**4. Advanced skin tissue engineering**

### **4.1 Dynamic bioreactors**

Tissue engineering in general, including skin tissue engineering, can be markedly improved by cultivation of cell-material constructs in dynamic bioreactors. These systems not only improve the supply of oxygen and nutrients to cells and waste removal, but also mechanically stimulate the cells with positive effects on their growth, differentiation, and phenotypic maturation.

First of all, the cell seeding can be improved in dynamic systems. In a study by Vitacolonna et al. [118], various methods of seeding fibroblasts on acellular dermal matrix were compared, namely static cell seeding after previous degassing of the matrix using a low-pressure syringe system, orbital shaker seeding, centrifugal seeding, and their combinations. Centrifugal seeding combined with matrix degassing significantly increased the seeding efficiency and homogeneity compared to the other methods.

Also the subsequent proliferation and other performance of cells can be markedly influenced by the dynamic cultivation. For example, human epidermal stem cells cultured on microcarriers in a rotary bioreactor exhibited higher proliferation and viability than the cells cultured in static conditions [119]. Human fibroblasts on nanofibrous poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffolds, subjected to biaxial distension for periods of time in a dynamic bioreactor,

**45**

**Figure 5.**

*coated silicone membrane.*

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

developed elastin fibers, whereas the cells on the same scaffolds cultured under static conditions showed negligible elastin production [120]. Cyclic uniaxial stretching of human HaCaT keratinocytes on collagen-silicon sheets induced the production of metalloproteinase 9 (MMP-9), a proteolytic enzyme necessary for keratinocyte migration, in these cells [121]. Strain also improved the mechanical strength of an engineered skin containing electrospun collagen scaffolds, human dermal fibroblasts, and epidermal keratinocytes, which was probably a result of enhanced epidermal cell proliferation, differentiation, and increased ECM production [34]. The keratinocyte differentiation under mechanical tension can be attributed to up-regulation of h2-calponin, which associates with actin stress fibers and decreases the cell proliferation rate (for a review, see [122]). Another type of mechanical stimulation implicated in keratinocyte differentiation is pressure stress, which increases the concentration of intracellular calcium, a stimulator of keratino-

In our experiments, we have developed a custom perfusion dynamic culture system allowing cell cultivation on elastic silicone membranes and generating cyclic pressure stress. First, these membranes were treated with plasma in order to increase their wettability and their ability to attach thin films made of nanofibrillar cellulose [124]. Afterwards, the porcine adipose tissue-derived stem cells were seeded on this surface. After 7 days of mechanical stimulation, a multilayered cell structure was observed in dynamic conditions, whereas in static conditions, only a

Increased concentration of calcium in keratinocytes and their differentiation can be also achieved by other means than mechanical stimulation, namely by stimulation with laser beam [125] or monodirectional pulsed electric current [126]. Electrical stimulation also enhanced the migration and proliferation of fibroblasts,

*Color-coded projection of porcine adipose tissue-derived stem cells cultivated on thin nanocellulose film structure in static (left) and dynamic conditions (middle). Fluorescence staining of nuclei (DAPI) and F-actin (Phalloidin). Right: Custom built culture chambers creating controlled mechanical stress and strain with perfusion. Below, formation of a multilayered structure of cells creating opaque layer on the nanocellulose-*

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

cyte differentiation [35, 123].

cell monolayer was formed (**Figure 5**).

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

developed elastin fibers, whereas the cells on the same scaffolds cultured under static conditions showed negligible elastin production [120]. Cyclic uniaxial stretching of human HaCaT keratinocytes on collagen-silicon sheets induced the production of metalloproteinase 9 (MMP-9), a proteolytic enzyme necessary for keratinocyte migration, in these cells [121]. Strain also improved the mechanical strength of an engineered skin containing electrospun collagen scaffolds, human dermal fibroblasts, and epidermal keratinocytes, which was probably a result of enhanced epidermal cell proliferation, differentiation, and increased ECM production [34]. The keratinocyte differentiation under mechanical tension can be attributed to up-regulation of h2-calponin, which associates with actin stress fibers and decreases the cell proliferation rate (for a review, see [122]). Another type of mechanical stimulation implicated in keratinocyte differentiation is pressure stress, which increases the concentration of intracellular calcium, a stimulator of keratinocyte differentiation [35, 123].

In our experiments, we have developed a custom perfusion dynamic culture system allowing cell cultivation on elastic silicone membranes and generating cyclic pressure stress. First, these membranes were treated with plasma in order to increase their wettability and their ability to attach thin films made of nanofibrillar cellulose [124]. Afterwards, the porcine adipose tissue-derived stem cells were seeded on this surface. After 7 days of mechanical stimulation, a multilayered cell structure was observed in dynamic conditions, whereas in static conditions, only a cell monolayer was formed (**Figure 5**).

Increased concentration of calcium in keratinocytes and their differentiation can be also achieved by other means than mechanical stimulation, namely by stimulation with laser beam [125] or monodirectional pulsed electric current [126]. Electrical stimulation also enhanced the migration and proliferation of fibroblasts,

#### **Figure 5.**

*Applications of Nanobiotechnology*

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].

Tissue engineering in general, including skin tissue engineering, can be markedly improved by cultivation of cell-material constructs in dynamic bioreactors. These systems not only improve the supply of oxygen and nutrients to cells and waste removal, but also mechanically stimulate the cells with positive effects on

First of all, the cell seeding can be improved in dynamic systems. In a study by Vitacolonna et al. [118], various methods of seeding fibroblasts on acellular dermal matrix were compared, namely static cell seeding after previous degassing of the matrix using a low-pressure syringe system, orbital shaker seeding, centrifugal seeding, and their combinations. Centrifugal seeding combined with matrix degassing significantly increased the seeding efficiency and homogeneity compared to

Also the subsequent proliferation and other performance of cells can be markedly influenced by the dynamic cultivation. For example, human epidermal stem cells cultured on microcarriers in a rotary bioreactor exhibited higher proliferation and viability than the cells cultured in static conditions [119]. Human fibroblasts on nanofibrous poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffolds, subjected to biaxial distension for periods of time in a dynamic bioreactor,

**4. Advanced skin tissue engineering**

their growth, differentiation, and phenotypic maturation.

**4.1 Dynamic bioreactors**

the other methods.

**44**

*Color-coded projection of porcine adipose tissue-derived stem cells cultivated on thin nanocellulose film structure in static (left) and dynamic conditions (middle). Fluorescence staining of nuclei (DAPI) and F-actin (Phalloidin). Right: Custom built culture chambers creating controlled mechanical stress and strain with perfusion. Below, formation of a multilayered structure of cells creating opaque layer on the nanocellulosecoated silicone membrane.*

expression of ECM proteins in these cells, and differentiation of these cells towards myofibroblasts, i.e., processes critical for wound healing [127]. The positive effect of electrical current on fibroblasts can be further combined with light stimulation of the fibroblast proliferation, e.g., on nanofibrous PCL scaffolds electrospun with a semiconductive polymer, namely poly(N,N-bis(2-octyldodecyl)-3,6-di(thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione-alt-thieno[3,2-b]thiophene) (PDBTT), subjected to the illumination from a red light-emitting diode [128]. Also magnetic stimulation can be effectively used in skin tissue engineering. For example, multilayered sheets of keratinocytes were obtained by cultivation of keratinocytes loaded with magnetite cationic liposomes in a magnetic field. After removal of the magnet, the sheets were released from the cultivation plates, and were harvested with a magnet. This technology was termed "magnetic force-based tissue engineering" [129].
