**4.2 Air-liquid interface**

In most experimental studies dealing with skin tissue engineering *in vitro*, keratinocytes are submerged in the cell culture media. However, in physiological skin *in vivo*, keratinocytes are exposed to air, at least their uppermost layer, i.e., *stratum corneum*, which is an impermeable barrier of cornified cell layers. Therefore, in advanced tissue engineering, keratinocytes should be exposed to the air-liquid interface (**Figure 6**) in order to achieve their phenotypic maturation and creation of the *stratum corneum* and the other epidermal layers, namely the basal, spinous, and granular layers [130].

The early differentiation of human amnion epithelial cells towards keratinocytes, manifested by formation desmosomes, was more pronounced in cells cultured at the air-liquid interface than in cells submerged in the culture medium [131]. In another study, human ADSCs were transdifferentiated towards keratinocytes in a medium containing retinoic acid, hydrocortisone, ascorbic acid, and bone morphogenetic protein-4 (BMP-4). This medium enabled high expression of pancytokeratin in conventional 2D cultures, especially if the cells were grown on type IV collagen. When the cell cultures were lifted to air-liquid interface, significant stratification was observed, particularly on growth supports coated with type IV collagen or fibronectin, and epidermal differentiation markers, such as involucrin and cytokeratins 1 and 14, were induced [132].

At the air-liquid interface, the keratinocytes or cells differentiating towards keratinocytes have been cultured on various substrates, e.g., acellular dermis [133], porous sponge-like gelatin scaffolds incorporated with chrondroitin-6-sulfate and hyaluronic acid [134], de-epithelialized human amniotic membrane [135], collagen IV and fibronectin [132] and fibrin in the form of layer [131], hydrogel or clot [136]. On the mentioned substrates, keratinocytes were grown either alone or in combination with fibroblasts submerged in the culture medium. In a study by Wang et al. [134], fibroblasts were grown inside the porous gelatin-based scaffolds submerged in the medium, while keratinocytes were grown on the top of the scaffolds, exposed to the air-liquid interface. Similarly, on the de-epithelialized amniotic membrane,

**Figure 6.**

*The principle of cell cultivation in a conventional cell culture system (A) and at the air-liquid interface (B).*

**47**

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

fibroblasts were cultured on the lower side of the membrane, submerged in the culture medium, while keratinocytes were grown on the upper side at the air-liquid interface [135]. In a study by Keck et al. [136], even a three-layered skin substitute was created. For the hypodermal layer, ADSCs and mature adipocytes were seeded within a fibrin hydrogel. On this layer, a fibrin clot with incorporated fibroblasts was placed for construction of the dermal layer. Keratinocytes were then added on the top of the two-layered construct and cultured at the air-liquid interface in order

Regarding the use of nanofibrous scaffolds for cultivation of keratinocytes at the air-liquid interface, synthetic and nature-derived scaffolds were used, namely electrospun PCL scaffolds [137, 138], electrospun polystyrene scaffolds [45] and fibrous sheets obtained after culturing human fibroblasts with ascorbic acid [139]. PCL scaffolds were used for construction of a three-dimensional *in vitro* skin model. The scaffolds were seeded with keratinocytes and melanocytes isolated from human scalp skin and cultured at the air-liquid interface. The keratinocytes contained a number of keratin fibrils and membrane-coated granules and formed a multilayered concentric structure, the surface of which became distinctly keratinized at the air-liquid interface. Cells with characteristic of melanocytes showed scattered distribution within the construct [137]. PCL scaffolds loaded with wound healing drugs, namely dexpanthenol and metyrapone, were used for a cell-based wound healing assay for rapid and predictive evaluation of wound therapeutics *in vitro*, using human HaCaT keratinocytes cultured at the air-liquid interface [138]. Interesting results were obtained on electrospun polystyrene scaffolds. In the absence of serum, keratinocytes, fibroblasts, and endothelial cells did not grow when cultured alone. However, when fibroblasts were cocultured with keratinocytes and endothelial cells, expansion of keratinocytes and endothelial cells occurred even in the absence of serum. Furthermore, the cells displayed native spatial three-dimensional organization when cultured at the air-liquid interface, even when all three cell types were introduced at random to the scaffolds [45]. The fibrous sheets produced by fibroblasts were used for creation of reconstructed human skin *in vitro*. After seeding the sheets with keratinocytes and the cell maturation *in vitro*, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiation markers and basement mem-

The cultivation of keratinocytes at the air-liquid interface was also combined with cultivation of these cells in dynamic bioreactors, which further improved their growth and phenotypic maturation. Uniaxial strain stress (deformation of the cultivation substrate by 5–20%) further enhanced proliferation and epidermal differentiation of keratinocytes cultured at the air-liquid interface on electrospun collagen scaffolds containing fibroblasts in comparison with keratinocytes on

Also the perfusion with cell culture media showed beneficial effects on tissueengineered skin constructs at the air-liquid interface. In a perfusion system with various growth supports for cells, such as acellular human dermis, Azowipes, electrospun polystyrene, and an electrospun composite of polystyrene and poly-dllactide fibers, human dermal fibroblast and endothelial cells showed greater viability under submerged conditions than at the air-liquid interface, whereas keratinocytes favored cultivation at the air-liquid interface. In addition, the viability of keratinocytes and fibroblasts was higher under continuous perfusion than under batch-feed perfusion, and on electrospun scaffolds than on acellular dermis [46]. In a recent study, a reconstructed skin model *in vitro*, containing a collagen matrix incorporated with fibroblasts and keratinocytes cultured at the air-liquid interface, was exposed to a continuous flow of cultivation medium (from 1.25 to

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

to create the epidermal layer [136].

brane proteins [139].

unstrained cell-material constructs [34].

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

fibroblasts were cultured on the lower side of the membrane, submerged in the culture medium, while keratinocytes were grown on the upper side at the air-liquid interface [135]. In a study by Keck et al. [136], even a three-layered skin substitute was created. For the hypodermal layer, ADSCs and mature adipocytes were seeded within a fibrin hydrogel. On this layer, a fibrin clot with incorporated fibroblasts was placed for construction of the dermal layer. Keratinocytes were then added on the top of the two-layered construct and cultured at the air-liquid interface in order to create the epidermal layer [136].

Regarding the use of nanofibrous scaffolds for cultivation of keratinocytes at the air-liquid interface, synthetic and nature-derived scaffolds were used, namely electrospun PCL scaffolds [137, 138], electrospun polystyrene scaffolds [45] and fibrous sheets obtained after culturing human fibroblasts with ascorbic acid [139].

PCL scaffolds were used for construction of a three-dimensional *in vitro* skin model. The scaffolds were seeded with keratinocytes and melanocytes isolated from human scalp skin and cultured at the air-liquid interface. The keratinocytes contained a number of keratin fibrils and membrane-coated granules and formed a multilayered concentric structure, the surface of which became distinctly keratinized at the air-liquid interface. Cells with characteristic of melanocytes showed scattered distribution within the construct [137]. PCL scaffolds loaded with wound healing drugs, namely dexpanthenol and metyrapone, were used for a cell-based wound healing assay for rapid and predictive evaluation of wound therapeutics *in vitro*, using human HaCaT keratinocytes cultured at the air-liquid interface [138].

Interesting results were obtained on electrospun polystyrene scaffolds. In the absence of serum, keratinocytes, fibroblasts, and endothelial cells did not grow when cultured alone. However, when fibroblasts were cocultured with keratinocytes and endothelial cells, expansion of keratinocytes and endothelial cells occurred even in the absence of serum. Furthermore, the cells displayed native spatial three-dimensional organization when cultured at the air-liquid interface, even when all three cell types were introduced at random to the scaffolds [45].

The fibrous sheets produced by fibroblasts were used for creation of reconstructed human skin *in vitro*. After seeding the sheets with keratinocytes and the cell maturation *in vitro*, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiation markers and basement membrane proteins [139].

The cultivation of keratinocytes at the air-liquid interface was also combined with cultivation of these cells in dynamic bioreactors, which further improved their growth and phenotypic maturation. Uniaxial strain stress (deformation of the cultivation substrate by 5–20%) further enhanced proliferation and epidermal differentiation of keratinocytes cultured at the air-liquid interface on electrospun collagen scaffolds containing fibroblasts in comparison with keratinocytes on unstrained cell-material constructs [34].

Also the perfusion with cell culture media showed beneficial effects on tissueengineered skin constructs at the air-liquid interface. In a perfusion system with various growth supports for cells, such as acellular human dermis, Azowipes, electrospun polystyrene, and an electrospun composite of polystyrene and poly-dllactide fibers, human dermal fibroblast and endothelial cells showed greater viability under submerged conditions than at the air-liquid interface, whereas keratinocytes favored cultivation at the air-liquid interface. In addition, the viability of keratinocytes and fibroblasts was higher under continuous perfusion than under batch-feed perfusion, and on electrospun scaffolds than on acellular dermis [46]. In a recent study, a reconstructed skin model *in vitro*, containing a collagen matrix incorporated with fibroblasts and keratinocytes cultured at the air-liquid interface, was exposed to a continuous flow of cultivation medium (from 1.25 to

*Applications of Nanobiotechnology*

tissue engineering" [129].

**4.2 Air-liquid interface**

granular layers [130].

and cytokeratins 1 and 14, were induced [132].

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

In most experimental studies dealing with skin tissue engineering *in vitro*, keratinocytes are submerged in the cell culture media. However, in physiological skin *in vivo*, keratinocytes are exposed to air, at least their uppermost layer, i.e., *stratum corneum*, which is an impermeable barrier of cornified cell layers. Therefore, in advanced tissue engineering, keratinocytes should be exposed to the air-liquid interface (**Figure 6**) in order to achieve their phenotypic maturation and creation of the *stratum corneum* and the other epidermal layers, namely the basal, spinous, and

The early differentiation of human amnion epithelial cells towards keratinocytes, manifested by formation desmosomes, was more pronounced in cells cultured at the air-liquid interface than in cells submerged in the culture medium [131]. In another study, human ADSCs were transdifferentiated towards keratinocytes in a medium containing retinoic acid, hydrocortisone, ascorbic acid, and bone morphogenetic protein-4 (BMP-4). This medium enabled high expression of pancytokeratin in conventional 2D cultures, especially if the cells were grown on type IV collagen. When the cell cultures were lifted to air-liquid interface, significant stratification was observed, particularly on growth supports coated with type IV collagen or fibronectin, and epidermal differentiation markers, such as involucrin

At the air-liquid interface, the keratinocytes or cells differentiating towards keratinocytes have been cultured on various substrates, e.g., acellular dermis [133], porous sponge-like gelatin scaffolds incorporated with chrondroitin-6-sulfate and hyaluronic acid [134], de-epithelialized human amniotic membrane [135], collagen IV and fibronectin [132] and fibrin in the form of layer [131], hydrogel or clot [136]. On the mentioned substrates, keratinocytes were grown either alone or in combination with fibroblasts submerged in the culture medium. In a study by Wang et al. [134], fibroblasts were grown inside the porous gelatin-based scaffolds submerged in the medium, while keratinocytes were grown on the top of the scaffolds, exposed to the air-liquid interface. Similarly, on the de-epithelialized amniotic membrane,

*The principle of cell cultivation in a conventional cell culture system (A) and at the air-liquid interface (B).*

**46**

**Figure 6.**

7.5 ml/h) at its basal side. Histological examination confirmed the formation of a significantly thicker *stratum corneum* compared to the control constructs cultivated under static conditions. Moreover, the keratinocyte differentiation markers involucrin and filaggrin, as well as the tight junction proteins claudin 1 and occludin, showed increased expression in the dynamically cultured skin models. However, the skin barrier function of the dynamically cultivated skin models was not enhanced compared with the skin models cultivated under static conditions [36]. Similar results were obtained in a study by Kalyanaraman et al. [140], performed on engineered skin substitutes based on collagen-glycosaminoglycan sponges, containing fibroblasts in their inside and keratinocytes on their surface, which were exposed to the air-liquid interface. Perfusion of these construct with the medium at the flow rate of 5 ml/min increased the metabolic activity of fibroblasts and maintained the epidermal barrier created by keratinocytes similarly as in static controls, while higher flow rates of 15 ml/min, and particularly 50 ml/ min, decreased the cell metabolic activity, increased the degradation of the scaffolds and decreased the epidermal barrier function, manifested by its increased hydration [140].
