Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Aug 28;2(32):5256-5264.
doi: 10.1039/C4TB00614C.

A simple material model to generate epidermal and dermal layers in vitro for skin regeneration

Ching-Ting Tsao  1 , Matthew Leung  1 , Julia Yu-Fong Chang  2 , Miqin Zhang  1
Affiliations

A simple material model to generate epidermal and dermal layers in vitro for skin regeneration

Ching-Ting Tsao et al. J Mater Chem B. .

Abstract

There is an urgent need for a rationally-designed, cellularized skin graft capable of reproducing the micro-environmental cues necessary to promote skin healing and regeneration. To address this need, we developed a composite scaffold, namely, CA/C-PEG, composing of a porous chitosan-alginate (CA) structure impregnated with a thermally reversible chitosan-poly(ethylene glycol) (C-PEG) gel to incorporate skin cells as a bi-layered skin equivalent. Fibroblasts were encapsulated in C-PEG to simulate the dermal layer while the keratinocytes were seeded on the top of CA/C-PEG composite scaffold to mimic the epidermal layer. The CA scaffold provided mechanical support for the C-PEG gel and the C-PEG gel physically segregated the keratinocytes from fibroblasts in the construct. Three different tissue culture micro-environments were tested: CA scaffolds without C-PEG cultured in cell culture medium without air-liquid interface (-gel-interface), CA scaffolds impregnated with C-PEG and cultured in cell culture medium without air-liquid interface (-gel-interface), and CA scaffolds impregnated with C-PEG cultured in cell culture medium with air-liquid interface (-gel- interface). We found that the presence of C-PEG increased the cellular proliferation rates of both keratinocytes and fibroblasts, and the air-liquid interface induced keratinocyte maturation. This CA/C-PEG composite scaffold design is able to recapitulate micro-environments relevant to skin tissue engineering, and may be a useful tool for future skin tissue engineering applications.

Keywords: ECM; PEG; alginate; chitosan; hydrogel; skin.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Schematic illustration of co-culture of HaCaT and hFF cells in (a) CA scaffolds (−gel− interface, labeled in yellow) (b) CA scaffolds with C-PEG gel (−gel−interface, labeled in grey), and (c) CA scaffolds with C-PEG gel and air-liquid interface (−gel−interface, labeled in grey with air-liquid interface created by transwell insert). (d) in situ gelation of hFF/C-PEG gel mixture within CA scaffold; this side-view picture emphasizes the C-PEG gel on of the surface of CA scaffold. (e) Top-down view of C-PEG gel overlaid with HaCaT epithelial cells after 2 weeks of culture in the Transwell insert. Both scale bars represent 1 cm.
Fig. 2
Fig. 2
Proliferation analysis of HaCaT and hFF cells in 3 different microenvironments over 14 days as determined by cell sorting. * indicates a statistical significance in cell numbers of hFF and HaCaT (p < 0.05) as compared to respective cells at the −gel−interface condition and at each respective time point.
Fig. 3
Fig. 3
SEM images of HaCaT/hFF cells cultured in 3 different microenvironments for 2 weeks: − gel−interface (a, d, g), −gel−interface (b, e, h), and −gel−interface (c, f, i). For HaCaT cells, the black boxes in low magnification images (a, b, c, scale bar = 100 μm) identify the areas in high magnification images (d, e, f, scale bar = 25 μm). For hFF cells in g, h, i, scale bar = 15 μm.
Fig. 4
Fig. 4
Histological analysis of HaCaT cells cultured in 3 different microenvironments for 2 weeks: −gel−interface (a, d), −gel−interface (b, e), and −gel−interface (c, f). For HaCaT cells, the black boxes in low magnification images (a, b, c, scale bar = 20 μm) identify the areas in high magnification images (d, e, f, scale bar = 5 μm).
Fig. 5
Fig. 5
Fluorescent images of HaCaT/hFF cells cultured in 3 different microenvironments for 2 weeks: −gel−interface (a, d), −gel−interface (b, e), and −gel−interface (c, f). Cellular nuclei are blue, HaCat and hFF cells express red and green fluorescence, respectively. Scale bar = 25 μm.
Fig. 6
Fig. 6
RNA transcription of Keratin 5, and Keratin 10 by HaCaT cells cultured in 3 different microenvironments for 2 weeks: −gel−interface, −gel−interface, and −gel−interface. Results are normalized to β-actin mRNA. *indicates a statistical significance (p < 0.05) of −gel− interface as compared to other conditions.
Fig. 7
Fig. 7
RNA transcription of Collagen I and III, Fibronectin, and Vimentin expressed by hFF cells cultured in 3 different microenvironments for 2 weeks: −gel−interface, −gel−interface, and − gel−interface. Results are normalized to β-actin mRNA. * indicates a statistical significance (p < 0.05) of −gel−interface as compared to other conditions.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wendt H, Hillmer A, Reimers K, Kuhbier JW, Schafer-Nolte F, Allmeling C, Kasper C, Vogt PM. PLoS One. 2011;6:e21833. - PMC - PubMed
    1. Seet WT, Manira M, Khairul Anuar K, Chua KH, Ahmad Irfan AW, Ng MH, Aminuddin BS, Ruszymah BH. PLoS One. 2012;7:e40978. - PMC - PubMed
    1. Papini R. BMJ. 2004;329:158–160. - PMC - PubMed
    1. Rnjak J, Li Z, Maitz PK, Wise SG, Weiss AS. Biomaterials. 2009;30:6469–6477. - PubMed
    1. Ojeh NO, Frame JD, Navsaria HA. Tissue Eng. 2001;7:457–472. - PubMed

LinkOut - more resources