**3. Nature-derived nanofibers degradable in the human tissues**

Nature-derived polymers degradable in human tissues include, in particular, polymers that are synthesized in the human body and usually act as components of ECM. These polymers are proteins (collagen and its derivative gelatin, elastin, fibrinogen and fibrin, keratin) or polysaccharides in non-sulfated form (hyaluronic acid) and sulfated form (heparin-like glycosaminoglycans). In addition, some natural polymers synthesized by other organisms, such as bacteria, fungi, insects, crustaceans or plants, are degradable in human tissues, because they are susceptible to enzymes present in human tissues, such as lysozyme and esterases. These polymers include chitosan, gellan gum, zein, and PHBV.

**Collagen** is the main structural protein in the extracellular space in a wide range of tissues in the body. Skin contains type I collagen, one of the most abundant collagens in the human body. Type I collagen is also abundant in tendons, ligaments, and vasculature, and it is a main component of the organic part of bone. Type I collagen is a fibrillar type of collagen; it is composed of amino acid chains forming triplehelices of elongated fibrils. That is why the nanofibrous collagen scaffolds closely mimic the architecture of the native ECM and are advantageous for tissue engineering. In addition, collagen has been reported to be relatively poorly immunogenic, even if used in allogeneic and xenogeneic forms, for example, recombinant human collagen or bovine and porcine collagen. However, mammalian collagen is associated with the risk of disease transmission, for example, bovine spongiform encephalopathy (for a review, see [89–91]). This risk can be reduced by the use of fish collagen, which became to be popular in tissue engineering, including skin tissue engineering and wound healing. In addition, the fish collagen enables an easier recovery of intact collagen triple helices than the mammalian collagen [92]. Fish collagen can be obtained from the skin, scales and bones of freshwater fish, such as tilapia [91–94], and marine fish, such as hoki fish (*Macruronus novaezelandiae*) [92, 95], or *Arothron stellatus,* also known as "stellate puffer," "starry puffer" or "starry toadfish" [96]. Nanofiber electrospun from tilapia skin collagen promoted the proliferation of human HaCaT keratinocytes, and stimulated epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in these cells. Moreover, the tilapia collagen nanofibers accelerated wound healing *in vivo* in rat models [91–94]. Beneficial effects on wound healing were also observed in nanofibrous meshes electrospun from collagen obtained from *Arothron stellatus* [96] and from fish scale collagen peptides [90].

Collagen is one of the most widely used natural proteins for creation of nanofibrous scaffolds for skin tissue engineering and wound healing. However, these scaffolds are usually mechanically weak, and therefore they need crosslinking or blending with synthetic polymers. Collagen crosslinking with conventionally used agents, particularly glutaraldehyde, is associated with the risk of the scaffold cytotoxicity. More benign crosslinkers used recently include, for example, citric acid [95] or quaternary ammonium organosilane, a multifunctional crosslinking agent, which improved the electrospinnability of collagen by reducing its surface tension, endowed the collagen nanofibers with potent antimicrobial activity and promoted the adhesion and metabolic activity of primary human dermal fibroblasts without any cytotoxicity, at least in a lower concentration of 0.1% w/w [97].

Synthetic polymers used for combination with collagen in nanofibrous scaffolds included PLA [98], PLGA [99, 100], and particularly PCL, which was either blended with collagen [101–104] or served as substrate for subsequent deposition of collagen [105]. Collagen has also been combined with natural polymers, such as silk fibroin [73] or chitosan in a form of blends [106] or in a form of bilayered scaffolds, where collagen was electrospun onto the chitosan scaffolds [107]. Collagen was also

*Current and Future Aspects of Nanomedicine*

scaffolds [66].

adhesion receptors [64].

The degradation behavior of fibroin scaffolds depends on the preparation method and structural characteristics, such as processing condition, pore size, and silk fibroin concentration (for a review, see [65]). For example, threedimensional porous scaffolds prepared from silk fibroin using all-aqueous process degraded within 2–6 months after implantation into muscle pouches of rats, while the scaffolds prepared using an organic solvent, hexafluoroisopropanol (HFIP), persisted beyond 1 year. It was probably due to a lower original silk fibroin concentration, larger pore size, and a higher and more homogeneous cellular infiltration of aqueous-derived scaffolds than in HFIP-derived

For skin tissue engineering and wound healing, silk fibroin has been combined

with various synthetic and natural polymers and other bioactive substances. The polymers included, for example, PCL, [67], poly(L-lactic acid)-*co*-poly(εcaprolactone) (PLACL, [68]), carboxyethyl chitosan, PVA, [69], chitin [70], cellulose-based materials modified by oxidation [71] or with lysozyme [72], collagen [73], gelatin [74], and hyaluronan [75]. The bioactive substances were, for example, growth factors, such as epidermal growth factor [64], vitamins, such as vitamin C [68], vitamin E [76], and pantothenic acid (vitamin B5; [77]), antioxidants, such as grape seed extract ([78]) or quinone-based chromenopyrazole [79], antibiotics, such as ciprofloxacin [64], tetracycline [68] or gentamycin [62], and other antimicrobial and wound healing agents, such as silver nanoparticles, dandelion leaf extract [63], *Aloe vera* [80], or astragaloside IV [74]. In order to enlarge the pore size in nanofibrous scaffolds for cell penetration, silk fibroin was electrospun together with so-called "sacrificial" crystals of ice [67] or NaCl [81, 82], that is, crystals which are removed after the electrospinning process. An interesting combination is silk fibroin with decellularized human amniotic membrane, which was used for developing a three-dimensional bi-layered scaffold for burn treatment. Adipose tissue-derived mesenchymal stem cells seeded on this scaffold increased expression of two main pro-angiogenesis factors, vascular endothelial growth factor, and basic fibroblast growth factor [83]. Also the transplantation of bone marrow-derived mesenchymal stem cells and epidermal stem cells into wounds using nanofibrous silk fibroin scaffolds supported re-epithelization, collagen synthesis, as well as the skin appendages regeneration [84]. Another interesting approach is to use silk fibroin produced by other species than *Bombyx mori*, namely by the moths *Antheraea assama* and *Philosamia ricini.* This "non-mulberry" silk fibroin possesses inherent Arg-Gly-Asp (RGD) motifs in its protein sequence, which facilitates binding of cells through their integrin

**Sericin** has also been applied in skin tissue engineering and wound healing, although in a lesser extent than silk fibroin. Sericin shows antioxidant, UV-protective, heat-protective, moisture-retaining, and antimicrobial properties,

(e.g., from *Antheraea mylitta*) than in sericin produced by *Bombyx mori*. The reason is that wild moths like *Antheraea mylitta* are exposed to a hostile environment in nature than *Bombyx mori* raised in captive conditions. Similarly as non-mulberry silk fibroin, also sericin has been reported to be more supportive for cell adhesion than mulberry sericin (for a review, see [85]). Sericin enhanced the proliferation and epidermal differentiation of human mesenchymal stem cells on gelatin/hyaluronan/chondroitin sulfate nanofibrous scaffolds [86]. Similarly, sericin improved the growth of murine L929 fibroblasts and human HaCaT keratinocytes cultured on the PVA nanofibrous scaffolds [87] and also the growth of L929 fibroblasts on chitosan nanofibrous scaffolds, together with antibacterial properties of these

which have been reported to be more pronounced in non-mulberry sericin

**76**

scaffolds [88].

grafted on the surface of composite electrospun PVA/gelatin/alginate nanofibers [41]. Collagen-based or collagen-containing nanofibers have been loaded with a wide range of bioactive substances, such as vitamin C, vitamin D3, hydrocortisone, insulin, triiodothyronine, and epidermal growth factor [100], transforming growth factor-β1 [102], plant extracts such as *Coccinia grandis* leaf extract [96], or lithospermi radix extract [107], antibiotics such as gentamicin [103], or bioactive glass [93, 104]. Collagen and PCL and bioactive glass nanoparticles were applied for delivery of endothelial progenitor cells into wounds in order to promote their vascularization and healing [104].

**Gelatin** is a derivative of collagen, obtained by denaturing its triple helical structure. Specifically, it is a mixture of peptides and proteins produced by partial hydrolysis of collagen extracted from the skin, bone, and connective tissue of animals, such as cattle, pigs, chicken, and also fish. Gelatin can be defined as a complex mixtures of oligomers of the α subunits joined by covalent bonds, and intact and partially hydrolyzed α-chains of varying molecular weight (for a review, see [89, 92]). Properties of gelatin, including its spinnability, depend on the source of collagen, animal species, age, type of collagen, type of conversion of collagen to gelatin (i.e., acidic *vs.* basic hydrolysis), and particularly on the conformation of gelatin molecules [108].

Similarly as collagen, also gelatin is the most promising for skin tissue engineering and wound healing applications in combination with various synthetic and natural polymers. For example, gelatin was combined with polyurethane [109], PLA [11, 17], and particularly with PCL, where it was incorporated into core-shell PCL/gelatin nanofibers as the core polymer [110] or electrospun independently of PCL using a double-nozzle technique, which resulted in creation of two types of nanofibers in the scaffolds, either mixed [111] or arranged in separate gelatin and PCL layers [112]. Gelatin was also combined with a copolymer of lactic acid and caprolactone P(LLA-CL) in the form of blends [113] or in the form of coaxial nanofibers with P(LLA-CL)/gelatin shell and albumin core containing epidermal growth factor, insulin, hydrocortisone, and retinoic acid [114]. Natural proteins combined with gelatin included dextran [31], pullulan [38], alginate [41], silk fibroin [74], and hyaluronan with chondroitin sulfate [86].

For combination with synthetic and natural polymers, for example, with PCL [115], and chitosan and keratin [116], gelatin was also used in the form of photocrosslinkable gelatin methacrylate hydrogel (GelMA). On PCL nanofibers, GelMA showed a higher decoration level in comparison with native gelatin [116]. Selfstanding nanofibrous matrices electrospun from GelMA enabled tuning of their water retention capacity, stiffness, strength, elasticity, and degradation by changing the exposure time to UV light [117].

**Elastin** is a protein found in the ECM, that maintains its elasticity [118]. It is the second main protein-based component of native skin ECM. The presence of elastin in composite electrospun nanofibrous scaffolds, containing gelatin, cellulose acetate and elastin, changed the fiber morphology from straight to ribbon-like structure, and decreased the swelling ratio and degradation rate of the scaffolds. In addition, elastin-containing scaffolds supported the attachment and proliferation of human fibroblasts [119].

**Fibrin** is a provisional ECM protein, which accumulates in wounds after injury to initiate hemostasis and healing. Fibrin is formed via the polymerization of fibrinogen monomers in the presence of thrombin, and this process can be simulated *in vitro* [120]. Fibrin forms a fine nanofibrous mesh, which is mechanically weak and needs to be deposited on some supportive structure, for example, synthetic nanofibrous meshes made of poly(L-lactide) (PLLA) [121]. In our experiments, fibrin was deposited on PLLA in the form of two types of coating, depending on the mode of

**79**

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Nature-Derived…*

fibrin preparation. Fibrin either covered the individual fibers in the membrane (F1 nanocoating), or covered the individual fibers and also formed a fine homogeneous nanofibrous mesh on the surface of the membrane (F2 nanocoating), depending on the mode of fibrin preparation. The fibroblasts on the F1 nanocoating remained in their typical spindle-like shape, while the cells on the F2 nanocoating were polygonal with a higher proliferation rate [122]. F2 nanocoating was then used for development of a bilayer skin construct. First, a nanofibrous PLLA mesh was coated with fibrin and seeded with human dermal fibroblasts. After reaching confluence, the fibroblasts were covered with a collagen hydrogel and were allowed to migrate into this hydrogel and to proliferate inside. After sufficient colonization of the hydrogel with fibroblasts and formation of a structure resembling the skin dermis, human epidermal keratinocytes were seeded on the top of the collagen hydrogel (**Figure 2**) [123]. Also fibrinogen was used for modification of synthetic polymeric nanofibers in order to enhance the cell adhesion and growth. Nanofibrous scaffolds electrospun from blends of PCL and fibrinogen improved the adhesion, proliferation, and epidermal differentiation of adipose tissue-derived stem cells (ADSCs) in comparison with pure PCL scaffolds. Composite PCL/fibrinogen scaffolds seeded with ADSCs also markedly improved healing of full-thickness excisional wounds created in rats in comparison with acellular dermal matrix or acellular dermal matrix with ADSCs [124]. **Keratin** is a fibrous structural protein, present in skin appendages, such as hair,

*Developing a bilayer construct of keratinocytes and fibroblasts on a PLLA nanofibrous membrane with fibrin* 

wool, feather, nails, horns, claws, hooves, and in the outer (cornified) layer of epidermis [125, 126]. Keratin protects epithelial cells from damage and stress and is

In most studies dealing with keratin-containing nanofibers, keratin was combined with other natural or synthetic polymers in order to improve the spinnability of keratin, or to improve the bioactivity of the co-electrospun polymer. For example, in a study by Cruz-Maya *et al*. [127], blending keratin with PCL improved the stability of the electrospinning process, promoted the formation of nanofibers without defects, such as beads and ribbons, typically observed in the fabrication of keratin nanofibers. At the same time, keratin markedly increases the fiber hydrophilicity compared with pure PCL, which improved the adhesion and proliferation of human mesenchymal stem cells [127]. Similarly, co-electrospinning of keratose (i.e., oxidative keratin) with PVA resulted in nanofibers with uniform fibrous structure, suitable hydrophilicity and mechanical properties [125]. Properties of electrospun keratin nanofibers were also improved by incorporation of hydrotalcites, intended for delivery of diclofenac. These nanofibers displayed a reduced swelling

insoluble in water and organic solvents.

*DOI: http://dx.doi.org/10.5772/intechopen.88602*

*and collagen hydrogel. Left: schematic design; right: real construct.*

**Figure 2.**

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Nature-Derived… DOI: http://dx.doi.org/10.5772/intechopen.88602*

**Figure 2.**

*Current and Future Aspects of Nanomedicine*

vascularization and healing [104].

gelatin molecules [108].

hyaluronan with chondroitin sulfate [86].

the exposure time to UV light [117].

grafted on the surface of composite electrospun PVA/gelatin/alginate nanofibers [41]. Collagen-based or collagen-containing nanofibers have been loaded with a wide range of bioactive substances, such as vitamin C, vitamin D3, hydrocortisone, insulin, triiodothyronine, and epidermal growth factor [100], transforming growth factor-β1 [102], plant extracts such as *Coccinia grandis* leaf extract [96], or lithospermi radix extract [107], antibiotics such as gentamicin [103], or bioactive glass [93, 104]. Collagen and PCL and bioactive glass nanoparticles were applied for delivery of endothelial progenitor cells into wounds in order to promote their

**Gelatin** is a derivative of collagen, obtained by denaturing its triple helical structure. Specifically, it is a mixture of peptides and proteins produced by partial hydrolysis of collagen extracted from the skin, bone, and connective tissue of animals, such as cattle, pigs, chicken, and also fish. Gelatin can be defined as a complex mixtures of oligomers of the α subunits joined by covalent bonds, and intact and partially hydrolyzed α-chains of varying molecular weight (for a review, see [89, 92]). Properties of gelatin, including its spinnability, depend on the source of collagen, animal species, age, type of collagen, type of conversion of collagen to gelatin (i.e., acidic *vs.* basic hydrolysis), and particularly on the conformation of

Similarly as collagen, also gelatin is the most promising for skin tissue engineering and wound healing applications in combination with various synthetic and natural polymers. For example, gelatin was combined with polyurethane [109], PLA [11, 17], and particularly with PCL, where it was incorporated into core-shell PCL/gelatin nanofibers as the core polymer [110] or electrospun independently of PCL using a double-nozzle technique, which resulted in creation of two types of nanofibers in the scaffolds, either mixed [111] or arranged in separate gelatin and PCL layers [112]. Gelatin was also combined with a copolymer of lactic acid and caprolactone P(LLA-CL) in the form of blends [113] or in the form of coaxial nanofibers with P(LLA-CL)/gelatin shell and albumin core containing epidermal growth factor, insulin, hydrocortisone, and retinoic acid [114]. Natural proteins combined with gelatin included dextran [31], pullulan [38], alginate [41], silk fibroin [74], and

For combination with synthetic and natural polymers, for example, with PCL [115], and chitosan and keratin [116], gelatin was also used in the form of photocrosslinkable gelatin methacrylate hydrogel (GelMA). On PCL nanofibers, GelMA showed a higher decoration level in comparison with native gelatin [116]. Selfstanding nanofibrous matrices electrospun from GelMA enabled tuning of their water retention capacity, stiffness, strength, elasticity, and degradation by changing

**Elastin** is a protein found in the ECM, that maintains its elasticity [118]. It is the second main protein-based component of native skin ECM. The presence of elastin in composite electrospun nanofibrous scaffolds, containing gelatin, cellulose acetate and elastin, changed the fiber morphology from straight to ribbon-like structure, and decreased the swelling ratio and degradation rate of the scaffolds. In addition, elastin-containing scaffolds supported the attachment and proliferation of human

**Fibrin** is a provisional ECM protein, which accumulates in wounds after injury to initiate hemostasis and healing. Fibrin is formed via the polymerization of fibrinogen monomers in the presence of thrombin, and this process can be simulated *in vitro* [120]. Fibrin forms a fine nanofibrous mesh, which is mechanically weak and needs to be deposited on some supportive structure, for example, synthetic nanofibrous meshes made of poly(L-lactide) (PLLA) [121]. In our experiments, fibrin was deposited on PLLA in the form of two types of coating, depending on the mode of

**78**

fibroblasts [119].

*Developing a bilayer construct of keratinocytes and fibroblasts on a PLLA nanofibrous membrane with fibrin and collagen hydrogel. Left: schematic design; right: real construct.*

fibrin preparation. Fibrin either covered the individual fibers in the membrane (F1 nanocoating), or covered the individual fibers and also formed a fine homogeneous nanofibrous mesh on the surface of the membrane (F2 nanocoating), depending on the mode of fibrin preparation. The fibroblasts on the F1 nanocoating remained in their typical spindle-like shape, while the cells on the F2 nanocoating were polygonal with a higher proliferation rate [122]. F2 nanocoating was then used for development of a bilayer skin construct. First, a nanofibrous PLLA mesh was coated with fibrin and seeded with human dermal fibroblasts. After reaching confluence, the fibroblasts were covered with a collagen hydrogel and were allowed to migrate into this hydrogel and to proliferate inside. After sufficient colonization of the hydrogel with fibroblasts and formation of a structure resembling the skin dermis, human epidermal keratinocytes were seeded on the top of the collagen hydrogel (**Figure 2**) [123].

Also fibrinogen was used for modification of synthetic polymeric nanofibers in order to enhance the cell adhesion and growth. Nanofibrous scaffolds electrospun from blends of PCL and fibrinogen improved the adhesion, proliferation, and epidermal differentiation of adipose tissue-derived stem cells (ADSCs) in comparison with pure PCL scaffolds. Composite PCL/fibrinogen scaffolds seeded with ADSCs also markedly improved healing of full-thickness excisional wounds created in rats in comparison with acellular dermal matrix or acellular dermal matrix with ADSCs [124].

**Keratin** is a fibrous structural protein, present in skin appendages, such as hair, wool, feather, nails, horns, claws, hooves, and in the outer (cornified) layer of epidermis [125, 126]. Keratin protects epithelial cells from damage and stress and is insoluble in water and organic solvents.

In most studies dealing with keratin-containing nanofibers, keratin was combined with other natural or synthetic polymers in order to improve the spinnability of keratin, or to improve the bioactivity of the co-electrospun polymer. For example, in a study by Cruz-Maya *et al*. [127], blending keratin with PCL improved the stability of the electrospinning process, promoted the formation of nanofibers without defects, such as beads and ribbons, typically observed in the fabrication of keratin nanofibers. At the same time, keratin markedly increases the fiber hydrophilicity compared with pure PCL, which improved the adhesion and proliferation of human mesenchymal stem cells [127]. Similarly, co-electrospinning of keratose (i.e., oxidative keratin) with PVA resulted in nanofibers with uniform fibrous structure, suitable hydrophilicity and mechanical properties [125]. Properties of electrospun keratin nanofibers were also improved by incorporation of hydrotalcites, intended for delivery of diclofenac. These nanofibers displayed a reduced swelling

ratio and a slower degradation profile compared to keratin-based non-woven nanofibrous mats containing free diclofenac [126]. Keratin was also a component of core-shell nanofibers, prepared by coaxial electrospinning of chitosan, PCL and keratin with *Aloe vera* extracts encapsulated inside the polymer nanofibers. This construct increased the adhesion and growth of L929 fibroblasts and was intended for wound healing applications [128]. Importantly, keratin was a component of bilayer scaffolds for skin tissue engineering, composed of human hair keratin/ chitosan nanofiber mat and gelatin methacrylate (GelMA) hydrogel. Human dermal fibroblasts were encapsulated and grown in the hydrogel matrix, while human HaCaT keratinocytes formed a layer on the top of the scaffolds, mimicking dermis and epidermis of skin tissue [116]. Another bilayer scaffolds was constructed using polyurethane wound dressing as an outer layer, and electrospun gelatin/keratin nanofibrous mat as an inner layer [109].

**Hyaluronic acid**, also called hyaluronan, is an anionic, non-sulfated linear glycosaminoglycan. It is distributed widely throughout connective, neural, and epithelial tissues, including skin, where it is a major component of ECM. Therefore, hyaluronic acid has been widely used for skin tissue engineering and wound healing, and it is approved for clinical application [33].

Hyaluronic acid stimulated infiltration of nanofibrous scaffold composed of hyaluronan, silk fibroin and PCL [75], and can help to promote cell proliferation [129]. Electrospinning of pure hyaluronic acid is not simple because of solubility characteristics of this polymer. Hyaluronic acid is well-soluble in water but lesssoluble in most organic solvents, which can be solved by mixtures of solvents as water/ethanol or water/dimethylformamide [130]. Increasing of evaporation and decreasing of solution surface tension by the solvent mixing helps to electrospinning process. Another possibility is electrospinning of hyaluronic acid together with a suitable water-soluble polymer such as PVA [131] or PEO. The solution of pure hyaluronic acid [132] or with relatively small amount of carrier PEO was successfully spun into nanofibrous material by air-assisted electrospinning technology, that is, electroblowing [133]. For creation of nanofibrous scaffolds, hyaluronic acid was also used in combination with PCL [134], PLA [135] or gelatin, chondroitin sulfate and sericin [86].

**Sulfated glycosaminoglycans** have been relatively rarely used as components of nanofibers for skin tissue engineering and wound healing in comparison with hyaluronic acid. This group of polysaccharides include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate (for a review, see [33]). From these polysaccharides, only heparin, heparan sulfate, and chondroitin sulfate were used as components of nanofibers for skin regenerative therapies. For example, heparin coatings on PLLA nanofibers increased the infiltration of the scaffolds with endothelial cells *in vitro,* and enhanced epidermal skin cell migration across the wound in a full-thickness dermal wound model in rats *in vivo* [136]. In a recent study by Yergoz *et al*. [137], a heparin-like nanofibrous hydrogel promoted regeneration of full thickness burn injury in mice. Chondroitin sulfate was used as a component of electrospun gelatin/PVA/chondroitin sulfate nanofibrous scaffolds, which supported the proliferation of human dermal fibroblasts [138], of electrospun nanofibrous composite scaffolds made of cationic gelatin/hyaluronan/chondroitin sulfate loaded with sericin, which promoted the differentiation of human mesenchymal stem cells toward epithelial lineage [86], and of electrospun gelatin/chondroitin sulfate nanofibrous scaffolds, which accelerated healing of full-thickness skin excision wounds in rats [139].

**Chitosan** is a linear polysaccharide composed of randomly distributed β-(1 → 4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine

**81**

**Figure 3.**

*solution.*

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Nature-Derived…*

(acetylated unit). It can be prepared by alkali treatment from chitin, a poly-Nacetyl-D-glucosamine polysaccharide, the major structural component of the exoskeleton of crustaceans (crab, shrimp), and of the cell wall of fungi and yeast [140]. Chitosan is known as biocompatible, antimicrobial, and biodegradable. In the human organism, it can be degraded by lysozyme, a hydrolytic enzyme present in various secretions such as saliva, tears, mucus, and human milk, and also in cytoplasmic granules of macrophages and polymorphonuclear neutrophils. Chitosan breakdown by lysozymes happens via the removal of glycosidic bonds between polysaccharide units in the polymer. Glucosamine and saccharide are the products of this process, which can be metabolized or stored as proteoglycans in the body. However, electrospinning of chitosan is difficult due to its polycationic characters. Due to the presence of amine groups in the chitosan molecule, acidic aqueous solutions are the best solvents for this polymer. The best candidates for solvent system seem to be mixture of acetic acid (AA) and formic acid (FA) or trifluoroacetic acid (TFA); however, TFA is highly toxic. Electrospinning of pure chitosan has very low productivity because it requires very concentrated polymeric solutions [141]. Therefore, for creation of nanofibrous scaffolds for skin tissue engineering, chitosan has been mixed with other natural or synthetic polymers, such as collagen [142], gelatin [143], keratin [116], cellulose [144], pectin [54, 55], silk fibroin [69], PHBV [145], PCL [142], PLA [146], PLGA [147], PEO, [148], and also with PVA, which was used in our studies (**Figure 3**). Chitosan has also been mixed with various nanoparticles, such as halloysite nanotubes [149], graphene oxide [150] or nanodiamonds [144]. The reason of all these mixtures was to improve the stability, spinnability, wettability, mechanical properties, and biofunctionality of chitosancontaining scaffolds for skin tissue engineering. Combination of chitosan with various polymers also enabled creation of bilayer scaffolds for reconstruction of two main skin layers, that is, epidermis containing keratinocytes and dermis containing fibroblasts [116, 142]. In order to enhance the antimicrobial and wound healing activity of chitosan, this polymer was electrospun together with extract from Henna leaves [151]. In addition, chitosan nanoparticles have been incorporated in nanofibrous scaffolds as carriers for controlled drug delivery, for example, delivery of growth factors, cytokines and angiogenic factors, such as platelet-derived growth factor [152], granulocyte colony-stimulating factor [153] or angiogenin [147].

*Scanning electron microscopy of nanofibrous layers produced by needle electrospinning from PVA/chitosan* 

*DOI: http://dx.doi.org/10.5772/intechopen.88602*

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Nature-Derived… DOI: http://dx.doi.org/10.5772/intechopen.88602*

#### **Figure 3.**

*Current and Future Aspects of Nanomedicine*

nanofibrous mat as an inner layer [109].

ing, and it is approved for clinical application [33].

full-thickness skin excision wounds in rats [139].

**Chitosan** is a linear polysaccharide composed of randomly distributed β-(1 → 4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine

ratio and a slower degradation profile compared to keratin-based non-woven nanofibrous mats containing free diclofenac [126]. Keratin was also a component of core-shell nanofibers, prepared by coaxial electrospinning of chitosan, PCL and keratin with *Aloe vera* extracts encapsulated inside the polymer nanofibers. This construct increased the adhesion and growth of L929 fibroblasts and was intended for wound healing applications [128]. Importantly, keratin was a component of bilayer scaffolds for skin tissue engineering, composed of human hair keratin/ chitosan nanofiber mat and gelatin methacrylate (GelMA) hydrogel. Human dermal fibroblasts were encapsulated and grown in the hydrogel matrix, while human HaCaT keratinocytes formed a layer on the top of the scaffolds, mimicking dermis and epidermis of skin tissue [116]. Another bilayer scaffolds was constructed using polyurethane wound dressing as an outer layer, and electrospun gelatin/keratin

**Hyaluronic acid**, also called hyaluronan, is an anionic, non-sulfated linear glycosaminoglycan. It is distributed widely throughout connective, neural, and epithelial tissues, including skin, where it is a major component of ECM. Therefore, hyaluronic acid has been widely used for skin tissue engineering and wound heal-

Hyaluronic acid stimulated infiltration of nanofibrous scaffold composed of hyaluronan, silk fibroin and PCL [75], and can help to promote cell proliferation [129]. Electrospinning of pure hyaluronic acid is not simple because of solubility characteristics of this polymer. Hyaluronic acid is well-soluble in water but lesssoluble in most organic solvents, which can be solved by mixtures of solvents as water/ethanol or water/dimethylformamide [130]. Increasing of evaporation and decreasing of solution surface tension by the solvent mixing helps to electrospinning process. Another possibility is electrospinning of hyaluronic acid together with a suitable water-soluble polymer such as PVA [131] or PEO. The solution of pure hyaluronic acid [132] or with relatively small amount of carrier PEO was successfully spun into nanofibrous material by air-assisted electrospinning technology, that is, electroblowing [133]. For creation of nanofibrous scaffolds, hyaluronic acid was also used in combination with PCL [134], PLA [135] or gelatin, chondroitin sulfate and

**Sulfated glycosaminoglycans** have been relatively rarely used as components of nanofibers for skin tissue engineering and wound healing in comparison with hyaluronic acid. This group of polysaccharides include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate (for a review, see [33]). From these polysaccharides, only heparin, heparan sulfate, and chondroitin sulfate were used as components of nanofibers for skin regenerative therapies. For example, heparin coatings on PLLA nanofibers increased the infiltration of the scaffolds with endothelial cells *in vitro,* and enhanced epidermal skin cell migration across the wound in a full-thickness dermal wound model in rats *in vivo* [136]. In a recent study by Yergoz *et al*. [137], a heparin-like nanofibrous hydrogel promoted regeneration of full thickness burn injury in mice. Chondroitin sulfate was used as a component of electrospun gelatin/PVA/chondroitin sulfate nanofibrous scaffolds, which supported the proliferation of human dermal fibroblasts [138], of electrospun nanofibrous composite scaffolds made of cationic gelatin/hyaluronan/chondroitin sulfate loaded with sericin, which promoted the differentiation of human mesenchymal stem cells toward epithelial lineage [86], and of electrospun gelatin/chondroitin sulfate nanofibrous scaffolds, which accelerated healing of

**80**

sericin [86].

*Scanning electron microscopy of nanofibrous layers produced by needle electrospinning from PVA/chitosan solution.*

(acetylated unit). It can be prepared by alkali treatment from chitin, a poly-Nacetyl-D-glucosamine polysaccharide, the major structural component of the exoskeleton of crustaceans (crab, shrimp), and of the cell wall of fungi and yeast [140]. Chitosan is known as biocompatible, antimicrobial, and biodegradable. In the human organism, it can be degraded by lysozyme, a hydrolytic enzyme present in various secretions such as saliva, tears, mucus, and human milk, and also in cytoplasmic granules of macrophages and polymorphonuclear neutrophils. Chitosan breakdown by lysozymes happens via the removal of glycosidic bonds between polysaccharide units in the polymer. Glucosamine and saccharide are the products of this process, which can be metabolized or stored as proteoglycans in the body.

However, electrospinning of chitosan is difficult due to its polycationic characters. Due to the presence of amine groups in the chitosan molecule, acidic aqueous solutions are the best solvents for this polymer. The best candidates for solvent system seem to be mixture of acetic acid (AA) and formic acid (FA) or trifluoroacetic acid (TFA); however, TFA is highly toxic. Electrospinning of pure chitosan has very low productivity because it requires very concentrated polymeric solutions [141]. Therefore, for creation of nanofibrous scaffolds for skin tissue engineering, chitosan has been mixed with other natural or synthetic polymers, such as collagen [142], gelatin [143], keratin [116], cellulose [144], pectin [54, 55], silk fibroin [69], PHBV [145], PCL [142], PLA [146], PLGA [147], PEO, [148], and also with PVA, which was used in our studies (**Figure 3**). Chitosan has also been mixed with various nanoparticles, such as halloysite nanotubes [149], graphene oxide [150] or nanodiamonds [144]. The reason of all these mixtures was to improve the stability, spinnability, wettability, mechanical properties, and biofunctionality of chitosancontaining scaffolds for skin tissue engineering. Combination of chitosan with various polymers also enabled creation of bilayer scaffolds for reconstruction of two main skin layers, that is, epidermis containing keratinocytes and dermis containing fibroblasts [116, 142]. In order to enhance the antimicrobial and wound healing activity of chitosan, this polymer was electrospun together with extract from Henna leaves [151]. In addition, chitosan nanoparticles have been incorporated in nanofibrous scaffolds as carriers for controlled drug delivery, for example, delivery of growth factors, cytokines and angiogenic factors, such as platelet-derived growth factor [152], granulocyte colony-stimulating factor [153] or angiogenin [147].

Nanofibrous scaffolds promising for skin tissue engineering and wound healing were also prepared directly from chitin, which was electrospun either alone with further modifications with fibronectin, laminin and particularly with type I collagen [154], or in combination with silk fibroin [70].

**Gellan gum** is a water-soluble anionic polysaccharide produced by the bacterium *Sphingomonas elodea* (formerly *Pseudomonas elodea*). The repeating unit of the polymer is a tetrasaccharide, which consists of two residues of D-glucose, one of residue of L-rhamnose and one residue of D-glucuronic acid. For skin tissue engineering and wound healing, gellan gum was electrospun with PVA in order to decrease its viscosity and repulsive forces between the polyanions along the polymer chains and to increase the stability, uniformity, and structural consistency of the nanofibers in aqueous environment. The nanofibrous scaffolds were further stabilized by crosslinking with various physical, chemical, and ionic methods, such as by heat, UV irradiation, methanol, glutaraldehyde, and by calcium chloride [155]. These scaffolds supported the adhesion and growth of human dermal 3T3-L1 fibroblasts [155, 156] and human HaCaT keratinocytes [157] and provided a better support for these cells than conventionally proposed gellan-based hydrogels and dry films. In addition, these scaffolds were endowed with antimicrobial activity by incorporation with amoxicillin, and accelerated healing of full-thickness skin excision wound in rats in comparison with non-treated wounds [157]. Similarly as chitosan, gellan gum has been reported to be degradable by lysozyme [158]. Three-dimensional printed gellan gum scaffolds also showed degradation *in vitro* in phosphate-buffered saline (PBS) or in simulated body fluid, and the degradation rate could be modulated by changing the ratio of surface area per mass of the scaffolds [159].

**Zein** is the major storage protein of corn, composed of amino acids such as leucine, glutamic acid, alanine and proline, and showing good biocompatibility, flexibility, microbial resistance, and antioxidant activity [61]. Zein has been shown to degradable *in vitro* in PBS and also *in vivo* when implanted subcutaneously in rats in the form of rod-like implants [160]. However, similarly as in many other natural polymers, the application of pure zein nanofibers is limited because of poor mechanical properties of these fibers. Therefore, for skin tissue engineering and wound dressing applications, zein has been mixed with various synthetic and nature-derived polymers, such as polyurethane [161], PLA [162], PCL, hyaluronic acid, chitosan [163], and polydopamine [164], and impregnated with TiO2 nanoparticles [164] or Ag nanoparticles [161] in order to enhance the antimicrobial activity of the scaffolds.

**Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)** (PHBV), is a polymer produced naturally by bacteria as a storage compound under growth-limiting conditions. It is a thermoplastic linear aliphatic polyester of polyhydroxyalkanoate type. PHBV is approved by the FDA for medical use. PHBV is biodegradable by bacterial enzymes, but it is also susceptible to hydrolytic degradation in water environment, although this degradation is relatively slow. When degradation of porous PHBV scaffolds for tissue engineering was simulated *in vitro* in PBS at 37°C, it lasted several months [165]. In the human body, however, the degradation of PHBV can be accelerated by nonspecific esterase and lysozyme enzymes, both present in cells of the immune system (for a review, see [165]). For biomedical application, PHBV is often used as an alternative to synthetic polymers, but it has several drawbacks, such as relatively high cost, brittleness, relatively difficult processing, and also hydrophobicity, which can hamper the cell adhesion and growth. However, PHBV is piezoelectric, which can stimulate the adhesion, growth, and phenotypic maturation of cells. Pure electrospun PHBV meshes supported the adhesion, growth, and epidermal differentiation of bone marrow mesenchymal stem cells, which was induced by an appropriate composition of cell culture media, containing epidermal

**83**

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Nature-Derived…*

Nanofibrous scaffolds made of nature-derived polymers hold a great promise for skin tissue engineering and wound healing. These scaffolds are created from biological matrices, and from this point of view, they resemble the extracellular matrix more closely than synthetic polymers. Some of these polymers, such as collagen, gelatin, elastin, keratin, nonsulfated and sulfated glycosaminoglycans, and also nonmulberry silk fibroin, contain motifs that are recognized and bound by cell adhesion receptors. Therefore, nature-derived polymers can increase the bioactivity of synthetic polymers, when combined with them in nanofibrous scaffolds. Conversely, synthetic polymers can improve the electrospinnability and mechanical properties of the natural polymers. Similarly as synthetic polymers, nature-derived polymers can be more or less degradable in human tissues. Degradable polymers include collagen, gelatin, elastin, keratin, glycosaminoglycans, but also chitosan, gellan gum and PHBV, that is, polymers produced by other than mammalian organisms. Polymers produced by other organisms, such as bacteria, fungi, algae, plants or insects, are usually nondegradable in human tissues, or their degradability is limited due to lack of appropriate enzymes. These polymers include glucans, such as cellulose or dextran, and other polysaccharides and proteins, such as pullulan, alginate, pectin, and silk fibroin. Well-degradable polymers are recommended as direct scaffolds for tissue engineering, while less-degradable polymers are suitable for "intelligent"

This review article was supported by the Grant Agency of the Czech Republic

growth factor, insulin, 3,3′,5-triiodo-L-thyronine (T3), hydrocortisone, and 1α, 25-dihydroxyvitamin (D3), and manifested by expression of genes for keratin, filaggrin, and involucrin, that is, an early, intermediate and late marker of keratinocyte differentiation, respectively [166]. In order to increase the attractiveness of electrospun PHBV nanofibers for the cell adhesion and growth, they were coated with collagen [167] blended with collagen [168], blended with chitosan [145] or blended with keratin [169]. Collagen-coated PHBV nanofibers alone or seeded with unrestricted somatic stem cells, isolated from umbilical cord, accelerated closure of excision wounds in rats *in vivo* compared to unmodified PHBV nanofibers [167]. Similar wound healing effect was also obtained with PHBV nanofibers blended with keratin [169]. Mechanical properties of PHBV nanofibers were improved by addition of graphene oxide nanoparticles in the electrospinning solution, which

also endowed these fibers with and antimicrobial activity [167].

wound dressing for drug delivery and cell delivery.

(grants No. 17-02448S and 17-00885S).

**Acknowledgements**

*DOI: http://dx.doi.org/10.5772/intechopen.88602*

**4. Conclusions**

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Nature-Derived… DOI: http://dx.doi.org/10.5772/intechopen.88602*

growth factor, insulin, 3,3′,5-triiodo-L-thyronine (T3), hydrocortisone, and 1α, 25-dihydroxyvitamin (D3), and manifested by expression of genes for keratin, filaggrin, and involucrin, that is, an early, intermediate and late marker of keratinocyte differentiation, respectively [166]. In order to increase the attractiveness of electrospun PHBV nanofibers for the cell adhesion and growth, they were coated with collagen [167] blended with collagen [168], blended with chitosan [145] or blended with keratin [169]. Collagen-coated PHBV nanofibers alone or seeded with unrestricted somatic stem cells, isolated from umbilical cord, accelerated closure of excision wounds in rats *in vivo* compared to unmodified PHBV nanofibers [167]. Similar wound healing effect was also obtained with PHBV nanofibers blended with keratin [169]. Mechanical properties of PHBV nanofibers were improved by addition of graphene oxide nanoparticles in the electrospinning solution, which also endowed these fibers with and antimicrobial activity [167].
