**Abstract**

Nanofibrous scaffolds belong to the most suitable materials for tissue engineering, because they mimic the fibrous component of the natural extracellular matrix. This chapter is focused on the application of nanofibers in skin tissue engineering and wound healing, because the skin is the largest and vitally important organ in the human body. Nanofibrous meshes can serve as substrates for adhesion, growth and differentiation of skin and stem cells, and also as an antimicrobial and moistureretaining barrier. These meshes have been prepared from a wide range of synthetic and nature-derived polymers. This chapter is focused on the use of nature-derived polymers. These polymers have good or limited degradability in the human tissues, which depends on their origin and on the presence of appropriate enzymes in the human tissues. Non-degradable and less-degradable polymers are usually produced in bacteria, fungi, algae, plants or insects, and include, for example, cellulose, dextran, pullulan, alginate, pectin and silk fibroin. Well-degradable polymers are usually components of the extracellular matrix in the human body or at least in other vertebrates, and include collagen, elastin, keratin and hyaluronic acid, although some polymers produced by non-vertebrate organisms, such as chitosan or poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate), are also degradable in the human body.

**Keywords:** skin replacements, wound dressings, nanofibers, electrospinning, epidermis, dermis, keratinocytes, fibroblasts, stem cells, vascularization, cell delivery, drug delivery, regenerative medicine

### **1. Introduction**

Nanofibrous scaffolds are one of the most promising materials for skin tissue engineering and wound dressing, because they resemble nanoarchitecture of the native extracellular matrix (for a review, see [1]). Therefore, they can serve as suitable carriers of cells for tissue engineering and also as suitable wound dressings, which are able to protect the wound from external harmful effects, mainly

microbial infection, and at the same time, they can keep appropriate moisture and gas exchange at the wound site.

Nanofibrous scaffolds for skin tissue engineering have been fabricated from a wide range of synthetic and nature-derived polymers, which can be either biostable or degradable within the human body. Biostable synthetic polymers used in nanofiber-based skin regenerative therapies include, for example, polyurethane [2], polydimethylsiloxane [3], polyethylene terephthalate [4], polyethersulfone [5], and also hydrogels such as poly(acrylic acid) (PAA, [6]), poly(methyl methacrylate) (PMMA, [7]), and poly[di(ethylene glycol) methyl ether methacrylate] (PDEGMA, [8]). Degradable synthetic polymers typically include poly(ε-caprolactone) (PCL, [9]) and its copolymers with polylactides (PLCL, [10]), polylactides (PLA, [11]) and their copolymers with polyglycolides (PLGA, [12]), and also so-called auxiliary polymers, such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO, [13]) or poly(vinyl alcohol) (PVA, [14]), which facilitated the electrospinning process and improved the mechanical properties and wettability of the chief polymer. However, the synthetic polymers, although they are well-chemically defined and tailorable, are often bioinert, hydrophobic and thus not promoting cell adhesion, and also not well-adhering to the wound site. Therefore, they need to be combined with other bioactive substances, particularly nature-derived polymers.

This chapter is focused on nature-derived polymers used for fabrication of nanofibrous scaffolds for skin tissue engineering and wound healing. The advantages of most of these polymers are their better bioactivity, flexibility, wettability, and adhesion to the wound site. Similarly as synthetic polymers, also nature-derived polymers can be divided into polymers with none or limited degradability, when implanted into human tissues, and polymers well-degradable in human tissues. The first group includes glucans, such as cellulose, schizophyllan, dextran, starch, and other polysaccharides and proteins, such as pullulan, xylan, alginate, pectin, gum tragacanth, gum arabic, silk fibroin, and sericin. The second group of polymers degradable in human tissues includes collagen and its derivative gelatin, elastin, keratin, glycosaminoglycans such as hyaluronic acid, heparin and chondroitin sulfate, and also polymers not produced in the human body, namely chitosan, gellan gum, zein, and poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV).

Some of the polymers degradable in human tissues, such as collagen, gelatin, elastin, keratin, and glycosaminoglycans, contain specific cell-binding motifs in their molecules, for example, specific amino acid sequences in proteins and oligosaccharide domains in glycosaminoglycans, which are recognized by cell adhesion receptors of integrin and non-integrin families (for a review, see [15, 16]). These molecules are often used in allogeneic or xenogeneic form, thus they can be associated with pathogen transmission or immune reaction. However, some synthetic polymers, for example PLA and PCL, have been reported to induce a more pronounced inflammatory reaction than gelatin [17].

This review chapter summarizes earlier and recent knowledge on skin tissue engineering and wound dressing applications, based on nanofibrous scaffolds made of nature-derived polymers, including our results.

## **2. Nature-derived nanofibers with none or limited degradability in the human tissues**

Nature-derived nondegradable polymers or polymers with limited degradability in human tissues include polymers not occurring in the human body and synthesized by other organisms, such as plants, algae, fungi, insects, and bacteria.

**71**

**Figure 1.**

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

Nanofibrous cellulose can be prepared in three basic forms: bacterial cellulose, which contains cellulose nanofibrils, synthesized by bacteria, nanofibrillar cellulose prepared from plants, particularly from wood, by hydrolysis, oxidation, and mechanical disintegration, and cellulose nanofibers created by electrospinning (for a review, see [19]). For electrospinning, cellulose should be solved. Well-known solvent of cellulose is N-methylmorpholine-N-oxide (NMMO). Another possibility is N-alkylinidazolium-derivate ionic liquid and N,N-dimethylacetamide containing 8 wt% of LiCl. However, any of them did not prove to be a good solvent for needleless electrospinning. The most favorable solvent of cellulose was found to be trifluoroacetic acid (TFA). However, TFA causes severe skin burns and is toxic for aquatic organisms even in low concentrations [20]. These problems, which limit the use of cellulose for creation of electrospun scaffolds for biomedical applications, can be solved by substituting the natural cellulose by its derivatives. The mostly used derivative of cellulose is cellulose acetate (CA), mainly due to its easier solubility and biocompatibility. CA can be dissolved in several solvents, however the best ones for electrospinning proved to be acetic acid (AA), and mixtures of acetone and N,N-dimethylacetamide (DMAC). Some results of successfully spun fibers by needleless electrospinning in our experiments can be found in **Figure 1**, demonstrating differences in the fiber morphology. The 95% aqueous mixture of AA showed the best results in comparison with acetone/DMAC mixtures due to

All the mentioned forms of cellulose have been widely applied as wound dressings releasing various bioactive agents into wounds (antimicrobial, anti-inflammatory, antioxidative agents, cytokines, and growth and angiogenic factors), as transparent wound dressings for direct optical monitoring of wounds, for systemic

*Scanning electron microscopy of nanofibrous layers produced by wire needleless electrospinning using different solvents, namely 12 wt% of CA in acetone/DMAC (9:1) (left) or 14 wt% of CA in 95% AA (right).*

**Cellulose** is a typical natural polymer nondegradable in human tissues. Cellulose belongs to the group of glucans, that is, polysaccharides derived from D-glucose, linked by glycosidic bond. In the cellulose molecules, these glycosidic bonds are of the β-type, thus the cellulose is a β-glucan. It is structural polysaccharide consisting of a linear chain of several hundred to over ten thousand β(1 → 4) linked D-glucose units. Cellulose is synthesized by plants, algae, fungi, some species of bacteria (*Gluconacetobacter xylinus*), and also by some animals, namely tunicates

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

(*Styela clava*) (for a review, see [18, 19]).

production of smoother fibers and lower cytotoxicity.

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

**Cellulose** is a typical natural polymer nondegradable in human tissues. Cellulose belongs to the group of glucans, that is, polysaccharides derived from D-glucose, linked by glycosidic bond. In the cellulose molecules, these glycosidic bonds are of the β-type, thus the cellulose is a β-glucan. It is structural polysaccharide consisting of a linear chain of several hundred to over ten thousand β(1 → 4) linked D-glucose units. Cellulose is synthesized by plants, algae, fungi, some species of bacteria (*Gluconacetobacter xylinus*), and also by some animals, namely tunicates (*Styela clava*) (for a review, see [18, 19]).

Nanofibrous cellulose can be prepared in three basic forms: bacterial cellulose, which contains cellulose nanofibrils, synthesized by bacteria, nanofibrillar cellulose prepared from plants, particularly from wood, by hydrolysis, oxidation, and mechanical disintegration, and cellulose nanofibers created by electrospinning (for a review, see [19]). For electrospinning, cellulose should be solved. Well-known solvent of cellulose is N-methylmorpholine-N-oxide (NMMO). Another possibility is N-alkylinidazolium-derivate ionic liquid and N,N-dimethylacetamide containing 8 wt% of LiCl. However, any of them did not prove to be a good solvent for needleless electrospinning. The most favorable solvent of cellulose was found to be trifluoroacetic acid (TFA). However, TFA causes severe skin burns and is toxic for aquatic organisms even in low concentrations [20]. These problems, which limit the use of cellulose for creation of electrospun scaffolds for biomedical applications, can be solved by substituting the natural cellulose by its derivatives. The mostly used derivative of cellulose is cellulose acetate (CA), mainly due to its easier solubility and biocompatibility. CA can be dissolved in several solvents, however the best ones for electrospinning proved to be acetic acid (AA), and mixtures of acetone and N,N-dimethylacetamide (DMAC). Some results of successfully spun fibers by needleless electrospinning in our experiments can be found in **Figure 1**, demonstrating differences in the fiber morphology. The 95% aqueous mixture of AA showed the best results in comparison with acetone/DMAC mixtures due to production of smoother fibers and lower cytotoxicity.

All the mentioned forms of cellulose have been widely applied as wound dressings releasing various bioactive agents into wounds (antimicrobial, anti-inflammatory, antioxidative agents, cytokines, and growth and angiogenic factors), as transparent wound dressings for direct optical monitoring of wounds, for systemic

#### **Figure 1.**

*Scanning electron microscopy of nanofibrous layers produced by wire needleless electrospinning using different solvents, namely 12 wt% of CA in acetone/DMAC (9:1) (left) or 14 wt% of CA in 95% AA (right).*

*Current and Future Aspects of Nanomedicine*

gas exchange at the wound site.

3-hydroxyvalerate) (PHBV).

tory reaction than gelatin [17].

**human tissues**

of nature-derived polymers, including our results.

microbial infection, and at the same time, they can keep appropriate moisture and

Nanofibrous scaffolds for skin tissue engineering have been fabricated from a wide range of synthetic and nature-derived polymers, which can be either biostable or degradable within the human body. Biostable synthetic polymers used in nanofiber-based skin regenerative therapies include, for example, polyurethane [2], polydimethylsiloxane [3], polyethylene terephthalate [4], polyethersulfone [5], and also hydrogels such as poly(acrylic acid) (PAA, [6]), poly(methyl methacrylate) (PMMA, [7]), and poly[di(ethylene glycol) methyl ether methacrylate] (PDEGMA, [8]). Degradable synthetic polymers typically include poly(ε-caprolactone) (PCL, [9]) and its copolymers with polylactides (PLCL, [10]), polylactides (PLA, [11]) and their copolymers with polyglycolides (PLGA, [12]), and also so-called auxiliary polymers, such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO, [13]) or poly(vinyl alcohol) (PVA, [14]), which facilitated the electrospinning process and improved the mechanical properties and wettability of the chief polymer. However, the synthetic polymers, although they are well-chemically defined and tailorable, are often bioinert, hydrophobic and thus not promoting cell adhesion, and also not well-adhering to the wound site. Therefore, they need to be combined with other

This chapter is focused on nature-derived polymers used for fabrication of nanofibrous scaffolds for skin tissue engineering and wound healing. The advantages of most of these polymers are their better bioactivity, flexibility, wettability, and adhesion to the wound site. Similarly as synthetic polymers, also nature-derived polymers can be divided into polymers with none or limited degradability, when implanted into human tissues, and polymers well-degradable in human tissues. The first group includes glucans, such as cellulose, schizophyllan, dextran, starch, and other polysaccharides and proteins, such as pullulan, xylan, alginate, pectin, gum tragacanth, gum arabic, silk fibroin, and sericin. The second group of polymers degradable in human tissues includes collagen and its derivative gelatin, elastin, keratin, glycosaminoglycans such as hyaluronic acid, heparin and chondroitin sulfate, and also polymers not produced in the human body, namely chitosan, gellan gum, zein, and poly(3-hydroxybutyrate-*co*-

Some of the polymers degradable in human tissues, such as collagen, gelatin, elastin, keratin, and glycosaminoglycans, contain specific cell-binding motifs in their molecules, for example, specific amino acid sequences in proteins and oligosaccharide domains in glycosaminoglycans, which are recognized by cell adhesion receptors of integrin and non-integrin families (for a review, see [15, 16]). These molecules are often used in allogeneic or xenogeneic form, thus they can be associated with pathogen transmission or immune reaction. However, some synthetic polymers, for example PLA and PCL, have been reported to induce a more pronounced inflamma-

This review chapter summarizes earlier and recent knowledge on skin tissue engineering and wound dressing applications, based on nanofibrous scaffolds made

**2. Nature-derived nanofibers with none or limited degradability in the** 

Nature-derived nondegradable polymers or polymers with limited degradability in human tissues include polymers not occurring in the human body and synthesized by other organisms, such as plants, algae, fungi, insects, and bacteria.

bioactive substances, particularly nature-derived polymers.

**70**

transdermal drug delivery (analgesics, antiphlogistics, corticoids, and antihypertensives) and for construction of epidermal electronics for monitoring wound healing or physiological status of the organism. Non-degradable nanocellulose has also been used as a temporary carrier for delivery of keratinocytes, dermal fibroblasts, and mesenchymal stem cells into wounds (for a review, see [19]).

However, for use as direct scaffolds for skin tissue engineering, cellulose should be rendered degradable in human tissues. Cellulose is degradable by cellulase enzymes (exoglucanases and endoglucanases), which hydrolyze 1,4-beta-D-glycosidic linkages. These enzymes are not synthesized in human tissues, but they can be incorporated into cellulose scaffolds in order to degrade them gradually [21, 22]. These enzymes are believed to be non-toxic for mammalian cells [23, 24]. Moreover, the final product of cellulose degradation by these enzymes is glucose, which is a natural nutrient for the cells, by contrast with the acidic by-products of the standard currently used biodegradable PLA or PLGA scaffolds [25]. Another possibility how to use cellulase enzymes in skin tissue engineering (and in tissue engineering in general) is cell sheet technology. First, cells can be grown on the top of non-degradable cellulose substrates. After reaching the cell confluence, self-standing cell sheets can be released by exposure of the cellulose substrates to cellulases. Unlike the proteolytic enzymes conventionally used for detaching cells from their growth supports, cellulases do not disintegrate the extracellular matrix (ECM) formed by cells and do not cleave extracellular parts of cell adhesion receptors binding the ECM [26]. The cell sheets can be then replanted in the wound bed.

Another interesting approach how to render the cellulose degradable was metabolic engineering of *Gluconacetobacter xylinus*, which then produced modified cellulose molecules with intercalated N-acetylglucosamine (GlcNAc) residues, susceptible to degradation with lysozyme, present in the human body. After subcutaneous implantation in mice, the modified cellulose was completely degraded within 20 days [27, 28].

Other approaches how to render the cellulose degradable, at least partially, is its oxidation and other chemical modifications of cellulose, such as its conversion into regenerated cellulose or 2,3-dialdehydecellulose. In addition, cellulose of animal origin, that is, from tunicates, degraded more quickly than plant cellulose. For example, when cellulose films from *Styela clava* were implanted subcutaneously into rats for 90 days, they lost almost 24% of their initial weight, while the films prepared from wood pulp cellulose lost only less than 10% (for a review, see [19]).

**Schizophyllan** is another β-glucan used for potential wound healing application. It is an extracellular β-1,3 beta-glucan with β-1,6 branching, produced by the fungus *Schizophyllum commune*. In blends with PVA, it was used for electrospinning of nanofibrous scaffolds, which provided a suitable growth support for human dermal fibroblast. In experimental wound models *in vivo*, schizophyllan attracted macrophages, necessary for the first physiological phase of wound healing, that is, inflammation. Schizophyllan and other 1,3-β-glucans also increased collagen deposition, cellularity, formation of granulation tissue, and vascularity at the wound site [29].

Other glucans used for fabrication of nanofibrous scaffolds for skin tissue engineering and wound healing include dextran, starch and pullulan. According to the type of their glycosidic bonds, these polysaccharides belong to α-glucans.

**Dextran** is a branched complex glucan, in which the D-glucose units are linked by α-1,6 glycosidic bonds with branches from α-1,3 linkages. Dextran is of microbial origin; it can be produced, for example, by some lactic acid bacteria from sucrose. Dextran was used as a component of nanofibrous polyurethane-based wound dressings, in which dextran promoted neovascularization of the wound site, and also served as carrier for β-estradiol, an endogenous estrogen, a potent anti-inflammatory

**73**

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

agent, and mitogen for keratinocytes. In addition, the presence of dextran made the polyurethane dressing softer, more flexible, more wettable, and well-adherent to the wound and promoted hemostatic activity of the dressing. *In vitro*, the presence of dextran and β-estradiol enhanced the proliferation of 3T3-L1 fibroblasts on the

Dextran was also used as component of a bilayer scaffold for skin tissue engineer-

(ε-caprolactone-*co*-lactide) and poloxamer (i.e., Pluronic), and the lower part was made of a hydrogel composed of dextran and gelatin without the addition of a chemical crosslinking agent. The lower dextran/gelatin hydrogel layer provided a highly swollen three-dimensional environment similar to extracellular matrix (ECM) of soft tissues. Both part of the scaffolds supported the growth of adiposetissue-derived stem cells; however, the number of these cells on the hydrogels decreased with increasing

Dextran is degradable by dextranases, enzymes hydrolyzing (1 → 6)-alpha-Dglycosidic linkages. This enzyme is produced mainly by bacterial and fungi, but it was also detected in animal and human tissues, namely liver and spleen. Therefore, dextran is often chosen for biomedical applications, particularly drug delivery, because it is slowly degradable in human organism. Dextran molecules with Mw higher than 40 kDa are sequestered in the liver and spleen, and then hydrolyzed by endo- and exodextranases. Dextran molecules with Mw lower than 40 kDa can be eliminated through renal clearance [32]. However, dextran hydrogels implanted subcutaneously or intramuscularly into rats did not show signs of degradation 6 weeks post-implantation and were surrounded by a thin fibrous capsule and some macrophages and giant cells, which is a response typical for a number of non-

**Starch** is another α-glucan, containing both α-1,4- and α-1,6 glycosidic bonds. It serves an energy storage polysaccharide in plants, and from this point of view, it is considered to be an analogue of glycogen, energy storage polysaccharide in animals. Starch consists of two types of molecules, namely linear amylose and branched amylopectin (for a review, see [33]). Electrospun starch-based nanofibrous meshes were proposed for wound healing applications. The electrospinning of starch was facilitated by addition of PVA, that is, a noncytotoxic, water-soluble, biocompatible synthetic polymer which reduced the repulsive forces produced in starch solution. The scaffolds then promoted the proliferation of mouse L929 fibroblasts [34]. Starch is degradable by amylases, that is, hydrolases that act on α-1,4-glycosidic bonds. Amylases occur in three forms, namely α-, β-, and γ-amylases. These enzymes are synthesized by microorganisms (bacteria and fungi), plants, and with exception of β-amylases, also in animals. Alpha-amylases are present in human organism, but not currently in all tissues-they are important enzymes of gastrointestinal tract and are produced by salivary glands and pancreas. Interestingly, α-amylases were also found in brain, and their lower expression there is probably associ-

ated with the pathogenesis of Alzheimer's neurodegenerative disease [35].

films and fibers. It is degradable by pullulanase, a specific kind of glucanase, produced in bacteria and not present in human tissues. When pullulan hydrogels alone or in combination with dextran were implanted subcutaneously into rats, they induced inflammatory reaction and were surrounded by a fibrous capsule [36]. Nevertheless, pullulan is water-soluble and thus removable from human issues, and

**Pullulan** is also an α-glucan with both α-1,4- and α-1,6 glycosidic bonds. It is a linear polysaccharide consisting of maltotriose units, in which three glucose units in maltotriose are connected by an α-1,4 glycosidic bond, whereas consecutive maltotriose units are connected to each other by an α-1,6 glycosidic bond. Pullulan is produced from starch by the fungus *Aureobasidium pullulans*. It shows a high waterabsorbing capability, adhesive properties, and the capability to form strong resilient

ing. The upper part of the scaffolds was made of electrospun blend of poly

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

scaffolds [30].

content of dextran [31].

degradable materials [32].

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

agent, and mitogen for keratinocytes. In addition, the presence of dextran made the polyurethane dressing softer, more flexible, more wettable, and well-adherent to the wound and promoted hemostatic activity of the dressing. *In vitro*, the presence of dextran and β-estradiol enhanced the proliferation of 3T3-L1 fibroblasts on the scaffolds [30].

Dextran was also used as component of a bilayer scaffold for skin tissue engineering. The upper part of the scaffolds was made of electrospun blend of poly (ε-caprolactone-*co*-lactide) and poloxamer (i.e., Pluronic), and the lower part was made of a hydrogel composed of dextran and gelatin without the addition of a chemical crosslinking agent. The lower dextran/gelatin hydrogel layer provided a highly swollen three-dimensional environment similar to extracellular matrix (ECM) of soft tissues. Both part of the scaffolds supported the growth of adiposetissue-derived stem cells; however, the number of these cells on the hydrogels decreased with increasing content of dextran [31].

Dextran is degradable by dextranases, enzymes hydrolyzing (1 → 6)-alpha-Dglycosidic linkages. This enzyme is produced mainly by bacterial and fungi, but it was also detected in animal and human tissues, namely liver and spleen. Therefore, dextran is often chosen for biomedical applications, particularly drug delivery, because it is slowly degradable in human organism. Dextran molecules with Mw higher than 40 kDa are sequestered in the liver and spleen, and then hydrolyzed by endo- and exodextranases. Dextran molecules with Mw lower than 40 kDa can be eliminated through renal clearance [32]. However, dextran hydrogels implanted subcutaneously or intramuscularly into rats did not show signs of degradation 6 weeks post-implantation and were surrounded by a thin fibrous capsule and some macrophages and giant cells, which is a response typical for a number of nondegradable materials [32].

**Starch** is another α-glucan, containing both α-1,4- and α-1,6 glycosidic bonds. It serves an energy storage polysaccharide in plants, and from this point of view, it is considered to be an analogue of glycogen, energy storage polysaccharide in animals. Starch consists of two types of molecules, namely linear amylose and branched amylopectin (for a review, see [33]). Electrospun starch-based nanofibrous meshes were proposed for wound healing applications. The electrospinning of starch was facilitated by addition of PVA, that is, a noncytotoxic, water-soluble, biocompatible synthetic polymer which reduced the repulsive forces produced in starch solution. The scaffolds then promoted the proliferation of mouse L929 fibroblasts [34]. Starch is degradable by amylases, that is, hydrolases that act on α-1,4-glycosidic bonds. Amylases occur in three forms, namely α-, β-, and γ-amylases. These enzymes are synthesized by microorganisms (bacteria and fungi), plants, and with exception of β-amylases, also in animals. Alpha-amylases are present in human organism, but not currently in all tissues-they are important enzymes of gastrointestinal tract and are produced by salivary glands and pancreas. Interestingly, α-amylases were also found in brain, and their lower expression there is probably associated with the pathogenesis of Alzheimer's neurodegenerative disease [35].

**Pullulan** is also an α-glucan with both α-1,4- and α-1,6 glycosidic bonds. It is a linear polysaccharide consisting of maltotriose units, in which three glucose units in maltotriose are connected by an α-1,4 glycosidic bond, whereas consecutive maltotriose units are connected to each other by an α-1,6 glycosidic bond. Pullulan is produced from starch by the fungus *Aureobasidium pullulans*. It shows a high waterabsorbing capability, adhesive properties, and the capability to form strong resilient films and fibers. It is degradable by pullulanase, a specific kind of glucanase, produced in bacteria and not present in human tissues. When pullulan hydrogels alone or in combination with dextran were implanted subcutaneously into rats, they induced inflammatory reaction and were surrounded by a fibrous capsule [36]. Nevertheless, pullulan is water-soluble and thus removable from human issues, and

*Current and Future Aspects of Nanomedicine*

transdermal drug delivery (analgesics, antiphlogistics, corticoids, and antihypertensives) and for construction of epidermal electronics for monitoring wound healing or physiological status of the organism. Non-degradable nanocellulose has also been used as a temporary carrier for delivery of keratinocytes, dermal fibroblasts,

However, for use as direct scaffolds for skin tissue engineering, cellulose should

Other approaches how to render the cellulose degradable, at least partially, is its oxidation and other chemical modifications of cellulose, such as its conversion into regenerated cellulose or 2,3-dialdehydecellulose. In addition, cellulose of animal origin, that is, from tunicates, degraded more quickly than plant cellulose. For example, when cellulose films from *Styela clava* were implanted subcutaneously into rats for 90 days, they lost almost 24% of their initial weight, while the films prepared from

**Schizophyllan** is another β-glucan used for potential wound healing application. It is an extracellular β-1,3 beta-glucan with β-1,6 branching, produced by the fungus *Schizophyllum commune*. In blends with PVA, it was used for electrospinning of nanofibrous scaffolds, which provided a suitable growth support for human dermal fibroblast. In experimental wound models *in vivo*, schizophyllan attracted macrophages, necessary for the first physiological phase of wound healing, that is, inflammation. Schizophyllan and other 1,3-β-glucans also increased collagen deposition, cellularity, formation of granulation tissue, and vascularity at the

Other glucans used for fabrication of nanofibrous scaffolds for skin tissue engineering and wound healing include dextran, starch and pullulan. According to the type of their glycosidic bonds, these polysaccharides belong to α-glucans. **Dextran** is a branched complex glucan, in which the D-glucose units are linked by α-1,6 glycosidic bonds with branches from α-1,3 linkages. Dextran is of microbial origin; it can be produced, for example, by some lactic acid bacteria from sucrose. Dextran was used as a component of nanofibrous polyurethane-based wound dressings, in which dextran promoted neovascularization of the wound site, and also served as carrier for β-estradiol, an endogenous estrogen, a potent anti-inflammatory

wood pulp cellulose lost only less than 10% (for a review, see [19]).

be rendered degradable in human tissues. Cellulose is degradable by cellulase enzymes (exoglucanases and endoglucanases), which hydrolyze 1,4-beta-D-glycosidic linkages. These enzymes are not synthesized in human tissues, but they can be incorporated into cellulose scaffolds in order to degrade them gradually [21, 22]. These enzymes are believed to be non-toxic for mammalian cells [23, 24]. Moreover, the final product of cellulose degradation by these enzymes is glucose, which is a natural nutrient for the cells, by contrast with the acidic by-products of the standard currently used biodegradable PLA or PLGA scaffolds [25]. Another possibility how to use cellulase enzymes in skin tissue engineering (and in tissue engineering in general) is cell sheet technology. First, cells can be grown on the top of non-degradable cellulose substrates. After reaching the cell confluence, self-standing cell sheets can be released by exposure of the cellulose substrates to cellulases. Unlike the proteolytic enzymes conventionally used for detaching cells from their growth supports, cellulases do not disintegrate the extracellular matrix (ECM) formed by cells and do not cleave extracellular parts of cell adhesion receptors binding the ECM [26]. The cell sheets can be then replanted in the wound bed. Another interesting approach how to render the cellulose degradable was metabolic engineering of *Gluconacetobacter xylinus*, which then produced modified cellulose molecules with intercalated N-acetylglucosamine (GlcNAc) residues, susceptible to degradation with lysozyme, present in the human body. After subcutaneous implantation in mice, the modified cellulose was completely degraded within

and mesenchymal stem cells into wounds (for a review, see [19]).

**72**

20 days [27, 28].

wound site [29].

in combination with chitosan and tannic acid, it was used for fabrication of electrospun nanofibrous meshes promising for wound healing [37]. In combination with dextran and gelatin, pullulan was used for electrospinning of nanofibrous scaffolds promising for skin tissue engineering. These scaffolds, especially when crosslinked with trisodium trimetaphosphate, supported the adhesion and spreading of human dermal fibroblasts and formation of actin cytoskeleton in these cells [38].

**Xylan** is a plant polysaccharide belonging to the group of hemicelluloses, that is, polymers often associated with cellulose. While cellulose is made of glucose units, hemicelluloses contain many different sugar monomers. Xylans are polysaccharides made of β-1,4-linked xylose (i.e., a pentose sugar) residues with side branches of α-arabinofuranose and α-glucuronic acids, which contribute to crosslinking of cellulose microfibrils and lignin through ferulic acid residues. Xylans are considered as relatively available and cost-effective natural materials for tissue engineering. Electrospun nanofibers containing beech-derived xylan and PVA were tested as potential dermal substitutes for skin tissue regeneration. These scaffolds provided a good support for the adhesion and proliferation of human foreskin fibroblasts and for production of collagen by these cells [39]. Bagasse xylan was also a component of hydrogels endowed with shape memory, namely carboxymethyl xylang-poly(acrylic acid) hydrogels, applicable in tissue engineering and biosensorics, particularly for construction of electronic skin [40].

**Alginates,** for example, sodium alginate or calcium alginate, are salts of alginic acid, a linear polysaccharide composed of (1,4)-β-D-mannuronic acid and (1,3)-α-L-guluronic acid. Alginates are produced by various species of brown algae, and also by the bacterium *Pseudomonas aeruginosa*, a major pathogen found in the lungs of patients with cystic fibrosis. The structure of alginates is similar to glycosaminoglycans, an important component of ECM in human tissues including skin [41]. Alginates have a great ability to keep moisture in the wound site and to adhere to skin. However, alginates are poorly spinnable, and therefore, for skin tissue engineering and wound dressing applications, they were electrospun together with other polymers, such as PVA [41, 42] or PEO [43]. Poor mechanical properties of alginates have been compensated by the combination with chitosan [44] or PCL [44, 45]. In addition, alginates themselves are not adhesive for mammalian cells, which was compensated by their combination with collagen and gelatin, containing ligands for cell adhesion receptors [41]. Alginates were modified with a cell adhesive GRGDSP oligopeptide, which acts as ligand for integrin cell adhesion receptors [43]. Sodium alginate was used for attachment of arginine to the surface of chitosan nanofibers in order to increase healing capability of this wound dressing [46]. Alginate nanofibers supported by PCL were impregnated with an extract from *Spirulina*, a photosynthetic cyanobacterium producing bioactive molecules with anti-oxidant and anti-inflammatory effects [45]. Electrospun sodium alginate nanofibers containing silver nanoparticles were used for fabrication of an electronic skin capable of pressure sensing and endowed with antibacterial activity [47].

The degradability of alginate in human organism is limited. Alginate is naturally degraded by alginate lyases or alginate depolymerases, which have been isolated from marine algae, marine animals, bacteria, fungi, viruses, and other microorganisms, but are not present in the human organism. Degradability of alginate can be increased by its oxidation and at low pH. Also the hydrophilicity and water uptake capacity of alginate can help in its removal from the wound site (for a review, see [48]).

**Pectin** is a complex of structural polysaccharides present in the cell walls of terrestrial plants, rich in galacturonic acid. Pectin is known as gelling agent in food industry, but it is also widely used in medicine, for example, against digestive disorders, such as obstipation and diarrhea, for oral drug delivery, as a component of dietary fibers trapping cholesterol and carbohydrates, as a demulcent, that is, a

**75**

review, see [65]).

age plant protein [61].

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

mucoprotective agent, and also in wound healing preparations [49]. Pectin is known as a natural prophylactic substance against poisoning with toxic cations, and its hemostatic and curing effects are well-documented in healing ointments [50]. Pectin is degradable by enzymes produced by bacterial, fungal and plant cells, and not present in human tissues [51–53]. Thus, pectin is degradable, at least partly, only in the intestinal tract populated with bacteria. However, pectin is water-soluble and quickly dissolves in the water environment, including the tissues. Therefore, in order to increase its stability, it was combined with chitosan and TiO2 nanoparticles for wound dressing applications [50] or used for construction of composite chitosan-pectin scaffolds for skin tissue engineering. Blending chitosan with pectin markedly improved the mechanical properties of the scaffolds, such as their Young's modulus, strain at break and ultimate tensile strength, in comparison with pure chitosan scaffolds, although the proliferation of cells (i.e., fibroblasts) was slightly slower on pectincontaining scaffolds [54, 55]. The reason is that pectin does not contain cell binding domains. The cell adhesion on pectin nanofibers was markedly enhanced by oxidizing pectin with periodate to generate aldehyde groups, and then crosslinking the nanofibers with adipic acid dihydrazide to covalently connect pectin macromolecular chains with adipic acid dihydrazone linkers. In addition, the crosslinked pectin nanofibers exhibited excellent mechanical strength and enhanced body degradability [56].

Other polysaccharides explored for creation of nanofibrous scaffolds for skin tissue engineering and wound healing are gum tragacanth and gum arabic, both polysaccharides of plant origin, degradable by bacteria and fungi, for example, in soil [57, 58]. **Gum tragacanth** is a viscous water-soluble mixture of polysaccharides, mainly

tragacanthin and bassorin. Tragacanthin dissolves to give a colloidal hydrosol. Bassorin, representing 60–70% of the gum, is insoluble and swells to a gel.

tory, anti-infective, angiogenic, and healing properties [60].

forms a glue-like layer coating two singular filaments of fibroin.

Chemically, tragacanthin is a complex mixture of acidic polysaccharides containing D-galacturonic acid, D-galactose, L-fucose (6-deoyl-L-galactose), D-xylose, and L-arabinose. Bassorin is probably a methylated tragacanthin. A small amount of cellulose, starch, protein and ash are also present (https://colonygums.com/tragacanth). In order to improve electrospinning and mechanical properties of the gum tragacanth, it was combined with PVA and PCL [59]. Gum tragacanth is endowed with microbial resistance and wound healing activity, which was further enhanced by curcumin, a naturally occurring poly-phenolic compound with a broad range of favorable biological functions, including anti-cancer, anti-oxidant, anti-inflamma-

**Gum arabic**, also known as gum acacia, is a complex and water-soluble mixture of glycoproteins and polysaccharides consisting mainly of arabinose and galactose. For skin tissue engineering, it was electrospun with PCL and also with zein, a stor-

**Silk fibroin** is a water-insoluble elastic protein present in silk fibers produced by larvae of *Bombyx mori* and some other moth of the *Saturniidae* family, such as *Antheraea assama, Antheraea mylitta*, and *Philosamia ricini* [62–64]. Silk fibroin occurs in the fibers together with sericin, a water-soluble serine-rich protein, which

In biomaterial science, silk fibroin is considered to be degradable, but in mammalian organism, this degradation is long-lasting and can take more than 1 year. As a kind of biomaterial approved by the Food and Drug Administration (FDA) for medical use, silk is defined by United States Pharmacopeia as non-degradable for its negligible tensile strength loss *in vivo*. However, silk fibroin is susceptible to biological degradation by proteolytic enzymes such as chymotrypsin, actinase, carboxylase, proteases XIV, XXI and E, and collagenase IA. The final degradation products of silk fibroin are amino acids, which are easily absorbed *in vivo* (for a

*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*

mucoprotective agent, and also in wound healing preparations [49]. Pectin is known as a natural prophylactic substance against poisoning with toxic cations, and its hemostatic and curing effects are well-documented in healing ointments [50]. Pectin is degradable by enzymes produced by bacterial, fungal and plant cells, and not present in human tissues [51–53]. Thus, pectin is degradable, at least partly, only in the intestinal tract populated with bacteria. However, pectin is water-soluble and quickly dissolves in the water environment, including the tissues. Therefore, in order to increase its stability, it was combined with chitosan and TiO2 nanoparticles for wound dressing applications [50] or used for construction of composite chitosan-pectin scaffolds for skin tissue engineering. Blending chitosan with pectin markedly improved the mechanical properties of the scaffolds, such as their Young's modulus, strain at break and ultimate tensile strength, in comparison with pure chitosan scaffolds, although the proliferation of cells (i.e., fibroblasts) was slightly slower on pectincontaining scaffolds [54, 55]. The reason is that pectin does not contain cell binding domains. The cell adhesion on pectin nanofibers was markedly enhanced by oxidizing pectin with periodate to generate aldehyde groups, and then crosslinking the nanofibers with adipic acid dihydrazide to covalently connect pectin macromolecular chains with adipic acid dihydrazone linkers. In addition, the crosslinked pectin nanofibers exhibited excellent mechanical strength and enhanced body degradability [56].

Other polysaccharides explored for creation of nanofibrous scaffolds for skin tissue engineering and wound healing are gum tragacanth and gum arabic, both polysaccharides of plant origin, degradable by bacteria and fungi, for example, in soil [57, 58].

**Gum tragacanth** is a viscous water-soluble mixture of polysaccharides, mainly tragacanthin and bassorin. Tragacanthin dissolves to give a colloidal hydrosol. Bassorin, representing 60–70% of the gum, is insoluble and swells to a gel. Chemically, tragacanthin is a complex mixture of acidic polysaccharides containing D-galacturonic acid, D-galactose, L-fucose (6-deoyl-L-galactose), D-xylose, and L-arabinose. Bassorin is probably a methylated tragacanthin. A small amount of cellulose, starch, protein and ash are also present (https://colonygums.com/tragacanth). In order to improve electrospinning and mechanical properties of the gum tragacanth, it was combined with PVA and PCL [59]. Gum tragacanth is endowed with microbial resistance and wound healing activity, which was further enhanced by curcumin, a naturally occurring poly-phenolic compound with a broad range of favorable biological functions, including anti-cancer, anti-oxidant, anti-inflammatory, anti-infective, angiogenic, and healing properties [60].

**Gum arabic**, also known as gum acacia, is a complex and water-soluble mixture of glycoproteins and polysaccharides consisting mainly of arabinose and galactose. For skin tissue engineering, it was electrospun with PCL and also with zein, a storage plant protein [61].

**Silk fibroin** is a water-insoluble elastic protein present in silk fibers produced by larvae of *Bombyx mori* and some other moth of the *Saturniidae* family, such as *Antheraea assama, Antheraea mylitta*, and *Philosamia ricini* [62–64]. Silk fibroin occurs in the fibers together with sericin, a water-soluble serine-rich protein, which forms a glue-like layer coating two singular filaments of fibroin.

In biomaterial science, silk fibroin is considered to be degradable, but in mammalian organism, this degradation is long-lasting and can take more than 1 year. As a kind of biomaterial approved by the Food and Drug Administration (FDA) for medical use, silk is defined by United States Pharmacopeia as non-degradable for its negligible tensile strength loss *in vivo*. However, silk fibroin is susceptible to biological degradation by proteolytic enzymes such as chymotrypsin, actinase, carboxylase, proteases XIV, XXI and E, and collagenase IA. The final degradation products of silk fibroin are amino acids, which are easily absorbed *in vivo* (for a review, see [65]).

*Current and Future Aspects of Nanomedicine*

in combination with chitosan and tannic acid, it was used for fabrication of electrospun nanofibrous meshes promising for wound healing [37]. In combination with dextran and gelatin, pullulan was used for electrospinning of nanofibrous scaffolds promising for skin tissue engineering. These scaffolds, especially when crosslinked with trisodium trimetaphosphate, supported the adhesion and spreading of human

**Xylan** is a plant polysaccharide belonging to the group of hemicelluloses, that is, polymers often associated with cellulose. While cellulose is made of glucose units, hemicelluloses contain many different sugar monomers. Xylans are polysaccharides made of β-1,4-linked xylose (i.e., a pentose sugar) residues with side branches of α-arabinofuranose and α-glucuronic acids, which contribute to crosslinking of cellulose microfibrils and lignin through ferulic acid residues. Xylans are considered as relatively available and cost-effective natural materials for tissue engineering. Electrospun nanofibers containing beech-derived xylan and PVA were tested as potential dermal substitutes for skin tissue regeneration. These scaffolds provided a good support for the adhesion and proliferation of human foreskin fibroblasts and for production of collagen by these cells [39]. Bagasse xylan was also a component of hydrogels endowed with shape memory, namely carboxymethyl xylang-poly(acrylic acid) hydrogels, applicable in tissue engineering and biosensorics,

dermal fibroblasts and formation of actin cytoskeleton in these cells [38].

**Alginates,** for example, sodium alginate or calcium alginate, are salts of alginic acid, a linear polysaccharide composed of (1,4)-β-D-mannuronic acid and (1,3)-α-L-guluronic acid. Alginates are produced by various species of brown algae, and also by the bacterium *Pseudomonas aeruginosa*, a major pathogen found in the lungs of patients with cystic fibrosis. The structure of alginates is similar to glycosaminoglycans, an important component of ECM in human tissues including skin [41]. Alginates have a great ability to keep moisture in the wound site and to adhere to skin. However, alginates are poorly spinnable, and therefore, for skin tissue engineering and wound dressing applications, they were electrospun together with other polymers, such as PVA [41, 42] or PEO [43]. Poor mechanical properties of alginates have been compensated by the combination with chitosan [44] or PCL [44, 45]. In addition, alginates themselves are not adhesive for mammalian cells, which was compensated by their combination with collagen and gelatin, containing ligands for cell adhesion receptors [41]. Alginates were modified with a cell adhesive GRGDSP oligopeptide, which acts as ligand for integrin cell adhesion receptors [43]. Sodium alginate was used for attachment of arginine to the surface of chitosan nanofibers in order to increase healing capability of this wound dressing [46]. Alginate nanofibers supported by PCL were impregnated with an extract from *Spirulina*, a photosynthetic cyanobacterium producing bioactive molecules with anti-oxidant and anti-inflammatory effects [45]. Electrospun sodium alginate nanofibers containing silver nanoparticles were used for fabrication of an electronic skin capable of pressure sensing and endowed with antibacterial activity [47].

The degradability of alginate in human organism is limited. Alginate is naturally degraded by alginate lyases or alginate depolymerases, which have been isolated from marine algae, marine animals, bacteria, fungi, viruses, and other microorganisms, but are not present in the human organism. Degradability of alginate can be increased by its oxidation and at low pH. Also the hydrophilicity and water uptake capacity of

alginate can help in its removal from the wound site (for a review, see [48]). **Pectin** is a complex of structural polysaccharides present in the cell walls of terrestrial plants, rich in galacturonic acid. Pectin is known as gelling agent in food industry, but it is also widely used in medicine, for example, against digestive disorders, such as obstipation and diarrhea, for oral drug delivery, as a component of dietary fibers trapping cholesterol and carbohydrates, as a demulcent, that is, a

particularly for construction of electronic skin [40].

**74**

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 scaffolds [66].

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 adhesion receptors [64].

**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, which have been reported to be more pronounced in non-mulberry sericin (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 scaffolds [88].

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

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 poly-

**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]

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

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

any cytotoxicity, at least in a lower concentration of 0.1% w/w [97].

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

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

mers include chitosan, gellan gum, zein, and PHBV.

and from fish scale collagen peptides [90].

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