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Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device

View Article: PubMed Central - PubMed

ABSTRACT

Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes.

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Morphological comparison of the regenerated epidermis under co-stimulation with mechanical stress and air-exposure.SEM images showing epidermis morphology of 3D skin cultured under non-stretch or intermittent-stretch conditions. (a,b) Collagen fiber (arrow) was covered with smooth epidermis of HaCaT under air-exposure only condition. (d,e) Collagen fiber was covered with tough stratum corneum of HaCaT differentiated under co-stimulation with intermittent stretch and air-exposure. (c,f) Epidermal layer was labeled using FITC-conjugated nanoparticles. (c) Nanoparticles were deposited into the epidermal layer but distinguishable granular layer was not detected. (f) Spinous or granular layers were confirmed, stained with FITC-labeled nanoparticles. Inside figure is the rat skin which was deposited with FITC-labeled nanoparticles. But, Nanoparticles also were not penetrated to the dermal layer of rat skin. (Bar = 10 μm (a,d), 100 μm (b,e) and 50 μm (c,f)).
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f2: Morphological comparison of the regenerated epidermis under co-stimulation with mechanical stress and air-exposure.SEM images showing epidermis morphology of 3D skin cultured under non-stretch or intermittent-stretch conditions. (a,b) Collagen fiber (arrow) was covered with smooth epidermis of HaCaT under air-exposure only condition. (d,e) Collagen fiber was covered with tough stratum corneum of HaCaT differentiated under co-stimulation with intermittent stretch and air-exposure. (c,f) Epidermal layer was labeled using FITC-conjugated nanoparticles. (c) Nanoparticles were deposited into the epidermal layer but distinguishable granular layer was not detected. (f) Spinous or granular layers were confirmed, stained with FITC-labeled nanoparticles. Inside figure is the rat skin which was deposited with FITC-labeled nanoparticles. But, Nanoparticles also were not penetrated to the dermal layer of rat skin. (Bar = 10 μm (a,d), 100 μm (b,e) and 50 μm (c,f)).

Mentions: The top surface of the reconstructed skin was observed by SEM to confirm the formation of the stratum corneum. Collagen matrix revealed a fibrous structure after gelation (Fig. 2a,d). A tougher stratum corneum with higher dimension was observed in intermittent stretch condition (IS) (Fig. 2d,e). To confirm if the dermal layer of the fibroblast is sufficiently concentrated to resist permeation of nanoparticles, it was topically treated with fluorescein isothiocyanate (FITC)-labeled nanoparticles. The chitosan coated-nanoparticles containing ceramide were around 186 nm (data not shown). As a result, fluorescence was detected only in the epidermal layer of HaCaT culture, but not in the dermal layer (Fig. 2c,f), which implied that the reconstructed dermal layer was highly concentrated due to which nanoparticles are unable to or rarely permeate it similar to the in vivo dermal layer (inside figure, Fig. 2f). In the epidermal layer under IS, the fluorescent image showed the distinguishable morphology of spinous or granular layers. This result implied that HaCaT was keratinized successfully. In contrast, cells under non-stretch condition (NS) did not reveal the characteristic and distinguishable layers.


Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device
Morphological comparison of the regenerated epidermis under co-stimulation with mechanical stress and air-exposure.SEM images showing epidermis morphology of 3D skin cultured under non-stretch or intermittent-stretch conditions. (a,b) Collagen fiber (arrow) was covered with smooth epidermis of HaCaT under air-exposure only condition. (d,e) Collagen fiber was covered with tough stratum corneum of HaCaT differentiated under co-stimulation with intermittent stretch and air-exposure. (c,f) Epidermal layer was labeled using FITC-conjugated nanoparticles. (c) Nanoparticles were deposited into the epidermal layer but distinguishable granular layer was not detected. (f) Spinous or granular layers were confirmed, stained with FITC-labeled nanoparticles. Inside figure is the rat skin which was deposited with FITC-labeled nanoparticles. But, Nanoparticles also were not penetrated to the dermal layer of rat skin. (Bar = 10 μm (a,d), 100 μm (b,e) and 50 μm (c,f)).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037429&req=5

f2: Morphological comparison of the regenerated epidermis under co-stimulation with mechanical stress and air-exposure.SEM images showing epidermis morphology of 3D skin cultured under non-stretch or intermittent-stretch conditions. (a,b) Collagen fiber (arrow) was covered with smooth epidermis of HaCaT under air-exposure only condition. (d,e) Collagen fiber was covered with tough stratum corneum of HaCaT differentiated under co-stimulation with intermittent stretch and air-exposure. (c,f) Epidermal layer was labeled using FITC-conjugated nanoparticles. (c) Nanoparticles were deposited into the epidermal layer but distinguishable granular layer was not detected. (f) Spinous or granular layers were confirmed, stained with FITC-labeled nanoparticles. Inside figure is the rat skin which was deposited with FITC-labeled nanoparticles. But, Nanoparticles also were not penetrated to the dermal layer of rat skin. (Bar = 10 μm (a,d), 100 μm (b,e) and 50 μm (c,f)).
Mentions: The top surface of the reconstructed skin was observed by SEM to confirm the formation of the stratum corneum. Collagen matrix revealed a fibrous structure after gelation (Fig. 2a,d). A tougher stratum corneum with higher dimension was observed in intermittent stretch condition (IS) (Fig. 2d,e). To confirm if the dermal layer of the fibroblast is sufficiently concentrated to resist permeation of nanoparticles, it was topically treated with fluorescein isothiocyanate (FITC)-labeled nanoparticles. The chitosan coated-nanoparticles containing ceramide were around 186 nm (data not shown). As a result, fluorescence was detected only in the epidermal layer of HaCaT culture, but not in the dermal layer (Fig. 2c,f), which implied that the reconstructed dermal layer was highly concentrated due to which nanoparticles are unable to or rarely permeate it similar to the in vivo dermal layer (inside figure, Fig. 2f). In the epidermal layer under IS, the fluorescent image showed the distinguishable morphology of spinous or granular layers. This result implied that HaCaT was keratinized successfully. In contrast, cells under non-stretch condition (NS) did not reveal the characteristic and distinguishable layers.

View Article: PubMed Central - PubMed

ABSTRACT

Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes.

No MeSH data available.


Related in: MedlinePlus