**3. Nanofibers from synthetic degradable polymers**

Synthetic degradable polymers have been used as scaffolds for skin tissue engineering, but also as wound dressing releasing various bioactive molecules by a controllable manner. Degradable polymers typically used in these applications are aliphatic polyesters, such as poly-ɛ-caprolactone (PCL), polylactide (PLA), and poly(lactide-*co*-glycolide) (PLGA). These polymers were approved by the Food and Drug Administration of the United States of America (FDA) for many medical applications.

Poly-ɛ-caprolactone (PCL) has been used most frequently from the mentioned polymers. It is a semi-crystalline polymer with tunable mechanical properties, and has a good solubility in a variety of solvents, and hence it can be combined with variety of other polymers. In comparison to other polyesters, PCL is a slowly degrading polymer, which can be essential for specific applications [52], and products of its degradation are non-toxic in the nature [53]. The acidic products of polyester degradation can affect the healing processes after implantation and may lead to inflammation [54]. However, due to the slow degradation of PCL, this risk is significantly lower compared to PLA and PLGA, which degrade significantly faster [55]. Slower degradation of PCL in comparison with its copolymer with PLA (PLCL) was also confirmed in our study, where both polymers were exposed to enzymatic degradation using lipase and proteinase K enzymes [13] (**Figure 2**).

However, PCL is more hydrophobic than PLA and particularly PLGA, and thus it is less supportive for cell adhesion. Therefore, PCL was rarely electrospun alone, i.e., without other polymers and bioactive additives. Nevertheless, pure PCL nanofibrous scaffolds were successfully used for cultivation and differentiation of hair follicle stem cells, isolated from the bulge regions of rat whiskers [56, 57]. In addition, pure PCL scaffolds supported the proliferation of mesenchymal stem cells, fibroblasts, and keratinocytes better than pure PVA scaffolds [12]. In spite of this, for purposes of skin regenerative therapies, PCL was usually combined with natural polymers, such as collagen, which was either blended with PCL before electrospinning [6, 58], or deposited on PCL nanofibers [59]. Gelatin, a collagen-derived protein, was either blended with PCL [60], or incorporated into core-shell PCL/gelatin nanofibers as the core polymer [22]. Gelatin was also electrospun independently of PCL using a double-nozzle technique, which resulted in creation of two types of nanofibers in the scaffolds, either mixed [61] or arranged in separate gelatin and PCL layers [27]. Multilayered and blend structures were found to fit most of native skin requirements in comparison with all the other mentioned structures [27].

Other natural polymers for modification of PCL nanofibers included whey protein [62], hyaluronic acid [24], keratin [28, 63], chitosan [28], fibrinogen [64], or gum arabic, and a corn protein zein [65]. These natural polymers were blended with PCL in the electrospinning solution. Polymer-modified PCL nanofibrous scaffolds have been often further modified with growth factors, such as epidermal growth factor (EGF) immobilized on PCL/collagen nanofibers [58] or on PCL/gelatin nanofibers [60] and transforming growth factor-β1 (TGF-β1) added into PCL/ collagen electrospinning solution [6].

Other bioactive substances used for incorporation into PCL-based nanofibers included medicinal herbs such as *Aloe vera* [61], lawsone, i.e., 2-hydroxy-1,4-naphthoquinone extracted from Henna, endowed with antimicrobial, antiparasitic,

**39**

PCL/collagen nanofibers [6].

**Figure 2.**

*10 μm.*

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic…*

anticancer, and antioxidant activities [22], other plant extracts with wound healing effects, e.g., from *Calendula officinalis* [65] or *Indigofera aspalathoides, Azadirachta indica, Memecylon edule*, *and Myristica andamanica* [66], molybdenum oxide

*Scanning electron microscopy analyses of electrospun PCL (A, B) and PLCL (C, D) in their intact state (upper row, A, C) and after 2 days of enzymatic degradation (lower row, B, D). Magnification 5000×, scale bar* 

nanoparticles for treating skin cancer [67] or antibiotics, which can be combined with PCL by various manners, e.g., through whey protein [62] or through micelles coating

In our experiments, PCL electrospun nanofibrous membranes were impregnated with alaptide or l-arginine. Alaptide is a spirocyclic dipeptide, which was designed as an analogue of melanocyte-stimulating hormone release-inhibiting factor (MIF) and synthesized by Kasafirek et al. at the Research Institute for Pharmacy and Biochemistry in Prague, Czechoslovakia, in the 1980s of the twentieth century [68]. Alaptide showed a great potential for regeneration of the injured skin and also for enhanced transdermal penetration of drugs [69, 70]. Arginine is amino acid which is a precursor of nitric oxide, implicated in wound healing. Arginine promoted re-epithelization and vascularization of wounds [71] and supported proliferative, antiapoptotic, and immune defense functions of fibroblasts [72]. In our study, concentrations up to 2.5 wt.% of alaptide and up to 10 wt.% of l-arginine were used for fabrication of the membranes. Normal human dermal fibroblasts (NHDF) were cultivated on the membranes for 7 days. Alaptide-containing membranes were fully colonized by the cells up to the highest alaptide concentration (2.5 wt.%). However,

*DOI: http://dx.doi.org/10.5772/intechopen.88744*

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic… DOI: http://dx.doi.org/10.5772/intechopen.88744*

#### **Figure 2.**

*Applications of Nanobiotechnology*

applications.

Another thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM) was used for fabrication of nanofibers for transdermal delivery of drugs, namely

levothyroxine (T4), which helps to reduce deposits of adipose tissue [51].

Synthetic degradable polymers have been used as scaffolds for skin tissue engineering, but also as wound dressing releasing various bioactive molecules by a controllable manner. Degradable polymers typically used in these applications are aliphatic polyesters, such as poly-ɛ-caprolactone (PCL), polylactide (PLA), and poly(lactide-*co*-glycolide) (PLGA). These polymers were approved by the Food and Drug Administration of the United States of America (FDA) for many medical

Poly-ɛ-caprolactone (PCL) has been used most frequently from the mentioned polymers. It is a semi-crystalline polymer with tunable mechanical properties, and has a good solubility in a variety of solvents, and hence it can be combined with variety of other polymers. In comparison to other polyesters, PCL is a slowly degrading polymer, which can be essential for specific applications [52], and products of its degradation are non-toxic in the nature [53]. The acidic products of polyester degradation can affect the healing processes after implantation and may lead to inflammation [54]. However, due to the slow degradation of PCL, this risk is significantly lower compared to PLA and PLGA, which degrade significantly faster [55]. Slower degradation of PCL in comparison with its copolymer with PLA (PLCL) was also confirmed in our study, where both polymers were exposed to enzymatic degradation using lipase and proteinase K enzymes [13] (**Figure 2**).

However, PCL is more hydrophobic than PLA and particularly PLGA, and thus it is less supportive for cell adhesion. Therefore, PCL was rarely electrospun alone, i.e., without other polymers and bioactive additives. Nevertheless, pure PCL nanofibrous scaffolds were successfully used for cultivation and differentiation of hair follicle stem cells, isolated from the bulge regions of rat whiskers [56, 57]. In addition, pure PCL scaffolds supported the proliferation of mesenchymal stem cells, fibroblasts, and keratinocytes better than pure PVA scaffolds [12]. In spite of this, for purposes of skin regenerative therapies, PCL was usually combined with natural polymers, such as collagen, which was either blended with PCL before electrospinning [6, 58], or deposited on PCL nanofibers [59]. Gelatin, a collagen-derived protein, was either blended with PCL [60], or incorporated into core-shell PCL/gelatin nanofibers as the core polymer [22]. Gelatin was also electrospun independently of PCL using a double-nozzle technique, which resulted in creation of two types of nanofibers in the scaffolds, either mixed [61] or arranged in separate gelatin and PCL layers [27]. Multilayered and blend structures were found to fit most of native skin requirements in comparison with all the other mentioned structures [27]. Other natural polymers for modification of PCL nanofibers included whey protein [62], hyaluronic acid [24], keratin [28, 63], chitosan [28], fibrinogen [64], or gum arabic, and a corn protein zein [65]. These natural polymers were blended with PCL in the electrospinning solution. Polymer-modified PCL nanofibrous scaffolds have been often further modified with growth factors, such as epidermal growth factor (EGF) immobilized on PCL/collagen nanofibers [58] or on PCL/gelatin nanofibers [60] and transforming growth factor-β1 (TGF-β1) added into PCL/

Other bioactive substances used for incorporation into PCL-based nanofibers included medicinal herbs such as *Aloe vera* [61], lawsone, i.e., 2-hydroxy-1,4-naphthoquinone extracted from Henna, endowed with antimicrobial, antiparasitic,

**3. Nanofibers from synthetic degradable polymers**

**38**

collagen electrospinning solution [6].

*Scanning electron microscopy analyses of electrospun PCL (A, B) and PLCL (C, D) in their intact state (upper row, A, C) and after 2 days of enzymatic degradation (lower row, B, D). Magnification 5000×, scale bar 10 μm.*

anticancer, and antioxidant activities [22], other plant extracts with wound healing effects, e.g., from *Calendula officinalis* [65] or *Indigofera aspalathoides, Azadirachta indica, Memecylon edule*, *and Myristica andamanica* [66], molybdenum oxide nanoparticles for treating skin cancer [67] or antibiotics, which can be combined with PCL by various manners, e.g., through whey protein [62] or through micelles coating PCL/collagen nanofibers [6].

In our experiments, PCL electrospun nanofibrous membranes were impregnated with alaptide or l-arginine. Alaptide is a spirocyclic dipeptide, which was designed as an analogue of melanocyte-stimulating hormone release-inhibiting factor (MIF) and synthesized by Kasafirek et al. at the Research Institute for Pharmacy and Biochemistry in Prague, Czechoslovakia, in the 1980s of the twentieth century [68]. Alaptide showed a great potential for regeneration of the injured skin and also for enhanced transdermal penetration of drugs [69, 70]. Arginine is amino acid which is a precursor of nitric oxide, implicated in wound healing. Arginine promoted re-epithelization and vascularization of wounds [71] and supported proliferative, antiapoptotic, and immune defense functions of fibroblasts [72]. In our study, concentrations up to 2.5 wt.% of alaptide and up to 10 wt.% of l-arginine were used for fabrication of the membranes. Normal human dermal fibroblasts (NHDF) were cultivated on the membranes for 7 days. Alaptide-containing membranes were fully colonized by the cells up to the highest alaptide concentration (2.5 wt.%). However,

the highest arginine concentration (10 wt.%) appeared as cytotoxic (**Figure 3**). One of the possible explanations is a dual effect of nitric oxide, which can act either as antioxidant or as oxidative agent [73].

Polylactide (PLA) is a polymer obtained by the ring-opening polymerization of lactide, i.e., cyclic dimer of lactic acid, as the monomer. The lactide has two enantiomers, namely l-lactide and d-lactide. Polymerization of each enantiomer alone results in creation of poly-l-lactide (PLLA) or poly-d-lactide (PDLA). Polymerization of a racemic mixture of l-lactide and d-lactide, i.e., mixture containing equal amounts of both enantiomers, gives rise of poly dl-lactide (PDLLA).

For skin tissue engineering, similarly as in PCL, PLA was often combined with other polymers and biologically active molecules in order to tailor desirable properties of the scaffolds. For example, for enhancing the cell adhesion on nanofibrous PLA scaffolds, PLA was blended and electrospun together with gelatin. Composite scaffolds containing PLA and gelatin in a ratio of 7:3 were more suitable for the attachment and viability of fibroblasts than the scaffolds made either of PLA or of gelatin alone [9]. Similarly, composite nanofibrous scaffolds made by electrospinning of a blend of poly-l-lactic acid/poly-(α,β)-dl-aspartic acid/collagen (PLLA/ PAA/Col I&III) increased the proliferation of adipose tissue-derived stem cells (ADSCs), i.e., an important cell type used in skin tissue engineering, in comparison with pure PLLA or PLLA/PAA scaffolds [74]. In our experiments, electrospun PLLA meshes were modified by additional coating with fibrin or collagen. Fibrin coating supported better the growth of dermal fibroblasts, while the growth of keratinocytes was better on collagen [10].

In another design of bilayer scaffolds for skin tissue engineering, PLLA in the form of microporous disc was combined with superficial chitosan/PCL nanofibrous mat. The disc was seeded with dermal fibroblasts, while the mat was used as

#### **Figure 3.**

*Normal human dermal fibroblasts cultivated for 7 days on PCL nanofibrous membrane impregnated with alaptide or arginine. A—0.1 wt.% of alaptide, B—2.5 wt.% of alaptide, C—1 wt.% of arginine, 10 wt.% of arginine. The cells were stained for nuclei (blue) and actin (red) using DNA-binding dye DAPI and phalloidin conjugated with TRITC. The images were acquired using Olympus IX71 fluorescence microscope equipped with DP71 camera and lens 10× (N.A. = 0.3).*

**41**

mice [79].

[13, 50].

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic…*

substrate for keratinocytes. The porous structure of the scaffolds allowed humoral communication of both cell types, but the nanofibers prevented the direct inter-

Other interesting application of PLA nanofibers was skin tissue engineering for the infected wound site. PLA solution was electrospun together with highly porous silver microparticles (AgMPs) or high surface area silver nanoparticles (AgNPs) and used as substrates for co-culture of human epidermal keratinocytes and *Staphylococcus aureus*. The scaffolds with AgMPs showed a higher and steadier release of silver ions and lower cytotoxicity towards keratinocytes than AgNPs-

PLA nanofibers have also been widely used for wound dressing applications, where they were loaded with various bioactive molecules improving wound healing and preventing microbial infection. Examples include PLLA/zein nanofibrous mats loaded with *Rana chensinensis* skin peptides with antibacterial and antioxidative activity [76], electrospun PLLA nanofibrous membranes coated by an *Aloe vera* gel [77], nanofibrillar matrices prepared from blends of PCL and PDLLA loaded with ciprofloxacin [78] or composite electrospun membranes containing polylactide:poly(vinyl pyrrolidone)/polylactide:poly(ethylene glycol) (PLA:PVP/ PLA:PEG) core/shell fibers, designed for treatment of burns and loaded with

In spite of all these encouraging results, PLA and PCL can elicit inflammatory response. Although inflammation is the first physiological stage of wound healing, followed by proliferation and remodeling, excessive inflammation can delay the wound healing and can lead to ulceration, fibrosis, scar formation or entering the wound into a chronic state [79, 80]. The inflammatory response to PLA and PCL was reduced in electrospun co-axial scaffolds containing nanofibers with bioactive gelatin shells and biodegradable synthetic cores of PLA and PCL [81]. Another approach was the incorporation of PLA scaffolds with anti-inflammatory drugs. PLA nanofibers with 20 wt.% of ibuprofen promoted the viability and proliferation of human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) *in vitro*, reduced wound contraction in mice *in vivo*, and when seeded with HEK and HDF, also enhanced new blood vessel formation in wounds of nude mice [80]. In a study by Yaru et al. [82], PLA nanofibers were incorporated with salicylate, a signaling molecule in plants, but also exhibiting a wide spectrum of signaling activities in mammals, including antithrombotic, anti-inflammatory, antineoplastic, and antimicrobial actions [83]. In addition, electrospun nanofibrous PDLLA scaffolds were incorporated with microalga *Spirulina*, which has anti-inflammatory, antioxidant, antimicrobial, antiallergenic, anticancer, and antidiabetic effects. The scaffolds were seeded with mesenchymal stem cells derived from mouse kidneys and used for treatment of the third degree burns in

PLA and PCL can be combined in a poly(l-lactic acid-*co*-ε-caprolactone) copolymer, P(LLA-CL), also referred to as PLACL [84–86], PLLCL [26] or PLCL

As mentioned above, blend nanofibers of P(LLA-CL) and PDEGMA were prepared for controlled drug and cell delivery [50]. P(LLA-CL) was also blended with gelatin [84], silk fibroin, vitamin E, and curcumin [85] or with silk fibroin, tetracycline, and ascorbic acid [86], which increased the proliferation of human dermal fibroblasts on these nanofibrous scaffolds and secretion of collagen by these cells. Co-axial nanofibers with P(LLA-CL)/gelatin shell and albumin core containing EGF, insulin, hydrocortisone, and retinoic acid supported proliferation and epidermal differentiation of ADSCs better than nanofibers prepared by a blend spinning of all mentioned components [26]. In combination with poloxamer (Pluronic) 123,

*DOI: http://dx.doi.org/10.5772/intechopen.88744*

curcumin and HHC36 antimicrobial peptides [8].

mingling of these cell types [29].

loaded scaffolds [75].

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic… DOI: http://dx.doi.org/10.5772/intechopen.88744*

substrate for keratinocytes. The porous structure of the scaffolds allowed humoral communication of both cell types, but the nanofibers prevented the direct intermingling of these cell types [29].

Other interesting application of PLA nanofibers was skin tissue engineering for the infected wound site. PLA solution was electrospun together with highly porous silver microparticles (AgMPs) or high surface area silver nanoparticles (AgNPs) and used as substrates for co-culture of human epidermal keratinocytes and *Staphylococcus aureus*. The scaffolds with AgMPs showed a higher and steadier release of silver ions and lower cytotoxicity towards keratinocytes than AgNPsloaded scaffolds [75].

PLA nanofibers have also been widely used for wound dressing applications, where they were loaded with various bioactive molecules improving wound healing and preventing microbial infection. Examples include PLLA/zein nanofibrous mats loaded with *Rana chensinensis* skin peptides with antibacterial and antioxidative activity [76], electrospun PLLA nanofibrous membranes coated by an *Aloe vera* gel [77], nanofibrillar matrices prepared from blends of PCL and PDLLA loaded with ciprofloxacin [78] or composite electrospun membranes containing polylactide:poly(vinyl pyrrolidone)/polylactide:poly(ethylene glycol) (PLA:PVP/ PLA:PEG) core/shell fibers, designed for treatment of burns and loaded with curcumin and HHC36 antimicrobial peptides [8].

In spite of all these encouraging results, PLA and PCL can elicit inflammatory response. Although inflammation is the first physiological stage of wound healing, followed by proliferation and remodeling, excessive inflammation can delay the wound healing and can lead to ulceration, fibrosis, scar formation or entering the wound into a chronic state [79, 80]. The inflammatory response to PLA and PCL was reduced in electrospun co-axial scaffolds containing nanofibers with bioactive gelatin shells and biodegradable synthetic cores of PLA and PCL [81]. Another approach was the incorporation of PLA scaffolds with anti-inflammatory drugs. PLA nanofibers with 20 wt.% of ibuprofen promoted the viability and proliferation of human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) *in vitro*, reduced wound contraction in mice *in vivo*, and when seeded with HEK and HDF, also enhanced new blood vessel formation in wounds of nude mice [80]. In a study by Yaru et al. [82], PLA nanofibers were incorporated with salicylate, a signaling molecule in plants, but also exhibiting a wide spectrum of signaling activities in mammals, including antithrombotic, anti-inflammatory, antineoplastic, and antimicrobial actions [83]. In addition, electrospun nanofibrous PDLLA scaffolds were incorporated with microalga *Spirulina*, which has anti-inflammatory, antioxidant, antimicrobial, antiallergenic, anticancer, and antidiabetic effects. The scaffolds were seeded with mesenchymal stem cells derived from mouse kidneys and used for treatment of the third degree burns in mice [79].

PLA and PCL can be combined in a poly(l-lactic acid-*co*-ε-caprolactone) copolymer, P(LLA-CL), also referred to as PLACL [84–86], PLLCL [26] or PLCL [13, 50].

As mentioned above, blend nanofibers of P(LLA-CL) and PDEGMA were prepared for controlled drug and cell delivery [50]. P(LLA-CL) was also blended with gelatin [84], silk fibroin, vitamin E, and curcumin [85] or with silk fibroin, tetracycline, and ascorbic acid [86], which increased the proliferation of human dermal fibroblasts on these nanofibrous scaffolds and secretion of collagen by these cells. Co-axial nanofibers with P(LLA-CL)/gelatin shell and albumin core containing EGF, insulin, hydrocortisone, and retinoic acid supported proliferation and epidermal differentiation of ADSCs better than nanofibers prepared by a blend spinning of all mentioned components [26]. In combination with poloxamer (Pluronic) 123,

*Applications of Nanobiotechnology*

antioxidant or as oxidative agent [73].

keratinocytes was better on collagen [10].

the highest arginine concentration (10 wt.%) appeared as cytotoxic (**Figure 3**). One of the possible explanations is a dual effect of nitric oxide, which can act either as

Polylactide (PLA) is a polymer obtained by the ring-opening polymerization of lactide, i.e., cyclic dimer of lactic acid, as the monomer. The lactide has two enantiomers, namely l-lactide and d-lactide. Polymerization of each enantiomer alone results in creation of poly-l-lactide (PLLA) or poly-d-lactide (PDLA). Polymerization of a racemic mixture of l-lactide and d-lactide, i.e., mixture containing equal amounts of both enantiomers, gives rise of poly dl-lactide (PDLLA). For skin tissue engineering, similarly as in PCL, PLA was often combined with other polymers and biologically active molecules in order to tailor desirable properties of the scaffolds. For example, for enhancing the cell adhesion on nanofibrous PLA scaffolds, PLA was blended and electrospun together with gelatin. Composite scaffolds containing PLA and gelatin in a ratio of 7:3 were more suitable for the attachment and viability of fibroblasts than the scaffolds made either of PLA or of gelatin alone [9]. Similarly, composite nanofibrous scaffolds made by electrospinning of a blend of poly-l-lactic acid/poly-(α,β)-dl-aspartic acid/collagen (PLLA/ PAA/Col I&III) increased the proliferation of adipose tissue-derived stem cells (ADSCs), i.e., an important cell type used in skin tissue engineering, in comparison with pure PLLA or PLLA/PAA scaffolds [74]. In our experiments, electrospun PLLA meshes were modified by additional coating with fibrin or collagen. Fibrin coating supported better the growth of dermal fibroblasts, while the growth of

In another design of bilayer scaffolds for skin tissue engineering, PLLA in the form of microporous disc was combined with superficial chitosan/PCL nanofibrous mat. The disc was seeded with dermal fibroblasts, while the mat was used as

*Normal human dermal fibroblasts cultivated for 7 days on PCL nanofibrous membrane impregnated with alaptide or arginine. A—0.1 wt.% of alaptide, B—2.5 wt.% of alaptide, C—1 wt.% of arginine, 10 wt.% of arginine. The cells were stained for nuclei (blue) and actin (red) using DNA-binding dye DAPI and phalloidin conjugated with TRITC. The images were acquired using Olympus IX71 fluorescence microscope equipped with* 

**40**

**Figure 3.**

*DP71 camera and lens 10× (N.A. = 0.3).*

P(LLA-CL) was also used for electrospinning of nanofibrous scaffolds for direct delivery of ADSCs into wounds in order to promote their healing [87].

In our experiments, composite PCL/PLCL nanofibers were coated either with platelet lysate, or with platelet lysate incorporated in fibrin assemblies [88] in various concentrations. Results for human keratinocytes (HaCaT cells) indicated that the presence of platelet lysate increased the metabolic activity and phenotypic maturation of keratinocytes. The best results were observed when the nanofibers were coated with fibrin together with platelet lysate (**Figure 4**).

Poly(lactide-*co*-glycolide) (PLGA) is a copolymer obtained by the ring-opening co-polymerization of two different monomers, i.e., lactic acid and glycolic acid. In skin regenerative therapies, it was applied for both skin tissue engineering and wound dressing. For these applications, PLGA was combined with various natural and synthetic polymers and bioactive compounds. For example, using bovine serum albumin as a carrier protein, vitamin C, vitamin D3, hydrocortisone, insulin, triiodothyronine, and EGF were simultaneously blend-spun into PLGA-collagen nanofibers. All these factors concertedly increased proliferation of fibroblasts and keratinocytes, while maintaining the keratinocyte basal state. In addition, vitamin C maintained its ability to facilitate secretion of type I collagen by fibroblasts, EGF stimulated proliferation of skin fibroblasts, and insulin potentiated adipogenic differentiation of fibroblasts [11]. In PLGA nanofibers, EGF was also combined with the local anesthetic lidocaine in order to accelerate wound healing in a rat model [89]. Coating PLGA nanofibers with a self-assembled complex of poly(ethylene argininyl aspartate diglyceride) polycation, heparin, and cargo growth factors, i.e., vascular endothelial growth factor (VEGF) and/or transforming growth factor-beta3 (TGF-β3), enhanced proliferation of human dermal fibroblasts and formation of tubular structures from human umbilical vein endothelial cells *in vitro*. In addition, these nanofibers reduced necrosis, improved vascularization, and maintained well-composed skin appendages in a mouse skin flap model *in vivo* [25]. Growth factors, namely recombinant human EGF and recombinant human basic fibroblast growth factor (bFGF), were also encapsulated in PLGA microspheres and loaded into hybrid scaffolds of PLGA and polyethylene oxide [90]. Both growth factors had a synergistic effect on the proliferation of human skin fibroblasts and increased the expression of genes for collagen and elastin in these cells [90]. Composite nanofibrous membranes containing PLGA and cellulose nanocrystals and loaded with neurotensin accelerated healing of full-thickness skin wounds in spontaneously diabetic mice [91]. Nanofibers created by electrospinning the dispersion composed of polyethyleneimine-carboxymethyl chitosan/pDNA-angiogenin

#### **Figure 4.**

*Immunofluorescence staining of cytokeratin 10 (green), cytokeratin 14 (red), and nuclei (blue) of HaCaT cells after 7 days in culture grown on PCL/PLCL nanofibers. Cell on nanofibers without coating (A), coated with platelet lysate (B) and coated with fibrin assemblies with platelet lysate (C) are shown. Leica TCS SPE DM2500 confocal microscope.*

**43**

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic…*

nanoparticles, curcumin, PLGA, and cellulose nanocrystals showed antimicrobial and regenerative effects when transplanted into the infected full-thickness burn

The PLGA nanofibers were also modified with ECM components. In a study by Shtrichman et al. [93], the PLGA nanofibrous scaffolds were modified with ECM deposited on these scaffolds by mesenchymal progenitor cells, derived from human embryonic stem cells, and human induced pluripotent stem cells, originating from hair follicle keratinocytes, which were cultured on the scaffolds and removed by subsequent decellularization. Subcutaneous implantation of the ECM-modified scaffolds in rats then showed that this stem cell-derived construct is biocompatible, biodegradable, and holds great potential for tissue regeneration applications. In addition, ECM-derived proteins, such as collagen and gelatin, can be electrospun directly together with PLGA [94]. In our earlier study, PLGA nanofibers were modified with fibrin or collagen in a similar manner as PLLA [10]. The morphology of these coatings, and also the behavior of HaCaT keratinocytes and human dermal fibroblasts on the coated and uncoated nanofibers, were similar

PLGA-based nanofibrous meshes were also used for treatment of skin fibrosis and keloids, formed by abnormal proliferation of scar tissue at the site of cutaneous injury. Composite nanofibers of PLGA and poly(vinyl alcohol) loaded with kynurenine, a tryptophan metabolite, improved the dermal fibrosis in a rat model [95]. PLGA nanofibers releasing dexamethasone and green tea polyphenols significantly induced the degradation of collagen fibers in keloids on the back of nude mice [96]. Last but not least, PLGA nanofibers were also explored for transdermal delivery

of drugs with poor oral absorption and limited bioavailability, e.g., Daidzein, a promising candidate for treating cardiovascular and cerebrovascular diseases [97],

for skin regenerative therapies is poly(ethylene glycol) (PEG), also known as poly(ethylene oxide) (PEO), depending on its molecular weight. PEG usually refers to polymers with a molecular mass below 20,000 g/mol, while PEO refers to

Another important degradable polymer for fabrication of nanofibrous meshes

In nanofibrous scaffolds, PEG or PEO have been usually used as auxiliary components improving electrospinnability, mechanical properties, and wettability of other polymers. For example, PEO was used to enable electrospinning of casein (i.e., a protein extensively used for drug delivery), which does not possess sufficient viscoelasticity due to its extensive intermolecular interactions [99], or to improve the electrospinnability and mechanical properties of silk fibroin [100]. As mentioned above, PEO or PEG was electrospun together with PMMA for creation of nanofibers delivering kynurenic acid [95] or with PLGA for delivery of human recombinant EGF and bFGF [90]. Other interesting applications of PEO include creation of electrospun carboxymethylcellulose/PEO nanofibers for delivery of viable commensal bacteria for preventive diabetic foot treatment [101], creation of three-dimensional scaffolds composed of PCL-PEG-PCL tri-block copolymer and iron oxide (Fe3O4) nanoparticles for skin tissue engineering [102], creation of biodegradable nanofiber mats based on thermoresponsive multiblock poly(ester urethane)s comprising PEG, poly(propylene glycol) (PPG), and PCL, which showed improved hydrolytic degradation compared to pure PCL and excellent adhesion of human dermal fibroblasts [103]. The adhesion and growth of fibroblast were also improved after combination of PLCL with Pluronic, i.e., a copolymer of PEO and poly(propylene oxide) (PPO)

or for local delivery of anticancer drugs (for a review, see [98]).

polymers with a molecular mass above 20,000 g/mol.

arranged in a tri-block PEO-PPO-PEO structure [104].

Other auxiliary polymers used for creation of nanofibrous scaffolds are poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP). In some studies,

*DOI: http://dx.doi.org/10.5772/intechopen.88744*

wounds in rats [92].

on PLGA and PLLA [10].

*Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic… DOI: http://dx.doi.org/10.5772/intechopen.88744*

nanoparticles, curcumin, PLGA, and cellulose nanocrystals showed antimicrobial and regenerative effects when transplanted into the infected full-thickness burn wounds in rats [92].

The PLGA nanofibers were also modified with ECM components. In a study by Shtrichman et al. [93], the PLGA nanofibrous scaffolds were modified with ECM deposited on these scaffolds by mesenchymal progenitor cells, derived from human embryonic stem cells, and human induced pluripotent stem cells, originating from hair follicle keratinocytes, which were cultured on the scaffolds and removed by subsequent decellularization. Subcutaneous implantation of the ECM-modified scaffolds in rats then showed that this stem cell-derived construct is biocompatible, biodegradable, and holds great potential for tissue regeneration applications. In addition, ECM-derived proteins, such as collagen and gelatin, can be electrospun directly together with PLGA [94]. In our earlier study, PLGA nanofibers were modified with fibrin or collagen in a similar manner as PLLA [10]. The morphology of these coatings, and also the behavior of HaCaT keratinocytes and human dermal fibroblasts on the coated and uncoated nanofibers, were similar on PLGA and PLLA [10].

PLGA-based nanofibrous meshes were also used for treatment of skin fibrosis and keloids, formed by abnormal proliferation of scar tissue at the site of cutaneous injury. Composite nanofibers of PLGA and poly(vinyl alcohol) loaded with kynurenine, a tryptophan metabolite, improved the dermal fibrosis in a rat model [95]. PLGA nanofibers releasing dexamethasone and green tea polyphenols significantly induced the degradation of collagen fibers in keloids on the back of nude mice [96].

Last but not least, PLGA nanofibers were also explored for transdermal delivery of drugs with poor oral absorption and limited bioavailability, e.g., Daidzein, a promising candidate for treating cardiovascular and cerebrovascular diseases [97], or for local delivery of anticancer drugs (for a review, see [98]).

Another important degradable polymer for fabrication of nanofibrous meshes for skin regenerative therapies is poly(ethylene glycol) (PEG), also known as poly(ethylene oxide) (PEO), depending on its molecular weight. PEG usually refers to polymers with a molecular mass below 20,000 g/mol, while PEO refers to polymers with a molecular mass above 20,000 g/mol.

In nanofibrous scaffolds, PEG or PEO have been usually used as auxiliary components improving electrospinnability, mechanical properties, and wettability of other polymers. For example, PEO was used to enable electrospinning of casein (i.e., a protein extensively used for drug delivery), which does not possess sufficient viscoelasticity due to its extensive intermolecular interactions [99], or to improve the electrospinnability and mechanical properties of silk fibroin [100]. As mentioned above, PEO or PEG was electrospun together with PMMA for creation of nanofibers delivering kynurenic acid [95] or with PLGA for delivery of human recombinant EGF and bFGF [90]. Other interesting applications of PEO include creation of electrospun carboxymethylcellulose/PEO nanofibers for delivery of viable commensal bacteria for preventive diabetic foot treatment [101], creation of three-dimensional scaffolds composed of PCL-PEG-PCL tri-block copolymer and iron oxide (Fe3O4) nanoparticles for skin tissue engineering [102], creation of biodegradable nanofiber mats based on thermoresponsive multiblock poly(ester urethane)s comprising PEG, poly(propylene glycol) (PPG), and PCL, which showed improved hydrolytic degradation compared to pure PCL and excellent adhesion of human dermal fibroblasts [103]. The adhesion and growth of fibroblast were also improved after combination of PLCL with Pluronic, i.e., a copolymer of PEO and poly(propylene oxide) (PPO) arranged in a tri-block PEO-PPO-PEO structure [104].

Other auxiliary polymers used for creation of nanofibrous scaffolds are poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP). In some studies,

*Applications of Nanobiotechnology*

P(LLA-CL) was also used for electrospinning of nanofibrous scaffolds for direct

In our experiments, composite PCL/PLCL nanofibers were coated either with platelet lysate, or with platelet lysate incorporated in fibrin assemblies [88] in various concentrations. Results for human keratinocytes (HaCaT cells) indicated that the presence of platelet lysate increased the metabolic activity and phenotypic maturation of keratinocytes. The best results were observed when the nanofibers

Poly(lactide-*co*-glycolide) (PLGA) is a copolymer obtained by the ring-opening co-polymerization of two different monomers, i.e., lactic acid and glycolic acid. In skin regenerative therapies, it was applied for both skin tissue engineering and wound dressing. For these applications, PLGA was combined with various natural and synthetic polymers and bioactive compounds. For example, using bovine serum albumin as a carrier protein, vitamin C, vitamin D3, hydrocortisone, insulin, triiodothyronine, and EGF were simultaneously blend-spun into PLGA-collagen nanofibers. All these factors concertedly increased proliferation of fibroblasts and keratinocytes, while maintaining the keratinocyte basal state. In addition, vitamin C maintained its ability to facilitate secretion of type I collagen by fibroblasts, EGF stimulated proliferation of skin fibroblasts, and insulin potentiated adipogenic differentiation of fibroblasts [11]. In PLGA nanofibers, EGF was also combined with the local anesthetic lidocaine in order to accelerate wound healing in a rat model [89]. Coating PLGA nanofibers with a self-assembled complex of poly(ethylene argininyl aspartate diglyceride) polycation, heparin, and cargo growth factors, i.e., vascular endothelial growth factor (VEGF) and/or transforming growth factor-beta3 (TGF-β3), enhanced proliferation of human dermal fibroblasts and formation of tubular structures from human umbilical vein endothelial cells *in vitro*. In addition, these nanofibers reduced necrosis, improved vascularization, and maintained well-composed skin appendages in a mouse skin flap model *in vivo* [25]. Growth factors, namely recombinant human EGF and recombinant human basic fibroblast growth factor (bFGF), were also encapsulated in PLGA microspheres and loaded into hybrid scaffolds of PLGA and polyethylene oxide [90]. Both growth factors had a synergistic effect on the proliferation of human skin fibroblasts and increased the expression of genes for collagen and elastin in these cells [90]. Composite nanofibrous membranes containing PLGA and cellulose nanocrystals and loaded with neurotensin accelerated healing of full-thickness skin wounds in spontaneously diabetic mice [91]. Nanofibers created by electrospinning the dispersion composed of polyethyleneimine-carboxymethyl chitosan/pDNA-angiogenin

*Immunofluorescence staining of cytokeratin 10 (green), cytokeratin 14 (red), and nuclei (blue) of HaCaT cells after 7 days in culture grown on PCL/PLCL nanofibers. Cell on nanofibers without coating (A), coated with platelet lysate (B) and coated with fibrin assemblies with platelet lysate (C) are shown. Leica TCS SPE* 

delivery of ADSCs into wounds in order to promote their healing [87].

were coated with fibrin together with platelet lysate (**Figure 4**).

**42**

**Figure 4.**

*DM2500 confocal microscope.*

PVA is regarded as hydrolytically degradable [17, 105], while in other studies, it is considered non-degradable [106]. PVP has been reported to be hydrolytically degradable [105]. In addition, both PVA and PVP are hydrophilic and water soluble, and thus they can be removed from a composite polymeric mesh in water environment. This property of PVA, PVP, and also of PEG or PEO, can be used for creation of so-called "sacrificial fibers" in order to enlarge the pores in nanofibrous scaffolds for penetration of cells [107]; for a review, see [108, 109] or for tailoring the appropriate surface roughness of nanofibers inside the scaffolds. For example, PLLA was electrospun together with PVP in increasing concentrations, and after subsequent etching of PVP from the scaffolds in water environment, nano- and microfibers with increasing nanoscale surface roughness were obtained. Higher surface nanoroughness and porosity of PLLA fibers increased their hydrophilicity and their colonization with human dermal fibroblasts [32]. Other applications of PVA and PVP are similar to those of PEG or PEO, i.e., to increase spinnability of poorly spinnable substances used for skin regenerative therapies. For this purpose, PVA was combined with polysaccharides, such as gum tragacanth [110] or Schizophyllan [111], and PVP with *Aloe vera* [112]. Both PVA and PVP have been used to improve mechanical properties, wettability, and attractiveness for cell adhesion of various synthetic and natural polymers, particularly PCL [110] and chitosan [1]. PVA was used as emulsifier in fabrication of blended electrospun PLGA/chitosan nanofibers for potential skin reconstruction [113]. PVA and particularly PVP are important components of nanofibers delivering various biomolecules and drugs into skin, such as antibiotics (PVA [49], PVP [114]), kynurenine (PVA [48]), curcumin and HHC36 antimicrobial peptides (PVP [8]) or antimicrobial suberin fatty acids isolated from outer birch bark (PVP [115]). PVA and PVP were combined in electrospun nanofibrous membranes designed for sustained release of the antibiotic ciprofloxacin into wounds [116]. Nanofibers with cellulose acetate (CA) as the core material and PVP solution as the shell material were used for transdermal delivery of artemisinin, a potent antimalarial drug, which was incorporated into CA [117].
