Electrospinning of Collagen and Its Derivatives for Biomedical Applications Electrospinning of Collagen and Its Derivatives for Biomedical Applications

Collagen, gelatin and their derived polypeptides can act as multifunctional natural poly- mers with excellent physicochemical properties for biomedical applications. The use of electrospinning technology can convert collagen materials into nanofibrous materials that exhibit porous micro-nanostructures with good mechanical properties and excellent biocompatibility profiles. In this chapter, a systematic review of collagen electrospin - ning is presented and related applications are introduced including tissue engineering (e.g., artificial skin, artificial vasculature, cartilage repair, etc.), drug delivery, hemostatic dressings, periodontal restoration, biofilms, and wound dressings will now be discussed. wound dressing, nerve regeneration, periodontal regeneration, and vascular reconstruction.


Introduction
Electrospinning is an easy and inexpensive process that can be used to prepare nanofibers from almost any soluble or fusible polymer under the action of a high electrostatic field. These electrospun nanofibers often possess extremely high surface areas, high porosities, tunable pore structures, and superior mechanical properties, which means they can be processed into materials with a wide variety of structure and function design [1][2][3][4][5][6]. Due to these advantages, electrospun nanofibers have been used for a broad range of biomedical and industrial applications, such as protective clothing, wound dressings, drug delivery applications, and tissue engineering [7][8][9][10].
The emergence of electrospinning (also known as electrostatic spinning) originated more than 100 years ago, with Zeleny first reporting on a variant of the electrospinning process in

Devices used for collagen electrospinning
Electrospinning is carried out using a Taylor cone that is generated by applying a high voltage to a polymer or melt solution, which results in formation of a liquid jet that is formed from interaction of a continuously increasing electric field with the surface tension of a droplet surface. During this process, a liquid jet starts oscillating to generate an irregular high-frequency spiral motion that leads to stretching of a fiber that is accompanied by fast volatilization of the solvent. This results in nano-scale fibers either being formed in a random manner on a collecting device, or being cross-linked into a membrane when a move and rotate collecting device is employed for processing [36,37]. The electrospinning apparatus is comprised of three parts: a high-voltage direct-current power supply, a liquid supply unit, and a collecting device [38]. Depending on the nature of the liquid supply unit, the collagen electrospinning apparatus can be classified into two types: needle electrospinning or needleless electrospinning. The liquid supply unit of a needle electrospinning unit normally consists of a microinjection pump, a syringe, a single spinneret (or spinneret array), and a metal conducting wire that connects the spinneret to a high-voltage power source. The collecting device is comprised of a carrier, such as a metal plate, metal roller, metal disk, metal drum, or a ground metal conducting wire.
The key component of any needle electrospinning assembly is the spinneret, which serves to prevent the monomer solution from becoming too viscous and solidifying under the spinning conditions, thus preventing blockage of the needle and potential damage to the equipment. However, the limited throughput of the needle spinneret means that the efficiency of polymer production using this technique is generally low [39]. Needleless electrospinning techniques represent a better way for high efficiency of polymer nanofiber production that can solve many of the limitations associated with traditional needle electrospinning techniques [40][41][42]. There are many methods for generating the surface disturbance of spinning solutions that is required for needless electrospinning, including ultrasonic disturbance, agitation, and acoustic bubble disturbance [43]. The stability of these surface disturbance processes is related to the quality (diameter distribution and L/D ratio distribution) of the needleless electrospinning membrane materials, resonance factors, liquid levels, solution concentrations, and solution viscosities. Needleless electrospinning equipment employs various types of electric field distribution, which can sometimes lead to membranes being produced with uneven thickness. Therefore, it is important that new collecting devices are designed to solve these important performance problems.

Introduction of composite nanofibers electrospinning technology
To date, electrospinning is the common method available to prepare nanofibers directly, quickly, and continuously under mild conditions in a low-cost, fast, and efficient manner [44,45]. Depending on their applications, electrospinning nanofibers can be divided into two categories: single-component nanofibers and composite nanofibers [46]. Early reports described nanofibers that were prepared by electrospinning homogeneous polymers, with changes of starting materials, solution conditions, and electrospinning parameters used to prepare singlecomponent nanofibers with different morphologies and properties. However, many of these single-component nanofibers such as collagen [47][48][49], gelatin [50,51], elastin [52], and fibrinogen [53,54], have some limitations including weak mechanical properties, poor processability, poor moisture resistance, rapid degradation rate, and potential immunogenic properties [55,56]. Thus, composite or hybrid nanofibers with different compositions (e.g., organic/ organic, organic/inorganic) have been proposed as promising materials that exhibit physicochemical properties arising from both the host and guest materials [57]. For example, Chen et al. used electrospinning to prepare collagen/chitosan nanofiber membranes, which could promote the growth of dermal and epidermal layers [58]. Gu et al. used electrospinning to prepare porous biocompatible nanofibers mats from poly(l-lactide)/gelatin, which exhibited controlled evaporative water losses and promote fluid drainage, which made them potentially useful materials for wound dressing applications [59]. For the preparation of composite nanofibers, electrospinning technics can be divided into three fundamental types: (1) blend electrospinning; (2) mixing electrospinning; and (3) coaxial electrospinning. Blend electrospinning is the most commonly used method, which involves a process whereby a spinning solution is generated by mixing different polymers in a defined ratio. Mixing electrospinning refers to an electrospinning process that employs two or more separate liquid feeding devices containing different solutions. The electrostatic field results in each polymer being stretched into nanofibers which then overlap with each other to form composite nanofiber membranes. Coaxial electrospinning involves the use of a spinneret consisting of two or more capillary tubes with different inner diameters which results in a defined gap between the two capillary tubes. The same (or different) electrostatic field is applied to the inner and outer layers of electrospun solutions which results in solutions of the core and surface polymers being expelled from each coaxial nozzle to generate a concentric stratified flow. Because each of the electrospinning solutions has a short confluence time and low diffusion coefficient, they are stretched into coaxial composite nanofibers by the presence of the electric field force. In a comparative study, Chen et al. prepared a range of composite nanofiber membranes using blend electrospinning, mixing electrospinning, and coaxial electrospinning. They found that the composite nanofibers membranes prepared by coaxial electrospinning had high regularity, the membranes produced by blend electrospinning had good moisture resistance, while nanofiber membranes fabricated by using mixing electrospinning exhibited the highest mechanical strength [60].

Technical factors of collagenous electrospinning
There are various factors that can influence the morphology and structural properties of nanofibers produced in the electrospinning process. These include: (1) the properties of the electrospinning solution, such as concentration, viscosity, electrical conductivity, surface tension, and distribution of polymer molecular weight; (2) process parameters, such as electric voltage, spinning temperature, spinning speed, collection speed, and spinning distance; and (3) environmental parameters, such as temperature, moisture, air velocity, and atmospheric composition.
Kazanci investigated the role of temperature, solvent, and pH on the properties of collagentype I nanofibers that were prepared under electrospinning conditions. They found that decreasing the temperature by 10°C, the PP-II (folded) fraction ratio of the resultant fibers increased from 37 to 52.5%. Moreover, nanofibers obtained from acidic solutions contained 59% of PP-II, suggesting that the collagen structure was well preserved [61]. Dulnik et al. prepared electrospun polycaprolactone/gelatin and polycaprolactone/collagen nanofibers using various solvents (hexafluoroisopropanol and a mixture of acetic acid and formic acid). The result showed that electrospun PCL/gelatin and PCL/collagen nanofibers obtained by various solvents had similar morphologies, although there were some differences in their internal structures that affected their susceptibility toward biodegradation [62].
Lu et al. used water as a solvent to electrospin pure gelatin solution, finding that low concentrations of gelatin had low viscosity which were insufficient to produce continuous nanofibers, resulted in the formation of unwanted microbeads. When the concentration of gelatin used was >25%, the spinning solution became too viscous, which inhibited efficient electrospinning, resulted in a few nanofibers [63].
Wang et al. investigated the influence of solution concentration, salt concentration, solvent type, ambient temperature, and environmental humidity on the electrospinning of gelatin solutions. Their results showed that using polymers with high dielectric constants and solvents with low volatility solvents resulted in formation of nanofibers with small diameters. The diameters of the nanofibers were found to increase as the temperature rose. Low temperatures were not conducive to effective volatilization of solvent, leading to prolonged solidification time and the production of superfine nanofibers. The morphology of nanofibers was also found to be affected by ambient humidity. When relative humidity levels were increased to 45%, the resultant nanofiber membranes were shown to contain small areas of reticular formation and 'beads-on-a-string' structures. As the relative humidity was increased to 60%, the nanofiber membranes produced were contained uneven nanometer-diameter distributions with associated micro-bead structures. This study also revealed that the average diameter of nanofibers decreased with an increase in ambient humidity [64] (Figure 1).
An et al. studied the influence of formic acid concentration, gelatin concentration, and electric voltage on the electrospinning process used to prepare gelatin/polylactide composite nanofibers. They found that the use of low formic acid concentrations (50%) resulted in nanofibers, along with the formation of significant amounts of microbeads. Increasing formic acid concentration led to the formation of randomly arrayed ultrafine nanofibers, without any microbeads. For example, increasing the concentration of formic acid from 70 to 98%, resulted in an increase in the average diameter of gelatin nanofibers from 208 to 312 nm. Moreover, it was found that the concentration of gelatin affected the surface tension and viscosity of the spinning solution, with high surface tension favoring formation of microbeads, and high viscosity minimizing their formation. For example, when gelatin concentration was too low, discrete microbeads or beaded nanofibers were formed, while an increase in concentration of gelatin to >10% resulted in exclusive formation of smooth nanofibers. It was also found that the average diameter of nanofibers increased from 260 to 335 nm as the gelatin concentration rose from 10 to 23% [65]. Huang et al. had also reported that elastin-mimetic peptide polymers could be electrospun into nanofibers, who investigated the effect of polypeptide content and solution flow rate on the morphology and mechanical properties of the resultant nanofibers [66].
It is now clear that various factors in the electrospinning process play a crucial role in determining the 'spinnability' of a polymer solution and the physical and chemical properties of the resultant nanofibers. However, adjustment of these parameters can result in improvements to the electrospinning process, thus enabling the properties of functional nanofibers to be tuned to meet the needs for biomedical applications.

Applications of electrospun collagen
Electrospinning technology can be used to convert collagen materials into nanofibers materials that exhibit porous micro-nanostructures with good mechanical properties and excellent biocompatibility profiles. Potential uses of these nanofibers for biomedical applications include tissue engineering (e.g., artificial skin, artificial vasculature, cartilage repair, etc.), drug delivery, hemostatic dressings, periodontal restoration, biofilm, and wound dressings. Some applications of polymers derived from composite collagen and gelatin nanofibers, along with information on their preparation (type and solvent) and fiber diameter are provided in Table 1.

Tissue engineering applications
The three-dimensional microstructure of materials prepared from electrospinning collagenderived materials can be used to effectively stimulate tissue regeneration. The network structure of these materials serves to promote the integration and recruitment of new tissue into their fibers scaffolds, thus accelerating the growth of new tissue. The main function of the nanofiber scaffold is to provide a suitable three-dimensional environment that cells can adhere to and proliferate. Yu et al. used of electrospinning to prepare composite nanofiber scaffolds made of alginate, chitosan, hydroxyapatite, and collagen, whose porous structure was beneficial for cell infiltration and growth. What is more, the release of collagen from the scaffolds was, respectively, 17 and 2% of that from the collagen film after immersing in SBF and collagenase solution for 10 days, which indicated that the disintegration of scaffold for bone tissue engineering can be reduced comparing to conventional collagen scaffold. Therefore such a composite mat would be applicable as a scaffold for regenerating bone tissue [71] (Figure 2).
Choi et al. used electrospinning techniques to fabricate aligned polycaprolactone/collagen nanofiber meshes that were used to guide morphogenesis of skeletal muscle cells and enhance their cellular organization. Comparison with randomly oriented nanofibers, the results revealed that unidirectionally oriented nanofibers were better at inducing muscle cell alignment and myotubule formation [72] (Figure 3).

Lee et al. utilized electrospinning techniques to prepare PCL/collagen nerve conduits for complex
peripheral motor nerve regeneration studies using end-to-side neurorrhaphy techniques. Results revealed that axonal continuity was normally recovered 8 weeks after surgery, with the muscle function recovery occurring after 1-20 weeks, and recovery of donor nerve function occurring after 20 weeks. Therefore, the use of electrospun PCL/collagen nerve conduits appeared to have great potential as materials for complex peripheral motor nerve repair [73] (Figure 4).
Composite nanofiber scaffolds have been prepared by electrospinning polycaprolactone and gelatin, which were then modified using cell-derived factor-1α. Experiments revealed that these scaffolds not only had good biocompatibility profiles, but also accelerated the healing of skull injuries in mice [98]. Ren et al. used gelatin and silicone to prepare composite nanofiber scaffolds as bone repairing material incorporating Ca 2+ ions. Their results indicated that these composite materials could promote accumulation of bone apatite and the differentiation and  proliferation of osteoblasts [99] (Figure 5). Li et al. used electrospinning to prepare composite nanofiber scaffolds from polyaniline and gelatin, whose properties were dependent on the amount of polyaniline present. For example, when the amount of polyaniline was increased from 0 to 5% (w/w), the average diameter of the fibers decreased from 803 ± 121 to 61 ± 13 nm, and its elastic coefficient increased from 499 ± 207 to 1384 ± 105 MPa. These composite nanofiber scaffolds not only had excellent mechanical strength, but also had excellent biocompatibility in cell culturing experiments [100].
Blit et al. utilized the electrospinning to fabricate fibrous scaffolds which were subsequently surface modified with polypeptide and used them as membrane supports to culture smooth muscle cells (SMCs). Results showed that SMCs seeded onto these elastin-like polypeptide-4 membranes exhibited a spindle-like morphology, with good actin filament organization and smooth muscle myosin heavy chain expression. Therefore, these electrospun nanocomposite membranes were promising candidates for the fabrication of contractile tissue engineered SMC-rich vascular medial layers [101] (Figure 6).

Drug delivery applications
Materials for drug delivery are often employed to release drug molecules into body tissues over extended periods of time. However, traditional drug delivery materials often do not  control the rate of release of drugs effectively, while their poor drug absorption properties mean that they can only be used to deliver relatively low loadings of drug. Nanofibers are well suited as drug delivery vehicles, because their fine nanostructures are capable of absorbing significant amounts of drug molecules in a uniform manner. Gradual degradation of these nanofibers in the body then allows for gradual release of the drug in a controlled manner.
Polylactide-polyglycolide (PLGA) and collagen have been electrospun into sandwich structured drug-loaded membranes, with PLGA/collagen used for the surface layers, and PLGA/ drugs contained in their core layer. The ability of these sandwich structured membranes to release vancomycin, gentamicin, and lidocaine in vitro was investigated. These results showed that these membranes could deliver therapeutic concentrations of vancomycin and gentamicin to human fibroblasts, ranging from 37 to 100% and 30 to 100% over 3 and 4 week periods, respectively [74] (Figure 7).
Meng et al. used electrospinning to uniformly distribute the drug Fenbufen throughout the structure of a nanofiber membrane prepared from gelatin and polylactide. The effect of gelatin concentration, fiber orientation, cross-linking time, and buffer pH on the sustained release of Fenbufen was successfully determined [102] (Figure 8). Huang et al. used proanthocyanidins as a cross-linking agent to prepare nanofibers as drug delivery vectors to deliver vitamin C magnesium phosphate. This study showed that the presence of the proanthocyanidin resulted in an increase in drug loading, which was important in maintaining a consistent rate of drug release. In addition, gelatin nanofibers that were cross-linked by proanthocyanidins were also seen to promote the proliferation of L929 cells. Therefore, this type of drug-loaded nanofibers material could potentially be used for controlled drug delivery, as well as for the promotion of wound healing [103] (Figure 9).  Khadka et al. prepared electrospun composite nanofibers derived from a recombinant elastin-like peptide (ELP), or from a mixture of ELP with synthetic polypeptide and co-poly(lglutamic acid 4 , l-tyrosine 1 ) (PLEY). These materials contained numerous pores on their nanofibers surfaces, which ranged in diameter from <1 to 0.5 μm. Consequently, drugs were doped into the pores of these nanofibers materials and their use as potential drug delivery systems explored [104] (Figure 10).

Wound dressing applications
Collagen, gelatin, or polypeptide nanofiber membranes exhibit high porosities, small pore diameters, large surface areas, and fine microstructures that affords them good biocompatibility, biodegradability, biological adhesiveness, and moisture absorption properties. Therefore, these materials can be used to prepare dressings that keep wounds moist and which help prevent bacterial infection. Composite nanofiber materials prepared from gelatin and other materials such as fungicides, inflammatory drugs, and growth factors, can improve the performance of wound dressings. This is because they effectively improve the speed of wound hemostasis and healing by reducing its exposure to the external environment and protecting it from exposure to air.
Zine et al. prepared composite nanofibers by electrospinning a mixture of poly3-hydroxybutyric acid-co-3-hydroxyvaleric acid (PHBV), graphene oxide (GO), and collagen. GO was used to prepare these nanofibers to increase their mechanical strength and convey antibacterial activity against pathogenic bacteria such as Escherichia coli and Staphylococcu aureus. Collagen was included with the aim of enhancing cell proliferation, without affecting the composite materials mechanical strength or porosity. Subsequent biological results revealed that these nanofiber membranes generally had good wound healing properties [75] (Figure 11).   (Figure 12).
Dubsky et al. used gelatin and polycaprolactone to prepare composite electrospun nanofibers, with subsequent cell culture experiments showing that these nanofibers can promoted cell adhesion and proliferation effectively. These composite nanofibers and medical gauze were applied to wounds of injured mice, with control experiments showing that inclusion of the nanofibers resulted in faster healing rates [106].

Restorative materials for periodontal applications
The application of nanofiber membrane as periodontal restorative materials is an interdisciplinary field that spans the areas of tissue engineering and wound dressings. Periodontal repair requires membrane materials that prevent epithelial cells and connective tissue from growing into defect areas that can result in the creation of space for the co-migration and proliferation of periodontal ligament cells. Materials used for this dental application must be mechanically robust, exhibit good biodegradability profiles, and present robust three-dimensional nanostructures that are biocompatible with cell tissues. Therefore, collagen, gelatin, or polypeptide-derived nanofibers materials are potentially good choices for periodontal restorative applications.  mechanical properties and metronidazole benzoate (MET) included to prevent bacterial infection. n-HAp was incorporated into the PLA:GEL membrane to mimic the collagen-HAp matrix that was present in bone and enhance the composites osteoconductive behavior. This formulation afforded an electrospun FGM with excellent mechanical integrity, biodegradability, and good cell-membrane interactions that was explored as a periodontal restorative material [107] (Figure 13).
Mixtures of polycaprolactone and gelatin and ZnO in hexafluoropropanol had also been electrospun into nanofibers to afford ZnO-loaded electrospun membranes that possessed good biocompatibility, stretching ability, antibacterial activity, as potentially useful materials for periodontal regeneration [108] (Figure 14).

Outlook
Collagen and gelatin (and their derived polypeptides) are natural biopolymers that exhibit good biocompatibility, biodegradability, and low immunogenicity, as well as being excellent materials as hosts for cell and tissue growth. With the development of collagen in medical application, many processing methods are required to ensure that their application can be fully realized. The rapid evolution of electrospinning techniques is well suited to meet this need, enabling nanofiber membranes with three-dimensional pore structures, which imitated the microstructure of the extracellular matrix. Consequently, these electrospinning techniques have attracted increasing attention for applications in the fields of tissue engineering, drug delivery, wound dressing, nerve regeneration, periodontal regeneration, and vascular reconstruction. The organic solvent used in electrospinning processes has potential toxicity issues, so the development of approaches that allow electrospinning to be carried out under aqueous conditions is highly desirable. Collagen, gelatin, or polypeptide nanofibers have been used to prepare biopolymers for applications in many different biomedical fields, including wound repair, artificial skin, and drug delivery. In order to satisfy a large number of medical applications, it will be necessary to develop more efficient electrospinning technics that produce large amounts of material with optimal microscopic structures. Current problems need to be resolved, include low efficiency, low mechanical strength, and poor spinning reproducibility. In addition, a lot of effort should be done in spinneret design, collecting device optimization, and solution delivery techniques. If these issues can be overcome, it is anticipated that collagen-derived nanofiber materials will have a major role for biomedical and biotechnological applications.