Elsevier

European Polymer Journal

Volume 142, 5 January 2021, 110158
European Polymer Journal

Coaxial and emulsion electrospinning of extracted hyaluronic acid and keratin based nanofibers for wound healing applications

https://doi.org/10.1016/j.eurpolymj.2020.110158Get rights and content

Highlights

  • Electrospun fibers loaded with Hyarulonic Acid and Keratin were promising for wound dressing applications.

  • Both Hyarulonic Acid and Keratin were extracted successfully from the animal sources.

  • Hyaluronic Acid obtained from rooster comb.

  • Core-shell nano and micro fibers were fabricated.

  • Both emulsion and coaxial electrospinning techniques used for nano and micro fiber production.

Abstract

Novel composites based on poly(є-caprolactone)/polyethylene oxide loaded with hyaluronic acid(HA) and keratin(KR) were produced separately using emulsion and coaxial electrospinning methods. HA and KR were extracted from animal sources, characterized and loaded into coaxial fiber structures as bioactive agents, separately and together. Morphological, chemical, thermal, and mechanical characteristics of the fibers were investigated. According to the SEM results, diameters of smooth and beadless fibers fabricated via emulsion method were at nanoscale (sub-micron) while fibers of coaxial method were at micro scale. Benefitted electrospinning techniques demonstrated that hydrophobic and hydrophilic polymers can be advantageously combined. Core polymer specific FT-IR bands were not visible, their presence was proven with DSC analysis which confirms core–shell morphology of the fibers. In vitro studies exhibited spun mats did not have any cytotoxic effects and the HA and KR incorporated into the fiber structure synergistically increased cell viability and cell proliferation. This study demonstrated that the electrospun fibers containing HA and KR fabricated by both emulsion and coaxial methods can be efficient for wound healing applications.

Introduction

Wound healing is a complicated process of tissue regeneration in which the body responds to the lost cellular structures as a result of different traumatic injuries [1]. To speed up this process, dressings like gels and creams are mostly applied clinically to the wound area [1], [2]. However, clinical treatment can be a painful and costly process, especially for patients with diabetes-related ulcers [3]. Therefore, there is a need to develop innovative dressings containing bioactive components that will relieve recurrent painful procedures and increase the wound healing process.

Various methods like phase separation, template synthesis, melt blowing, self-assembly, and electrospinning have been used to produce polymeric fibers [1]. Among these methods, electrospinning draws more attention for wound healing applications as it is a simple, cost-effective and versatile method for producing drug-loaded fibers in nanometer-sized [4], [5]. The porous surface of nanofibers produced by this method can mimic the extracellular matrix (ECM) that fills the gaps between the cells and connects and supports them [6], [7]. Besides, these nanofibers can provide a high surface area, which creates a favorable environment for cell -attachment, growth, and proliferation [8], [9]. In addition, the nanofiber dressing prevents the passage of any substance that can cause bacterial or microbial infection and allows the transition of oxygen required for wound healing thanks to its porous structure [8], [10].

The electrospinning method allows the use of various natural and synthetic polymers to fabricate nanofibers. Among the synthetic polymers, poly(є-caprolactone) (PCL) is a suitable candidate for wound dressing applications due to its biocompatibility, biodegradability, structural stability, and mechanical properties [11], [12]. However, it is highly hydrophobic, and its degradation rate can be adjusted by mixing it with a hydrophilic polymer [13], [14]. Polyethylene oxide (PEO) is a hydrophilic, non-toxic, biocompatible, and biodegradable polymer [15], [16]. It rapidly degrades by interacting body fluid, therefore preferred as a drug carrier for wound dressing applications [15], [17]. It has been shown that PEO blending to PCL highly develops the surface features owing to the hydrophilic structure of PEO [13].

Natural polymers are incorporated into the structure of nanofibers made of synthetic polymers in wound dressing applications to enhance compatibility with tissue and promote cell growth and proliferation [18], [19]. Besides, natural polymers, which accelerate the wound healing process due to their structural similarities with ECM, have been shown to repair damaged tissues [20]. It cannot be electrospun alone owing to their molecular structure, therefore generally used together with synthetic polymers for nanofiber production [1].

Hyaluronic acid (HA) is a polysaccharide of the glycosaminoglycan group, which is naturally found in the ECM structure in tissue [21]. It exists in cockscomb, in synovial fluid, in the vitreous humour of the human eye, in the umbilical cord at high amounts [22], [23]. Ensuring the moisture balance of the region in wound healing facilitates cell proliferation and migration [24]. Since HA is a hygroscopic macromolecule, it provides control of hydration in the region during wound healing [25]. Keratin is known to be one of the most abundant proteins in nature and is preferred in tissue engineering research due to its biocompatibility, biodegradability, and biofunctionality [26], [27]. It is found in hair, feathers, wool, nails, horns, and hooves in mammals [28], [29]. The use of keratin in tissue-engineered scaffolds has been shown to promote cell adhesion and proliferation also enhance tissue biocompatibility of the material, both in vitro [29] and in vivo [30], [31].

Different methods have been developed for the incorporation of bioactive molecules into nanofibers by electrospinning such as blend, coaxial, and emulsion electrospinning. It has been indicated in previous studies that coaxial and emulsion electrospinning techniques show superior properties in preserving the properties of bioactive molecules used and drug release studies compared to the blend electrospinning [4], [32]. By using emulsion and coaxial techniques, core–shell structured fibers can be produced in which the drug / bioactive agent can be effectively loaded [33]. It has been a matter of discussion by the researchers which technique is superior because both techniques have their advantages and disadvantages. Emulsion electrospinning compared to coaxial, is a more common technique in terms of providing protein dissolution in a soft solvent, and it has a simpler setup [34], [35]. However, maintaining precise control over the placement of the drug in the core or shell of the structure is one of the advantages of using coaxial electrospinning instead of emulsion [33], [36].

In this study, nanofiber wound dressings containing bioactive substances that accelerate wound healing were produced for wounds such as burns and diabetes-related ulcers. Hyaluronic acid and keratin as bioactive substances were extracted from animal sources and characterization studies were performed. The obtained keratin and hyaluronic acid were incorporated into the core structure of poly(є-caprolactone) and polyethylene oxide polymers and the fibers were produced by emulsion and coaxial electrospinning techniques. The morphology, chemical bond structure, thermal behavior, and mechanical strength of the fibers produced by adding hyaluronic acid and keratin separately and together were investigated. The effects of bioactive substances and two different methods on fiber structures were investigated by detailed characterization tests.

Section snippets

Materials

Hexane, Urea, Sodium Dodecyl Sulfate (SDS), 2-Mercaptoethanol, Acetone, Sodium Acetate, Ethanol, Chloroform, and Amyl alcohol were purchased from Merck KGaA, Germany. Dichloromethane (DCM, ≥99.0% pure) was bought from ISOLAB, Germany. Poly(є-caprolactone) (PCL, the weight-average molecular weight of 80,000 g/mol) and Tween-80 (surfactant) were purchased from Sigma-Aldrich, UK. Polyethylene oxide (PEO, the weight-average molecular weight of 600 g/mol) and dialysis membrane (cut-off value 14 kDa

Results and discussion

As shown in Fig. 3a, the FT-IR spectrum of KR displayed characteristic peaks at 3274 cm−1 (amide A), 2924 cm−1 (amide B) 1624 cm−1 (amide I) ve 1514 cm−1 (amide II), 1207 cm− 1 (amide III) bands. These results agree with the findings of the previous studies [30], [37], [44]. HA spectrum showed single bondOH stretching vibrations at 3283 cm−1 and CH2 stretching vibrations at 2923 cm−1. The peaks at 1630 cm−1, 1449 cm−1, and 1077 cm−1 were attributed to the presence of amid II, C-0 group with C = O

Conclusion

In this study, electrospun PCL / PEO fibers loaded with HA and KR natural polymers that increase biocompatibility and bioactivity were produced using emulsion and coaxial electrospinning methods. HA and KR were extracted successfully from the animal sources and FTIR, SDS PAGE and H-NMR analysis have revealed that HA and KR bioactive polymers were successfully obtained by chemical treatment from rooster combs and animal hooves, respectively. According to SEM results, it was observed that the

CRediT authorship contribution statement

Sena Su: Conceptualization, Methodology. Tuba Bedir: Conceptualization, Methodology. Cevriye Kalkandelen: Data curation, Writing - original draft. Ahmet Ozan Başar: Visualization, Investigation. Hilal Turkoğlu Şaşmazel: Visualization, Investigation. Cem Bulent Ustundag: Validation. Mustafa Sengor: Writing - review & editing. Oguzhan Gunduz: Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the Turkish Scientific and Technical Research Council (TÜBİTAK) under Project Number: 218S270 and this work has also been supported by Marmara University Scientific Research Projects Coordination Unit under grant number FEN-B-121218-0614.

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