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. 2016 Jan 7;5(1):108-18.
doi: 10.1002/adhm.201500005. Epub 2015 Apr 16.

Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering

Xin Zhao  1   2   3 , Qi Lang  1   2 , Lara Yildirimer  1   2 , Zhi Yuan Lin  1   2 , Wenguo Cui  4 , Nasim Annabi  1   2   5 , Kee Woei Ng  6 , Mehmet R Dokmeci  1   2   5 , Amir M Ghaemmaghami  3 , Ali Khademhosseini  1   2   5   7
Affiliations

Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering

Xin Zhao et al. Adv Healthc Mater. .

Abstract

Natural hydrogels are promising scaffolds to engineer epidermis. Currently, natural hydrogels used to support epidermal regeneration are mainly collagen- or gelatin-based, which mimic the natural dermal extracellular matrix but often suffer from insufficient and uncontrollable mechanical and degradation properties. In this study, a photocrosslinkable gelatin (i.e., gelatin methacrylamide (GelMA)) with tunable mechanical, degradation, and biological properties is used to engineer the epidermis for skin tissue engineering applications. The results reveal that the mechanical and degradation properties of the developed hydrogels can be readily modified by varying the hydrogel concentration, with elastic and compressive moduli tuned from a few kPa to a few hundred kPa, and the degradation times varied from a few days to several months. Additionally, hydrogels of all concentrations displayed excellent cell viability (>90%) with increasing cell adhesion and proliferation corresponding to increases in hydrogel concentrations. Furthermore, the hydrogels are found to support keratinocyte growth, differentiation, and stratification into a reconstructed multilayered epidermis with adequate barrier functions. The robust and tunable properties of GelMA hydrogels suggest that the keratinocyte laden hydrogels can be used as epidermal substitutes, wound dressings, or substrates to construct various in vitro skin models.

Keywords: degradation; epidermis; keratinocytes; mechanical properties; photocrosslinkable gelatin.

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Figures

Figure 1
Figure 1
Compressive stress-strain characterization (A), compressive modulus (B), tensile stress-strain curves (C), tensile modulus (D), swelling ratio (E), representative photographs of morphology changes during in vitro degradation (F) and mass retention during degradation (G) of GelMA hydrogels of varying concentrations. Note that about 25% of 20% GelMA remained after 56 days of degradation study. * indicates p < 0.05.
Figure 2
Figure 2
Viability, adhesion and proliferation of HaCaT cells cultured on surfaces of GelMA with different concentrations. (A). Representative live/dead fluorescence images of HaCaT cells on GelMA surfaces of 5% (i), 10% (ii) and 20% (iii) after 7 days of culture. Green fluorescent cells are alive and red fluorescent cells indicate dead cells. (B). Representative phalloidin/DAPI fluorescence images of HaCaT cells on GelMA surfaces of 5% (i), 10% (ii) and 20% (iii) after 7 days of culture. Cell filaments are stained by phalloidin (red) and nuclei stained by DAPI (blue). (C) Quantification of the staining using NIH ImageJ software of the living and dead cells of the 2D cultures of GelMA at different concentrations. (D) Quantification of the staining using NIH ImageJ software of the sample area covered by cells of 2D cultures of GelMA with different concentrations. (E) Quantification of the staining using NIH ImageJ software of the number of cells on surfaces of GelMA with different concentrations.* indicates p < 0.05. (F). Fi is a representative phase contrast image of the cell monolayer developed on 20% GelMA after 7 days of culture and Fii is the corresponding image of immunocytochemical staining of E-cadherin (green) in HaCaT cell junctions and DAPI nucleic staining (blue). Prominent fluorescence of E-cadherin in adjacent cells was observed.
Figure 3
Figure 3
Reconstructed epidermis on hydrogel scaffolds. Examples of hematoxylin and eosin (H & E) stained sections of reconstructed epidermis on GelMA (A) and control collagen (B) scaffolds after 2 weeks (i) and 6 weeks (ii) of culture at air-liquid interface (ALI) and human epidermis (C). Flattening and stratification of HaCaT cells from the top surface of the reconstructed epidermis on either GelMA or collagen scaffolds can be clearly seen. Note the presence of GelMA (red arrow) and absence of collagen (black arrow) after 6 weeks of culture at ALI. Scale bar = 100 μm. (D) Quantification of the thickness of the reconstructed epidermis at different time of culture at ALI and human epidermis. E=epidermis; S=scaffolds. * indicates p < 0.05.
Figure 4
Figure 4
Expression of proteins of reconstructed epidermis on hydrogel scaffolds. Examples of ki 67 (red, proliferation maker), involucrin (green, differentiation marker) and DAPI (blue, nuclei) stained sections of reconstructed epidermis on GelMA (A) and collagen (B) scaffolds after 2 weeks (i) and 6 weeks (ii) of culture at ALI and human epidermis (C). Scale bar = 100 μm. (D) Quantification of the number of epidermis layers of the reconstructed epidermis at different time of culture at ALI and human epidermis.
Figure 5
Figure 5
Resistance measurements (A), rate of water loss (B), and relative water permeability of naked collagen, collagen covered with a reconstructed epidermis (Epidermis (C)), GelMA hydrogel (20%), and GelMA hydrogel covered with epidermis (Epidermis (G)). Note the significant influence on barrier function of an epidermal cover. * indicates p < 0.05.
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