the dope. The cross linking density of the dope gradually increased and percentage of free amine decreased with the increase in the ratio of keratin: alginate in the dope (Fig. 1c). The crosslinking density significantly increased (p < 0.0001) from 73.5% � 1.2–93% � 0.2 upon increasing the keratinalginate ratio from 1:1 to 1:10 with EDC concentration at 10 mM. A lower crosslinking density (85.2% � 0.2) was recorded for the dope formed with 1 mM EDC compared to 10 mM EDC when keratin: alginate ratio was kept at a constant at 1:10; on the other hand, there was no difference in the crosslinking degree upon further increasing the EDC concentration above 20 mM (Fig. S2). Therefore, the optimum EDC concentration which was used throughout in this study was 10 mM. Crosslinked dopes of keratin-alginate with 0.1% and 10% alginate were of very low and very high viscosities respectively which made it impossible to be drawn from needle through coagulating bath to fabri cate fibers (result not shown). 3.3. Fiber fabrication Keratin-alginate dope at different ratios was found to be spinnable
Fig. 3. Representative SEM images of (a) dual crosslinked keratin-alginate fibers and (d) alginate fibers, scale of 10 μm. Representative high magnification of (b) the dual crosslinked keratin-alginate fiber and (e) alginate fiber, scale of 1 μm. Cross-section of representative (c) keratin-alginate fiber, (f) alginate fiber, scale of 10 μm. (g) EDAX graph of the surface and cross-sections of the fibers. DCF-1:10, dual crosslinked fiber; Alg, alginate fiber. 5
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via wet spinning and drawn into uniformed diameter fibers in calcium chloride bath (Fig. S3). During wet spinning, following extrusion of dope into coagulation bath, calcium chloride act as an instantaneous coagu lant and immobilizes the polymer jet by ionic complexation, thereby stabilizing the jet to produce fiber (Fig. 2). Utilizing chicken feather as a source of keratin for fiber production will add value to the poultry in dustry, provide a sustainable supply of fibers, and the applications of waste feather will also mitigate current disposal problems, thereby benefit the environment. Though keratin and alginate combination are previously documented in literature [15,38] this is the first report of dual crosslinked keratin-alginate wet spun fibers. The fibers prepared from the crosslinked dopes (Dope-1:1, Dope-1:2, Dope-1:4 and Dope-1:10) in the calcium bath will be referred to as DCF-1:1, DCF-1:2, DCF-1:4 and DCF-1:10 respectively. The average diameter of dual crosslinked keratin-alginate fibers was found to be in the range of 30–130 μm (Table S1). For control, alginate dope was also extruded in the coagulation bath to form fibers and named Alg. The average
diameter of alginate fibers was found to be 31.5 μm (Table S1). 3.4. Characterization of fibers 3.4.1. Morphological analysis The morphology of the dual crosslinked and alginate fibers was investigated by scanning electron microscopy (Fig. 3). The dual cross linked fibers showed cylindrical morphology whereas the alginate fibers showed flattened morphology (Fig. 3 a, d). At higher magnification, the SEM images showed wrinkles all over the surface of the fibers (Fig. 3b, e). These wrinkles may be due to in-homogenous drying and shrinkage of the fibers as well as the roughness along the inner perimeter of the blunt needle hole [27]. At higher magnification, it can also be seen that the surface of alginate fibers are smoother than the dual crosslinked keratin-alginate fibers. Further, the fibers were fractured (Fig. 3c, f) to reveal the morphology and % of elements using EDX in its core and top surface. EDX mapping showed that calcium was present in both the
Fig. 4. Representative mechanical properties of the fabricated fibers, DCF-Keratin:Alginate (a) Stress-strain graph, (b) Tensile strength, (c) Elongation at break (%) (d) Elastic modulus (e) Toughness (f) Diagram of strength versus elongation for comparing a range of natural fibers and tissues such as cotton, jute, coir, sisal, lotus, heart valves, tendon, ligament, hard tissue (bone), and elastic cartilage (Wu et al., 2014). Significant differences between groups at * p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. DCF, dual crosslinked fiber; Alg, alginate fiber. 6
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fibers confirming calcium mediated coagulation for the preparation of the fibers (Fig. 3g). The presence of carbon, nitrogen, oxygen, and cal cium was evidenced in both alginate fibers and dual crosslinked fibers. In addition, sulphur and nitrogen was also present in the dual cross linked fibers indicating presence of keratin in them. Further, the core and top surface of fibers revealed differential % of calcium incorporation using EDX (Fig. 3g). Noticeably, the core region of the dual crosslinked fiber contains 5.6% calcium atomic wt. against the 17.2% calcium atomic wt. on the surface region indicating calcium incorporation in the fibers via surface diffusion mediated crosslinking reactions. Similar result of differential calcium content was also ob tained for the alginate fiber (Table S2).
3.4.3. Water absorption ratio of fibers Water absorption capacity influences the mechanical and diffusion properties of the fibers; thus, it is considered to be an important parameter for the fibers. In this study, it can be seen that the respective biopolymers ratio and crosslinking index had a significant influence on the water uptake capacity of the fibers (Fig. 5a). When the keratin: alginate ratio increased from 1:1 to 1:10 in the dual crosslinked fibers, the swelling ratio significantly increased (p < 0.0001) from 26.6% to 95.1%. Though with the increase in crosslinking index, the water ab sorption ratio and hydrophilicity should reduce, but in the case of DCF1:1 fibers as the crosslinking between alginate and keratin is low; the fibers were not mechanically strong to retain their form in the PBS environment which subsequently limited swelling and retention capac ity. Also, the increased proportion of carboxyl groups of alginate in DCF1:10 contributed to higher swelling. This is in agreement with a previous report [35]. Notably, the swelling of the alginate fibers (165.5% � 6.5) were significantly higher (p < 0.0001) than all the keratin-alginate fi bers. This is because dual crosslinking of the keratin-alginate fibers restricted them to inflate when they were incubated in aqueous conditions.
3.4.2. Mechanical characterization Dual crosslinked and alginate fibers were evaluated for mechanical testing with 100 N load cell with strain rate at 10 mm/min (Fig. 4). The stress-strain graph of the fibers shows linear elasticity at low strains, followed by a plastic region of non-linear increase in stress in response to a large deformation, and finally a breaking point at the largest strain (Fig. 4a and inset of 4a). Tensile strength of the fibers were calculated from the stress-strain curves and shown in Fig. 4b. It can be observed that higher crosslinking density led to higher tensile strength, where DCF-1:10 recorded significantly higher (p < 0.0001) tensile strength of 102.4 � 20.2 MPa compared to DCF-1:1 (9.2 � 0.8 MPa), DCF-1:2 (11.3 � 3.4 MPa) and DCF-1:4 (15.7 � 4.5). It is to be noted that while there was no difference in the crosslinking density between dope-1:4 and dope-1:10, the fiber DCF-1:10 had higher tensile strength than DCF-1:4. This may be because higher proportion of alginate incorporated in the dope-1:10 enabled higher ionic crosslinking with the calcium ions dur ing fiber formation. Thus it can be emphasized that both ionic as well as covalent crosslinking contributes to the mechanical properties of the fibers. Noticeably, the tensile strength for DCF-1:10 significantly increased (p < 0.05) by 27% than alginate fibers (80.7 � 8.2 MPa). The elongation at break (%) increased significantly (p < 0.01) with increase in keratin: alginate ratio from 11% for DCF-1:1–20% for DCF-1:4 and DCF-1:10 (Fig. 4c). The elongation at break (%) of alginate fibers at 25% was significantly higher (p < 0.05) than all the keratin-alginate fibers. Interestingly the % elongation of DCF-1:10 obtained in this study are higher than the reported values for cotton, silk and zein fibers [3] indicating better usability of the fibers. In general, fiber elasticity is commonly described by the extent of recovery of a fiber after being subjected to loading and unloading action. The modulus of the fibers gradually increased with increasing crosslinking density (Fig. 4d). The fiber DCF-1:10 displayed modulus of 21.9 � 0.8 MPa which was significantly higher (p < 0.0001) than the modulus of 2.1 � 0.2 MPa for DCF-1:1, 3.4 � 0.2 MPa for DCF-1:2 and 4.1 � 0.1 MPa for DCF-1:4. The above data suggests that higher keratin to alginate ratio improved the overall strength and elasticity of the crosslinked keratin-alginate fibers. Notably, DCF-1:10 displayed 20% higher modulus than alginate fibers, which can be attributed to the dual crosslinking of the fibers. Similarly, the toughness increased dramatically from 82.2 MJ m 3 for DCF-1:1–1152.9 MJ m 3 for DCF-1:10 (Fig. 4e), which was 33% higher than alginate fibers. Because the density of DCF-1:10 obtained is ~1.59 g cm 3, the weight-specific toughness or gravimetric toughness of the DCF-1:10 were calculated to be 724 J g 1 which is much higher than the reported values for Kevlar (78 J g 1) [41,42]. The mechanical properties of the dual crosslinked fibers may be further improved by increasing the concentration of ionic crosslinker. The strength vs. elongation of DCF-1:10 are compared with the natural fibers and human tissues as shown in Fig. 4f. Notably, though dual crosslinked fibers ex hibits lower strength than the other natural fibers, but their elongation at break % is much higher than the reported values for natural fibers [6, 43] which may render the dual crosslinked fibers less susceptible to wrinkles. More importantly, the mechanical properties of the DCF-1:10 are close to that of the living human tissues, especially ligament that makes them attractive for tissue engineering applications.
3.4.4. Thermogravimetric analysis The thermo-gravimetric analysis of keratin extracted from chicken feathers, alginate fiber (Alg) and a representative dual crosslinked keratin-alginate fiber as shown in Fig. 5b. In case of keratin, the initial weight loss can be attributed to moisture evaporation around 100 � C; the second weight loss was due to the decomposition of keratin started at 200 � C. A weight loss around 100 � C for the dual crosslinked fibers could be attributed to water evaporation, however, the dual crosslinked fibers decomposed at a much lower temperature compared to the extracted keratin. Alginate fibers exhibited a three-step decomposition, with the first step at 75 � C due to dehydration, the second step starting at 160 � C, and the third step at 500 � C due to calcium decomposition. A 50% mass loss was observed at 231 � C and 342.8 � C of alginate fibers and keratin extract respectively. For the dual crosslinked fibers, 50% mass loss was evidenced at the range of 175 � C to 300 � C. As keratin is organic, it was decomposed completely while the fibers evidenced inorganic residues possibly due to incorporation of the calcium chloride during fiber fabrication process which further supports our EDX data. 3.5. Textile structures The dual crosslinked fibers (DCF-1:10) were assembled into complex architectures to demonstrate our technology for producing natural polymer based textiles for sustainable clothing. Further, these fibers could also be used to fabricate reinforcing structures and as medical textile. Fig. 6a and b shows the fibers were flexible and easily formed twists and knots as evidenced from FE-SEM micrographs. Further, the fibers were assembled into simple and complex braided structures that proved the ability of the fibers to withstand the mechanical constraints imposed by regular textile processes (Fig. 6c and d). A braided structure offers a higher surface area providing superior adhesion possibilities for cells [44,45]. In our future work, we plan to use the fiber braids for ligament tissue engineering owing to the similarity in the mechanical properties and architecture with the tissue. 4. Conclusions In summary, we have developed dual crosslinked fibers from renewable and abundantly available biopolymers using environmen tally friendly processing method. The covalent crosslinking between keratin and alginate has been proven to effectively form amide linkages to form a dope. The dope when extruded in a coagulation bath was further stabilized by calcium ions to form dual crosslinked uniform sized fibers. The fibers prepared from different proportions of biopolymers displayed tunable tensile strength, modulus, toughness and swelling. 7
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Fig. 5. (a) Water absorption ratio of the fibers, and (b) Thermogravimetric analysis of Keratin, Alg and DCF-1:10. Significant differences between groups at * p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. DCF, dual crosslinked fiber; Alg, alginate fiber.
Fig. 6. The flexibility of the dual crosslinked fibers demonstrated by (a) Twist, scale of 10 μm (b) knot, scale of 10 μm and formation of braid by twisting of (c) 3 fibers, scale of 100 μm and (d) 3 � 3 fibers, scale of 100 μm.
The optimised fiber had improved mechanical properties and displayed controlled swelling compared to control alginate fibers. Further, the fi bers displayed flexibility as demonstrated from the twists and knots for assembly into complex textile architectures such as braids. Based on the promising results from this study, we are actively developing methods to design sustainable textile materials with robustness for organic ecofriendly clothing as well as for medical textile applications such as patches, implants, mesh forms, and for ligament tissue engineering.
reviewing and editing. Data availability The raw data required to reproduce these findings can be obtained upon request to [email protected]. Declaration of competing interest The authors declare no competing financial interests.
Author contribution Ashmita Mukherjee: Experimental work, Methodology, Writing original draft preparation. Yogesh H. Kabutare: Experimental work, Validation. Paulomi Ghosh: Supervision, Conceptualization, Writing -
Acknowledgements This work received funding from Department of Science and 8
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Polymer Testing 81 (2020) 106286
Technology -INSPIRE Faculty [DST/INSPIRE/04/2016/002483] and CSIR-Indian Institute of Chemical Biology, India. Y.H.K. thanks NIPER Kolkata for fellowship. We thank Prof. Samit Chattopadhyay and Dr. Indubhusan Deb for scientific suggestions.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.polymertesting.2019.106286. References [1] R. Geyer, J.R. Jambeck, K.L. Law, Production, use, and fate of all plastics ever made, Sci. Adv. 3 (7) (2017), e1700782. [2] O. Nechyporchuk, T. K€ ohnke, Regenerated casein–nanocellulose composite fibers via wet spinning, ACS Sustain. Chem. Eng. 7 (2019) 1419–1426. [3] A.J. Poole, J.S. Church, M.G. Huson, Environmentally sustainable fibers from regenerated protein, Biomacromolecules 10 (2009) 1–8. [4] M.M. Brooks, Regenerated protein fibres: a preliminary review, in: S. Eichhorn, J. W.S. Hearle, M. Jaffe, T. Kikutani (Eds.), Handbook of Textile Fibre Structure, Woodhead Publishing, 2009, pp. 234–265. [5] S.J. Gerbode, J.R. Puzey, A.G. McCormick, L. Mahadevan, How the cucumber tendril coils and overwinds, Science 337 (2012) 1087–1091. [6] M. Wu, H. Shuai, Q. Cheng, L. Jiang, Bioinspired green composite lotus fibers, Angew. Chem. Int. Ed. 53 (2014) 3358–3361. [7] F. Guo, N. Wang, Q. Cheng, L. Hou, J. Liu, Y. Yu, Y. Zhao, Low cost coir fiber composite with integrated strength and toughness, ACS Sustain. Chem. Eng. 4 (2016) 5450–5455. [8] K. Callegaro, A. Brandelli, D.J. Daroit, Beyond plucking: feathers bioprocessing into valuable protein hydrolysates, Waste Manag. 95 (2019) 399–415. [9] T. Korniłłowicz-Kowalska, J. Bohacz, Biodegradation of keratin waste: theory and practical aspects, Waste Manag. 31 (2011) 1689–1701. [10] A. Idris, R. Vijayaraghavan, U.A. Rana, D. Fredericks, A.F. Patti, D.R. MacFarlane, Dissolution of feather keratin in ionic liquids, Green Chem. 15 (2013) 525–534. [11] S. Członka, N. Sienkiewicz, A. Strąkowska, K. Strzelec, Keratin feathers as a filler for rigid polyurethanefoams on the basis of soybean oil polyol, Polym. Test. 72 (2018) 32–45. [12] J. Navarro, J. Swayambunathan, M. Lerman, M. Santoro, J.P. Fisher, Development of keratin-based membranes for potential use in skin repair, Acta Biomater. 83 (2019) 177–188. [13] A. Shavandi, T.H. Silva, A.A. Bekhit, A.E.A. Bekhit, Keratin: dissolution, extraction and biomedical application, Biomater. Sci. 5 (2017) 1699–1735. [14] Q. Li, L. Zhu, R. Liu, D. Huang, X. Jin, N. Che, Z. Li, X. Qu, H. Kang, Y. Huang, Biological stimuli responsive drug carriers based on keratin for triggerable drug delivery, J. Mater. Chem. B 22 (2012) 19964–19973. [15] Z. Sun, Z. Yi, H. Zhang, X. Ma, W. Su, X. Sun, X. Li, Bio-responsive alginate-keratin composite nanogels with enhanced drug loading efficiency for cancer therapy, Carbohydr. Polym. 175 (2017) 159–169. [16] M.A. Khosa, J. Wu, A. Ullah, Chemical modification, characterization, and application of chicken feathers as novel biosorbents, RSC Adv. 3 (2013) 20800–20810. [17] G. Galaburri, M.L.P. Ramos, J.M. L� azaro-Martínez, R.F. de Luis, M.I. Arriortua, M. E. Villanueva, G.J. Copello, pH and ion-selective swelling behaviour of keratin and keratose 3D hydrogels, Eur. Polym. J. 118 (2019) 1–9. [18] J.G. Rouse, M.E. Van Dyke, A review of keratin-based biomaterials for biomedical applications, Materials 3 (2010) 999–1014. [19] A. Aluigi, A. Varesano, A. Montarsolo, C. Vineis, F. Ferrero, G. Mazzuchetti, C. Tonin, Electrospinning of keratin/poly(ethylene oxide) blend nanofibers, J. Appl. Polym. Sci. 104 (2007) 863–870. [20] M. Zoccola, A. Aluigi, C. Vineis, C. Tonin, F. Ferrero, M.G. Piacentino, Study on cast membranes and electrospun nanofibers made from keratin/fibroin blends, Biomacromolecules 9 (2008) 2819–2825. [21] A. Vasconcelos, G. Freddi, A. Cavaco-Paulo, Biodegradable materials based on silk fibroin and keratin, Biomacromolecules 9 (2008) 1299–1305. [22] K. Katoh, M. Shibayama, T. Tanabe, K. Yamauchi, Preparation and properties of keratin poly(vinyl alcohol) blend fiber, J. Appl. Polym. Sci. 91 (2004) 756–762.
9
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Dual crosslinked keratin-alginate fibers formed via ionic complexation of amide networks with improved toughness for assembling into braids Ashmita Mukherjee, Yogesh H. Kabutare, Paulomi Ghosh * CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032, West Bengal, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Dual crosslinking Sustainable fibers Green process Textile Braids
With the dwindling of petroleum resources worldwide, there is an immediate need for a renewable, environment friendly, cost effective and sustainable bio-resource in the textile industry. Here, we report a dual crosslinked fiber (DCF) derived from renewable biopolymers. In this study, keratin was extracted from bio-waste of chicken feathers with a thiol content of 0.172 mM. The extracted keratin was used to prepare dope with alginate at different ratios and N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride via amide linkages. The formation of covalently crosslinked dope was evidenced from FTIR and ninhydrin assay. The dope was then extruded in calcium bath to produce fibers with uniform diameter wherein the calcium ions were used to ion ically crosslink the covalently crosslinked dope. The resulting dual crosslinked fibers were characterized in terms of chemical composition, surface morphology, mechanical properties, thermal degradation, and swelling. The strength, modulus and toughness of the dual crosslinked fibers were substantially improved by 27%, 20%, and 33% respectively than that of control alginate fiber. The gravimetric toughness of the optimised dual crosslinked fiber (724 J g 1) was much higher than the values reported for Kevlar (78 J g 1). We further assembled the dual crosslinked fibers into complex braided architectures using the textile techniques, demonstrating the flexibility of the fibers. We believe that this preliminary work of sustainable fiber production could open new insights into eco-friendly organic textile manufacturing and for tissue engineering applications.
1. Introduction Textile industry depends mostly on the synthetic polymers besides few natural polymers such as cotton, silk and jute. Synthetic and manmade fibers are however not sustainable because of its detrimental ef fect on environment coupled with global depletion of petroleum re serves and rising oil cost [1,2]. Therefore, it is necessary to consider producing fibers from alternative bio-resources with large availability [3,4]. In recent years, sustainable fibers derived from eco-friendly nat ural materials such as cucumber vine, lotus, and coconut coir have received increasing attention [5–7]. Chicken feathers that are otherwise disposed in landfills could be a prospective resource for keratin to pro duce alternative natural polymer based fibers for industrial and medical applications [8–10]. Keratins fabricated into different forms such as sponges, films, nanoparticles, membranes either alone or blended are highly attractive for industrial and biomedical use due to their intrinsic biocompatibility, and physical properties [11–17]. However, drawing of keratin fibers is still not actively pursued due to the inherent poor mechanical properties
of pure keratin. Therefore, many researchers have attempted to increase the processability of keratin by combining it with synthetic or natural polymers such as poly(ethylene oxide) (PEO), poly-(vinyl alcohol) (PVA), silk and cellulose for fiber formation [18–22]. For instance, Katoh et al. [22] used PVA to increase the viscosity of the aqueous keratin solutions to improve upon the fiber drawing capabilities for potential applications as absorbents for toxic substances. However, these fibers were predominantly petroleum-dependent because of high amount of PVA incorporated, and thus were non-sustainable, non-re newable, and had limited industrial potential. Wrzesniewska-Tosik et al. [23] also employed fabrication techniques to prepare keratin-cellulose fibers for possible application as hygienic fabrics. However, introduc tion of keratin into cellulose fibers deteriorated the overall mechanical strength of the fibrous materials. Recently, Xu and Yang [24] reported the preparation of keratin fibers of high tensile strength but other associated parameters such as elastic modulus, toughness, and swelling were not detailed. Thus, there is still a gap to produce sustainable ker atin based fibers with sufficient mechanical properties. This motivates us to use the crosslinking mechanism to produce mechanically robust
* Corresponding author. E-mail address: [email protected] (P. Ghosh). https://doi.org/10.1016/j.polymertesting.2019.106286 Received 6 October 2019; Received in revised form 21 November 2019; Accepted 7 December 2019 Available online 9 December 2019 0142-9418/© 2019 Elsevier Ltd. All rights reserved.
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keratin based fibers. Sodium alginate is a naturally occurring biocompatible polymer composed of blocks of (1–4)-linked β-D-mannuronic acid (M) and α-Lguluronic acid (G) monomers and known for having many biomedical applications including wound healing, drug delivery and tissue engi neering [25–27]. Alginate can be easily modified by enzymatic and chemical means to alter the composition and structure to enhance the functional properties for specific applications [28]. Alginate readily crosslink with divalent cations, usually calcium ions forming ionic linkages with carboxylic acid groups on the polymer backbone. Samorezov et al. [29] employed ionic- and photo-crosslinking to create alginate hydrogels with spatially controlled physical and chemical properties. According to the authors, these dual crosslinked alginate hydrogels, with tunable mechanical properties, has potential to be used in regenerative medicine. In recent years, various other polymeric de rivatives have been developed with dual crosslinking mechanism [30–32]. Nevertheless, to the best of our knowledge, covalently cross linked keratin-alginate forming fibers via ionic complexation and resulting in a dual crosslinked network are not yet documented in literature. Herein, we have extracted keratin from Gallus gallus domesticus (chicken) feathers and characterized it. Thereafter, we have chemically crosslinked keratin with alginate via N-(3-Dimethylaminopropyl)-N0 ethylcarbodiimide hydrochloride (EDC) to form dope. The dope pre pared from varying the amount of keratin, alginate and concentration of crosslinkers was used to prepare uniform diameter fibers via ionic crosslinking in a calcium bath. An elaborate comparative study was done to evaluate the effect of the dual crosslinking on tensile strength, elastic modulus, toughness, swelling, and thermal properties of the fi bers. We further demonstrated the flexibility of the dual crosslinked fi bers by assembling them into complex braided architectures using the regular textile techniques.
subsequently lyophilised to obtain the powdered form of keratin and was stocked in storage containers with lid at room temperature until further use. 2.3. Characterization of keratin The molecular weight of the extracted keratin was estimated by SDS PAGE according to a previously published report [34] and is provided in the supplementary information. Separation was performed at 80 V to run the sample from the stacking gel to the separating end and 120 V was maintained for 2 h to run the protein sample over the gel. Thereafter the gel was stained in the staining solution overnight, de-stained and then was placed in the UV light to observe the separation of protein strands in the gel. The FTIR of freeze-dried chicken feathers and extracted sample from the feathers was determined on a PerkinElmer system (Spectrum-100). The samples were powdered, mixed with KBr to form pellet and analysis was performed between a range of 4000 and 400 cm 1. The thiol content of the feather keratin was determined by Ellman’s assay following a previously published report [34]. Briefly, 0.1 M PBS-EDTA buffer of pH 8.0 was freshly prepared containing 50 μL of 4 mg/mL DTNB. Then, 250 μL of 1% w/v extracted keratin was pipetted to the 2.5 mL PBS-EDTA buffer and mixed well. The sample was then subjected to incubation at 25 � C for 15 min, and the absorbance was taken at 412 nm by UV Spectrophotometer (UV-10, Thermo). Standards were prepared using cysteine to make a standard curve for determina tion of the thiol content. 2.4. Preparation of dope For production of fibers, dope was prepared by carbodiimide cross linking of keratin with alginate. The crosslinking was carried out by varying the concentrations of crosslinker and proportion of the bio polymers (Table 1) according to a previously published report [35]. Briefly, keratin solution% (w/v) was added into the alginate–EDC mixture and stirred at room temperature for 24 h to obtain a clear viscous dope. Alginate dope, 1% (w/v) was also prepared and used as the control.
2. Experimental section 2.1. Materials Chicken feathers of Gallus gallus domesticus were collected from local market (Jadavpur, Kolkata, India). Ethanol (Himedia), urea (Merck), sodium-bisulfite (Lobachemie, India) sodium dodecyl sulphate (SDS, Qualigens), Whatman filter paper (GE Healthcare UK Limited) were used for the process of keratin extraction. 5, 50 -dithio-bis-(2-nitro benzoic acid) [DTNB i.e. Ellman’s Reagent] (Sigma, USA), L-cysteine hydrochloride (Sigma, USA), EDTA (Qualigens), sodium dihydrogen phosphate (Merck) were used for thiol content determination. For per forming SDS PAGE, phosphate buffered saline (PBS, Himedia), glycerol and TRIS HCl (Merck), TEMED (sigma), and the protein marker of the range 245–5 kDa (Puragen, Genetix) were used. Alginate (Himedia) and EDC (Sigma, USA) were used for dope and fiber preparation. Ninhydrin reagent (Sigma) was used for determining crosslinking density.
2.5. Characterization of dope The IR spectrum of lyophilised keratin-alginate crosslinked dope was determined by FTIR using potassium bromide pellet method. The crosslinking index was evaluated quantitatively by determining the amount of free amine using ninhydrin assay as per a previously pub lished report [35]. Briefly, 1 mL of 0.4% w/v ninhydrin aqueous solution was added to 10 mg of minced, pulverized sample (n ¼ 10), mixed and kept in a hot water bath (OVFU) at 90 � C for 20 min. The mixture was subsequently centrifuged after cooling down to room temperature. The mixture’s supernatant was used for determining the absorbance at 570 nm in a microplate reader MULTISKAN GO (Thermo Scientific). The degree of crosslinking was calculated using the formula below:
2.2. Extraction of feather keratin Chicken feathers were washed with warm water, dried at 50 � C in an incubator (RRB Scientific & Surgicals, India) for 24 h and chopped into tiny pieces with a scissor. Thereafter, the feathers were sterilized in ethanol for 15 min and were air dried. The feathers were then delipi dified and keratin was extracted via reduction process as per a previ ously published report [33]. Briefly, 2 g defatted feathers were added in 50 mL aqueous solution consisting of 0.5 M sodium bisulfite, 8 M urea and 0.08 M SDS. The mixture was kept at 50 � C in an incubator under shaking condition for 2 h. After that, the resulting mixture was subjected to centrifugation at 9000�g (REMI, model 8C, India) for 15 min to discard off the insoluble chicken feathers. The supernatant was filtered through a funnel containing filter paper and the filtrate was dialysed using cellulose membrane dialysis tube. The dialysed sample was
Table 1 Amounts of keratin, alginate and EDC for dope preparation. Dope code
K: A
Keratin (w/v) %
Alginate (w/v) %
EDC conc. (mM)
Dope-1:10 Dope-1:4 Dope-1:2 Dope-1:1 Dope-1-1:10 Dope20–1:10 Dope-Alg
1:10 1: 4 1: 2 1: 1 1:10 1:10
0.1 0.25 0.5 1 0.1 0.1
1 1 1 1 1 1
10 10 10 10 1 20
N/A
N/A
1
N/A
K: A, weight ratio of keratin to alginate; Dope-Alg, dope of 1% w/v alginate; N/ A, not applicable. 2
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Crosslinking index ð%Þ ¼
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TGA instrument in the temperature range of 50–800 � C, using aluminum crucibles, under dynamic nitrogen atmosphere (50 mL/min) with a heating rate of 10 � C/min following a published report [38].
ðNHo– NHtÞ � 100 NHo
Where, NHo and NHt are the absorbance of the lyophilised keratin before and after crosslinking, respectively.
2.8. Textile structures
2.6. Fiber fabrication
To determine flexibility for textile manufacture, the dual crosslinked fibers were twisted, knotted, and manually intertwined to form simple and complex braided structures. The textile structures were imaged by FE-SEM.
Fibers were prepared by wet spinning following a previously pub lished report with slight modifications [36]. The alginate and cross linked alginate-keratin dope were degassed by centrifuging at 5500 rpm for 10 min and then the solutions were transferred in the 20 mL syringe. The spinning was performed in a custom-made wet-spinning device at room temperature consisting of a syringe pump (Cole-Parmer, US), a polypropylene coagulation bath chamber, a fiber collection shaft and motor (Elmee, Kolkata, India) for controlling the fiber collection rate. The syringe containing dope of alginate (1%) or crosslinked keratin-alginate was loaded into the syringe pump system operated at a rate of 0.05 mL/min. The tip of the syringe was attached with 30 cm long silicon tubing (2 mm inner diameter X 4 mm outer diameter) fitted to a blunt tip metallic needle of 15 mm, 22 gauge (Supelco, Merck, SGE Luer Lock Needles) submerged into the coagulation bath containing 5% w/v CaCl2. The fabricated fibers were collected at a speed of 60 rpm in a rotating shaft fitted with a motor. Calcium ions induced an immediate coagulation of the alginate concurrently forming continuous micron sized fibers. Finally, the fibers were dried at room temperature.
2.9. Statistical analysis The data obtained in this study were presented as mean � standard deviation and statistically evaluated using Graph-pad prism software. There were significant differences between the experimental groups when the p value was less than 0.05. 3. Results and discussion 3.1. Extraction and characterization of keratin Keratin was extracted by the reduction method from bio-waste of chicken feathers that are abundantly and easily available. The extraction was performed using sodium bi-sulphite, a cheap reducing reagent; urea, a protein denaturant and SDS to prevent agglomeration of the protein chains. The yield of keratin from chicken feather was 74.2% by mass. The molecular weight of the extracted keratin determined by SDS PAGE was found to be around 10 KDa (Fig. 1a). This is very close to the result obtained in a previously published report [34]. The FTIR of the freeze-dried extracted sample from chicken feathers is shown in Fig. 1b. The positions of the bands in the sample indicated the conformations of the protein material: 1659 cm 1 for amide I, 1535 cm 1 for amide II, and 1238 cm 1 for amide III. The peak at 3309 cm 1 could be attributed to the stretching vibrations of O–H and N–H (Amide A), the bands at 2960 cm 1 and 2926 cm 1 could be ascribed to the CH3 stretching vibration (Amide B). The peak observed at 1453 cm 1 is due to bending vibrations of CH2 & CH3. The peaks at 1061 cm 1 and 1025 cm 1 could be due to asymmetric and symmetric S–O stretching vibra tions contributed by cysteine-S-sulphonated residues formed during extraction process. This is in agreement with a previous study [39]. The peaks at 985 cm 1 and 584 cm 1 were due to the characteristic ab sorptions of the C–S and S–S bonds. The IR spectrum of extracted keratin is similar to that of the chicken feather (Fig. S1). The thiol content in 1% w/v keratin sample as obtained from the Ellman’s assay was 0.172 � 0.09 mM. This is similar to a published report where 0.16 mM of thiol was estimated to be present in the extracted feather keratin [34].
2.7. Fiber characterization 2.7.1. Morphological analysis The surface morphology evaluation of the fibers was performed under Field Emission Scanning Microscope (FE-SEM, JEOL, JSM7500F). The fibers were mounted on a copper stub using carbon adhe sive tape and processed for Pd coating. EDX of the samples were also done for determining the elemental composition. 2.7.2. Mechanical characterization Alginate and keratin-alginate crosslinked fibers were evaluated for mechanical tests using 5 cm long fibers with pre-determined diameters measured in a brightfield microscope. Tensile tests of the fibers (n ¼ 10) were performed using a Universal testing machine (Instron 5500R) with a 100 N load cell at a strain rate of 10 mm/min at room temperature according to a published report [37]. The tensile strength, Young’s modulus, and elongation at break were obtained from at least five resultant stress–strain curves. Further, the area under stress-strain curve was used to calculate the toughness of the dual crosslinked and alginate fibers. The density of the fibers (~0.8 g) was measured in a gas pyc nometer (Micromeritics AccuPyc II 1340) using helium gas at a tem perature of 25 � C. Gravimetric toughness was determined by dividing the toughness value obtained by the density of the fibers.
3.2. Preparation of dope and characterization Though keratin is abundantly available, drawing of keratin fibers is rather difficult because of the inherent weak mechanical properties of the biopolymer. Therefore, keratin is covalently crosslinked with algi nate in this study to form a dope for continuous fiber fabrication in a suitable coagulating bath. FT-IR of the crosslinked dope was done to determine the observable changes in the functional groups after the crosslinking process (Fig. 1b). The FTIR spectrum of sodium alginate shows the peaks around 3431, 1603,1416, and 1030 cm 1 that could be attributed to the stretching of O–H,–COO (asymmetric),–COO (sym metric), and C–O–C, respectively. This is in agreement with a previous report [38]. The spectrum of crosslinked keratin-alginate dope shows the characteristic absorption bands of alginate. In the crosslinked dope, the band for Amide A and Amide B was still evidenced similar to the keratin sample. The peaks at 1622 cm 1 and 1417 cm 1 could be – O) asymmetric and symmetric stretching of ascribed to the carbonyl (C–
2.7.3. Water absorption ratio of fibers The water intake efficiency of the fibers (n ¼ 3) was evaluated by soaking dry fibers in PBS for a pre-determined time at 25 � C. After that excess PBS was drawn out. The weight of the fibers before and after the immersion in PBS was determined by weighing balance. The water ab sorption ratio (AR %) was calculated by the equation: AR% ¼
ðWw WdÞ � 100 Wd
Here, Wd and Ww are the weights of the fibers before and after im mersion in PBS [38]. 2.7.4. Thermogravimetric analysis Thermal degradation behavior of keratin, alginate and crosslinked keratin-alginate fibers were evaluated in NETZSCH, STA 449 F1 Jupiter 3
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Fig. 1. (a) SDS PAGE image for molecular weight determination of keratin, (b) FTIR graph of extracted keratin and dope and (c) Crosslinking density of dope prepared from various ratios of Keratin and alginate (Dope-K:A) and 10 mM EDC. Significant differences between groups at **p < 0.01, ***p < 0.001 and ****p < 0.0001. M, marker; S, sample.
Fig. 2. Schematic illustration showing chemistry of dope formation by covalent crosslinking of the biopolymers (keratin with alginate) and then ionic complexation by calcium ions to produce dual crosslinked fibers. 4
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alginate respectively. The peak at 1030 cm 1 could be assigned to C–O–C stretching. This agrees with previous reports [10,40]. The disappearance of the NH2 associated band at 1534 cm 1 and appearance of a new band of amide at 1615 cm 1 suggested successful crosslinking of the biopolymers. Mechanistically, crosslinking of carboxylic group of alginate with amine group of keratin using the crosslinker EDC leads to amide linkages [35]. In the dope, reduced intensities of the peaks be tween the regions. 1190–974/cm and 1480–1370/cm were evidenced, indicating a reduction of the mannuronate and guluronate groups, respectively, compared to that of sodium alginate [35]. The characteristic peak of amide III at 1208 cm 1 from keratin was evidenced in the dope spec trum, which confirmed the incorporation of keratin in dope. The scheme of crosslinking reaction is presented in Fig. 2. The crosslinking index was evaluated from the ninhydrin assay wherein the sample containing higher amount of unbound amine moi eties will result in a higher optical absorbance reading and will have low crosslinking index. Accordingly, a sample that has few free amine functional groups will demonstrate a lower absorbance and subse quently have high crosslinking index. The keratin: alginate ratio
significantly influenced the crosslinking index of the dope. The cross linking density of the dope gradually increased and percentage of free amine decreased with the increase in the ratio of keratin: alginate in the dope (Fig. 1c). The crosslinking density significantly increased (p < 0.0001) from 73.5% � 1.2–93% � 0.2 upon increasing the keratinalginate ratio from 1:1 to 1:10 with EDC concentration at 10 mM. A lower crosslinking density (85.2% � 0.2) was recorded for the dope formed with 1 mM EDC compared to 10 mM EDC when keratin: alginate ratio was kept at a constant at 1:10; on the other hand, there was no difference in the crosslinking degree upon further increasing the EDC concentration above 20 mM (Fig. S2). Therefore, the optimum EDC concentration which was used throughout in this study was 10 mM. Crosslinked dopes of keratin-alginate with 0.1% and 10% alginate were of very low and very high viscosities respectively which made it impossible to be drawn from needle through coagulating bath to fabri cate fibers (result not shown). 3.3. Fiber fabrication Keratin-alginate dope at different ratios was found to be spinnable
Fig. 3. Representative SEM images of (a) dual crosslinked keratin-alginate fibers and (d) alginate fibers, scale of 10 μm. Representative high magnification of (b) the dual crosslinked keratin-alginate fiber and (e) alginate fiber, scale of 1 μm. Cross-section of representative (c) keratin-alginate fiber, (f) alginate fiber, scale of 10 μm. (g) EDAX graph of the surface and cross-sections of the fibers. DCF-1:10, dual crosslinked fiber; Alg, alginate fiber. 5
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via wet spinning and drawn into uniformed diameter fibers in calcium chloride bath (Fig. S3). During wet spinning, following extrusion of dope into coagulation bath, calcium chloride act as an instantaneous coagu lant and immobilizes the polymer jet by ionic complexation, thereby stabilizing the jet to produce fiber (Fig. 2). Utilizing chicken feather as a source of keratin for fiber production will add value to the poultry in dustry, provide a sustainable supply of fibers, and the applications of waste feather will also mitigate current disposal problems, thereby benefit the environment. Though keratin and alginate combination are previously documented in literature [15,38] this is the first report of dual crosslinked keratin-alginate wet spun fibers. The fibers prepared from the crosslinked dopes (Dope-1:1, Dope-1:2, Dope-1:4 and Dope-1:10) in the calcium bath will be referred to as DCF-1:1, DCF-1:2, DCF-1:4 and DCF-1:10 respectively. The average diameter of dual crosslinked keratin-alginate fibers was found to be in the range of 30–130 μm (Table S1). For control, alginate dope was also extruded in the coagulation bath to form fibers and named Alg. The average
diameter of alginate fibers was found to be 31.5 μm (Table S1). 3.4. Characterization of fibers 3.4.1. Morphological analysis The morphology of the dual crosslinked and alginate fibers was investigated by scanning electron microscopy (Fig. 3). The dual cross linked fibers showed cylindrical morphology whereas the alginate fibers showed flattened morphology (Fig. 3 a, d). At higher magnification, the SEM images showed wrinkles all over the surface of the fibers (Fig. 3b, e). These wrinkles may be due to in-homogenous drying and shrinkage of the fibers as well as the roughness along the inner perimeter of the blunt needle hole [27]. At higher magnification, it can also be seen that the surface of alginate fibers are smoother than the dual crosslinked keratin-alginate fibers. Further, the fibers were fractured (Fig. 3c, f) to reveal the morphology and % of elements using EDX in its core and top surface. EDX mapping showed that calcium was present in both the
Fig. 4. Representative mechanical properties of the fabricated fibers, DCF-Keratin:Alginate (a) Stress-strain graph, (b) Tensile strength, (c) Elongation at break (%) (d) Elastic modulus (e) Toughness (f) Diagram of strength versus elongation for comparing a range of natural fibers and tissues such as cotton, jute, coir, sisal, lotus, heart valves, tendon, ligament, hard tissue (bone), and elastic cartilage (Wu et al., 2014). Significant differences between groups at * p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. DCF, dual crosslinked fiber; Alg, alginate fiber. 6
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fibers confirming calcium mediated coagulation for the preparation of the fibers (Fig. 3g). The presence of carbon, nitrogen, oxygen, and cal cium was evidenced in both alginate fibers and dual crosslinked fibers. In addition, sulphur and nitrogen was also present in the dual cross linked fibers indicating presence of keratin in them. Further, the core and top surface of fibers revealed differential % of calcium incorporation using EDX (Fig. 3g). Noticeably, the core region of the dual crosslinked fiber contains 5.6% calcium atomic wt. against the 17.2% calcium atomic wt. on the surface region indicating calcium incorporation in the fibers via surface diffusion mediated crosslinking reactions. Similar result of differential calcium content was also ob tained for the alginate fiber (Table S2).
3.4.3. Water absorption ratio of fibers Water absorption capacity influences the mechanical and diffusion properties of the fibers; thus, it is considered to be an important parameter for the fibers. In this study, it can be seen that the respective biopolymers ratio and crosslinking index had a significant influence on the water uptake capacity of the fibers (Fig. 5a). When the keratin: alginate ratio increased from 1:1 to 1:10 in the dual crosslinked fibers, the swelling ratio significantly increased (p < 0.0001) from 26.6% to 95.1%. Though with the increase in crosslinking index, the water ab sorption ratio and hydrophilicity should reduce, but in the case of DCF1:1 fibers as the crosslinking between alginate and keratin is low; the fibers were not mechanically strong to retain their form in the PBS environment which subsequently limited swelling and retention capac ity. Also, the increased proportion of carboxyl groups of alginate in DCF1:10 contributed to higher swelling. This is in agreement with a previous report [35]. Notably, the swelling of the alginate fibers (165.5% � 6.5) were significantly higher (p < 0.0001) than all the keratin-alginate fi bers. This is because dual crosslinking of the keratin-alginate fibers restricted them to inflate when they were incubated in aqueous conditions.
3.4.2. Mechanical characterization Dual crosslinked and alginate fibers were evaluated for mechanical testing with 100 N load cell with strain rate at 10 mm/min (Fig. 4). The stress-strain graph of the fibers shows linear elasticity at low strains, followed by a plastic region of non-linear increase in stress in response to a large deformation, and finally a breaking point at the largest strain (Fig. 4a and inset of 4a). Tensile strength of the fibers were calculated from the stress-strain curves and shown in Fig. 4b. It can be observed that higher crosslinking density led to higher tensile strength, where DCF-1:10 recorded significantly higher (p < 0.0001) tensile strength of 102.4 � 20.2 MPa compared to DCF-1:1 (9.2 � 0.8 MPa), DCF-1:2 (11.3 � 3.4 MPa) and DCF-1:4 (15.7 � 4.5). It is to be noted that while there was no difference in the crosslinking density between dope-1:4 and dope-1:10, the fiber DCF-1:10 had higher tensile strength than DCF-1:4. This may be because higher proportion of alginate incorporated in the dope-1:10 enabled higher ionic crosslinking with the calcium ions dur ing fiber formation. Thus it can be emphasized that both ionic as well as covalent crosslinking contributes to the mechanical properties of the fibers. Noticeably, the tensile strength for DCF-1:10 significantly increased (p < 0.05) by 27% than alginate fibers (80.7 � 8.2 MPa). The elongation at break (%) increased significantly (p < 0.01) with increase in keratin: alginate ratio from 11% for DCF-1:1–20% for DCF-1:4 and DCF-1:10 (Fig. 4c). The elongation at break (%) of alginate fibers at 25% was significantly higher (p < 0.05) than all the keratin-alginate fibers. Interestingly the % elongation of DCF-1:10 obtained in this study are higher than the reported values for cotton, silk and zein fibers [3] indicating better usability of the fibers. In general, fiber elasticity is commonly described by the extent of recovery of a fiber after being subjected to loading and unloading action. The modulus of the fibers gradually increased with increasing crosslinking density (Fig. 4d). The fiber DCF-1:10 displayed modulus of 21.9 � 0.8 MPa which was significantly higher (p < 0.0001) than the modulus of 2.1 � 0.2 MPa for DCF-1:1, 3.4 � 0.2 MPa for DCF-1:2 and 4.1 � 0.1 MPa for DCF-1:4. The above data suggests that higher keratin to alginate ratio improved the overall strength and elasticity of the crosslinked keratin-alginate fibers. Notably, DCF-1:10 displayed 20% higher modulus than alginate fibers, which can be attributed to the dual crosslinking of the fibers. Similarly, the toughness increased dramatically from 82.2 MJ m 3 for DCF-1:1–1152.9 MJ m 3 for DCF-1:10 (Fig. 4e), which was 33% higher than alginate fibers. Because the density of DCF-1:10 obtained is ~1.59 g cm 3, the weight-specific toughness or gravimetric toughness of the DCF-1:10 were calculated to be 724 J g 1 which is much higher than the reported values for Kevlar (78 J g 1) [41,42]. The mechanical properties of the dual crosslinked fibers may be further improved by increasing the concentration of ionic crosslinker. The strength vs. elongation of DCF-1:10 are compared with the natural fibers and human tissues as shown in Fig. 4f. Notably, though dual crosslinked fibers ex hibits lower strength than the other natural fibers, but their elongation at break % is much higher than the reported values for natural fibers [6, 43] which may render the dual crosslinked fibers less susceptible to wrinkles. More importantly, the mechanical properties of the DCF-1:10 are close to that of the living human tissues, especially ligament that makes them attractive for tissue engineering applications.
3.4.4. Thermogravimetric analysis The thermo-gravimetric analysis of keratin extracted from chicken feathers, alginate fiber (Alg) and a representative dual crosslinked keratin-alginate fiber as shown in Fig. 5b. In case of keratin, the initial weight loss can be attributed to moisture evaporation around 100 � C; the second weight loss was due to the decomposition of keratin started at 200 � C. A weight loss around 100 � C for the dual crosslinked fibers could be attributed to water evaporation, however, the dual crosslinked fibers decomposed at a much lower temperature compared to the extracted keratin. Alginate fibers exhibited a three-step decomposition, with the first step at 75 � C due to dehydration, the second step starting at 160 � C, and the third step at 500 � C due to calcium decomposition. A 50% mass loss was observed at 231 � C and 342.8 � C of alginate fibers and keratin extract respectively. For the dual crosslinked fibers, 50% mass loss was evidenced at the range of 175 � C to 300 � C. As keratin is organic, it was decomposed completely while the fibers evidenced inorganic residues possibly due to incorporation of the calcium chloride during fiber fabrication process which further supports our EDX data. 3.5. Textile structures The dual crosslinked fibers (DCF-1:10) were assembled into complex architectures to demonstrate our technology for producing natural polymer based textiles for sustainable clothing. Further, these fibers could also be used to fabricate reinforcing structures and as medical textile. Fig. 6a and b shows the fibers were flexible and easily formed twists and knots as evidenced from FE-SEM micrographs. Further, the fibers were assembled into simple and complex braided structures that proved the ability of the fibers to withstand the mechanical constraints imposed by regular textile processes (Fig. 6c and d). A braided structure offers a higher surface area providing superior adhesion possibilities for cells [44,45]. In our future work, we plan to use the fiber braids for ligament tissue engineering owing to the similarity in the mechanical properties and architecture with the tissue. 4. Conclusions In summary, we have developed dual crosslinked fibers from renewable and abundantly available biopolymers using environmen tally friendly processing method. The covalent crosslinking between keratin and alginate has been proven to effectively form amide linkages to form a dope. The dope when extruded in a coagulation bath was further stabilized by calcium ions to form dual crosslinked uniform sized fibers. The fibers prepared from different proportions of biopolymers displayed tunable tensile strength, modulus, toughness and swelling. 7
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Fig. 5. (a) Water absorption ratio of the fibers, and (b) Thermogravimetric analysis of Keratin, Alg and DCF-1:10. Significant differences between groups at * p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. DCF, dual crosslinked fiber; Alg, alginate fiber.
Fig. 6. The flexibility of the dual crosslinked fibers demonstrated by (a) Twist, scale of 10 μm (b) knot, scale of 10 μm and formation of braid by twisting of (c) 3 fibers, scale of 100 μm and (d) 3 � 3 fibers, scale of 100 μm.
The optimised fiber had improved mechanical properties and displayed controlled swelling compared to control alginate fibers. Further, the fi bers displayed flexibility as demonstrated from the twists and knots for assembly into complex textile architectures such as braids. Based on the promising results from this study, we are actively developing methods to design sustainable textile materials with robustness for organic ecofriendly clothing as well as for medical textile applications such as patches, implants, mesh forms, and for ligament tissue engineering.
reviewing and editing. Data availability The raw data required to reproduce these findings can be obtained upon request to [email protected]. Declaration of competing interest The authors declare no competing financial interests.
Author contribution Ashmita Mukherjee: Experimental work, Methodology, Writing original draft preparation. Yogesh H. Kabutare: Experimental work, Validation. Paulomi Ghosh: Supervision, Conceptualization, Writing -
Acknowledgements This work received funding from Department of Science and 8
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Technology -INSPIRE Faculty [DST/INSPIRE/04/2016/002483] and CSIR-Indian Institute of Chemical Biology, India. Y.H.K. thanks NIPER Kolkata for fellowship. We thank Prof. Samit Chattopadhyay and Dr. Indubhusan Deb for scientific suggestions.
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