Novel PLCL nanofibrous/keratin hydrogel bilayer wound dressing for skin wound repair
Graphical Abstract
Introduction
As the primary physical barrier between the human body and the external environment, the skin plays a crucial role in balancing the body temperature and againsting external bacterial and viral invasion [1], [2]. As the skin is often in direct contact with environments outside the body, it is prone to mechanical damage. In some severe skin wounds, the epidermis and dermis can be completely damaged, and the repair of such skin wounds is often a complex and lengthy process. Researchers have developed various bilayer wound dressings by simulating the anatomical structure of the wounded skin tissue where the epidermis and dermis are damaged [3], [4], [5]. According to the literature, hydrogels with high water content and three-dimensional functional network structure are widely used as wound dressings due to their excellent and tunable chemical, physical and biological properties [6], [7], [8], [9], [10], [11]. Nonetheless, hydrogels with high moisture contents often are brittle and have low mechanical strengths [12], [13], [14]. Therefore, some researchers have combined hydrogels with electrospun nanofibers because of electrospun nanofibers’ good mechanical strength, high porosity, and large surface area-to-volume ratio [4], [15], [16], [17]. Bilayer wound dressing prepared using nanofibers to simulate the epidermis and hydrogels to simulate the dermis has attracted much attention from researchers [18], [19], [20], [21].
The upper epidermis of the skin, which is composed of the basement membrane and keratinocytes and functions to protect against infection and external stimuli by covering the wound, can be simulated by the use of tightly bound structured films or nanofibers. The dermis, which requires a large amount of absorption and retention of wound exudates to promote the proliferation of fibroblasts and migration of keratinocytes under three-dimensional culture conditions, can be mimicked using porous hydrogels or sponges [22]. Common methods for preparation of bilayer wound dressing typically are immersing [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36] and coating techniques [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47]. In a research article, Kim and colleagues manufactured a bilayer scaffold of keratin/chitosan-blended nanofibers and gelatin-methacrylate (GelMA) hydrogel. The keratin/chitosan-blended nanofibrous mats were immersed into an 8% GelMA prepolymer solution of human dermal fibroblasts (HDF). The scaffold of keratin/chitosan-blended nanofibers and GelMA hydrogel has good mechanical properties and can influence the re-epithelialization during skin wound healing [34]. Recently, Ma and co-workers reported the development of a bi-layered scaffold from poly(lactic-co-glycolic acid) (Res-PLGA) nanofiber mat and alginate di-aldehyde (ADA)-gelatin (GEL) crosslinking hydrogel (ADA-GEL) by immersing the Res-PLGA electrospun nanofiber mat in ADA and gelatin solution. The Res-PLGA nanofiber mat and ADA-GEL hydrogel scaffold were found to promote wound healing [48]. Although the preparation method can improve the mechanical properties of the hydrogel/nanofiber bionic bi-layer dressing, it fails to consider the weak interfacial binding force between the hydrogels and the nanofibers. As a result, the material is prone to interlayer cracking. To solve the shortcomings of current hydrogel/electrospinning composite wound dressings, we first proposed a new method, low-pressure filtration-assisted method, to prepare bilayer wound dressing. The bilayer wound dressing manufactured by this method was conducive to solving the problem of weak interfacial adhesion between the two materials. The suction force of the pump produces negative pressure during filtration so that the keratin precursor can penetrate the nanofibers by air pressure above the flat bottom funnel, and this allowed the interface between the hydrogel and the nanofibers to become closer. The hydrogel precursor infiltrated into the pores of nanofibers, with a certain depth of infiltration. The deeply embedded hydrogel acted as a "rivet", thus enhancing the interface bonding between the hydrogel and nanofibers. Thus, compared with other methods (immersing and coating methods) the bilayer wound dressing manufactured by the low-pressure filtration-assisted method had the advantages of strong interfacial adhesion, and the nanofibers, and the hydrogels and the nanofibers were not prone to interlayer cracking.
Studies have shown that the sequences arginine-glycine-aspartic acid (RGD) and leucine-aspartic acid-valine (LDV) in keratin can promote cell adhesion and proliferation [34], [49], [50]. Keratin not only is a natural biomaterial that has good biocompatibility, but also is an essential component of skin; thus, it has gained considerable attention from researchers in recent years [51], [52]. PLCL is a polymer with good mechanical properties and biocompatibility that is widely recognized as a biological material [53]. In addition, the skin wound healing process is regulated by many cytokines. FGF-2 has been shown to promote cell infiltration, migration, proliferation, and microangiogenesis construction. Therefore, FGF-2 should be considered in the design of composite dressings to achieve the best possible repair effects through the synergistic effect between the biomimetic structure of materials and cytokines [54], [55].
In this study, non-FGF-2-loaded and FGF-2-loaded PLCL nanofibrous/keratin hydrogel bilayer wound dressings were designed. The performances of the keratin hydrogel and PLCL nanofibers were evaluated. The microstructure of the keratin protein hydrogel, as well as the swelling performance, mechanical properties, and microstructure of the PLCL nanofibers, were investigated. Two preparation methods, the low-pressure filtration-assisted method, and the coating method, were compared. Based on the comparison, we first developed a new low-pressure filtration-assisted method for the preparation of bionic bilayer dressing to enhance the interface bonding between the hydrogels and the nanofibers. We also assessed the cytotoxicity of the scaffold to L929 cells, and carried out hemolytic and coagulation tests to examine the biocompatibility of the dressings. In vitro degradation experiments were conducted to explore the degradation of the bilayer dressing. Finally, to demonstrate the wound healing effect of the bilayer scaffold, we implanted the bilayer scaffold into full-thickness excisional wounds and then evaluated its wound healing quality, re-epithelization, skin appendages (hair follicles) regeneration, collagen deposition, microangiogenesis, and ADSCs recruitment. The skin healing mechanism of the bilayer dressing was also preliminarily investigated.
Section snippets
Materials
Human hair was obtained from a local barber. The hair was cleaned upon received. Tris-HCl (pH 8.5), thiourea, and urea were purchased from Solarbio (Beijing, China). 2-Mercaptoethanol was purchased from Macklin (Shanghai, China). PLCL (Mn = 100,000, LA: CL = 75:25) was purchased from Daigang Biological Engineering Co., Ltd. (Jinan, China). N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)− 3-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
SDS-PAGE of keratin
The SDS-PAGE analysis of keratin is shown in Fig. 2A. According to the analysis, the majority of human keratins had molecular weights of around 40–60 kDa, while some had molecular weights of about 12–18 kDa. These values correspond to the molecular weights of the main components of human hair keratins and agree with those reported in the literature [64], [65].
Optimization of preparation process of keratin hydrogel and PLCL nanofibers
Photographs and cross-sectional SEM images of freeze-dried keratin hydrogels prepared using a crosslinking agent at different
Conclusion
In conclusion, we successfully prepared a novel bionic PLCL/keratin-FGF-2 bilayer wound dressing. The porous network and uniform pore size of keratin hydrogel provided sufficient space for cell growth, thus could promote cell proliferation and migration. The keratin hydrogel also had excellent swelling properties with the maximum water absorption of up to 874.09%; thus, it could quickly absorb wound exudate and create an environment suitable for wound healing. The keratin hydrogel with optimal
CRediT authorship contribution statement
Miaomiao Zhang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing. Shixin Xu: Methodology, Investigation, Supervision. Chen Du: Methodology, Investigation, Supervision. Ruoying Wang: Methodology, Investigation. Cuicui Han: Methodology, Investigation, Writing – review & editing. Yongan Che: Methodology, Investigation, Conceptualization. Wei Feng: Methodology, Investigation, Conceptualization. Chengwei Wang:
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 work was supported in part by the Key Research and Development Program of Shaanxi (Program No. 2022SF-200), Fund of Jiangsu Key Laboratory of Advanced Functional Polymers Design and Application in Soochow University (Program No. KJS2007), and Undergraduate Training Programs of Shaanxi Province for Innovation and Entrepreneurship (Program No. S202110699368).
Declaration of competing interest
The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported
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