A two-layer skin construct consisting of a collagen hydrogel reinforced by a fibrin-coated polylactide nanofibrous membrane

Abstract

Background:

Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells.

Methods:

The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh.

Results:

The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer.

Conclusion:

The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.

Keywords:

• full-thickness skin substitutes
• collagen hydrogel
• fibroblast and keratinocyte co-cultivation
• fibrin
• nanostructure

View correction statement:

A two-layer skin construct consisting of a collagen hydrogel reinforced by a fibrin-coated polylactide nanofibrous membrane [Corrigendum]

Acknowledgments

We further acknowledge the BioImaging Facility, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic (supported by Czech-BioImaging large RI project No. LM2015062, funded by the Ministry of Education, Youth and Sports, Czech Republic), and the Light Microscopy Core Facility, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic (supported by MEYS LM2015062, CZ.02.1.01/0.0/0.0/16_013/0001775, OPPK CZ.2.16/3.1.00/21547 and MEYS LO1419), for their support with the [confocal/widefield/superresolution imaging/image analysis] presented here. The group of Dr Tomas Suchy, from Institute of Rock Structure and Mechanics of the Czech Academy of Sciences, is acknowledged for providing us with isolated collagen of type I. Mr Robin Healey (Czech Technical University in Prague) is gratefully acknowledged for his language revision of the manuscript.

Disclosure

MB, JP, AB, VJ and LB received grants from the Grant Agency of the Czech Republic (grant No. 17-0244S). JP received a grant from the Grant Agency of Charles University in Prague (grant No. 756218). AZ and LV received funding from the project “Fighting Infectious Diseases”, awarded by the MEYS CR, financed from EFRR (grant No. CZ.02.1.01/0.0/0.0/16_019/0000787). MB received a postdoctoral fellowship awarded by the Czech Academy of Sciences (reg. No. L200111752). The authors report no other conflicts of interest in this work.

Supplementary materials

Video S1 The horizontal sections through the collagen hydrogel with keratinocytes adhered on the hydrogel surface and fibroblasts homogeneously immigrated inside the collagen hydrogel. The fibroblasts migrated from the membrane into the collagen hydrogel for a period of 11 days. Keratinocytes proliferated on the hydrogel for 7 days. Both cell types were stained with phalloidin–TRITC for the cell F-actin cytoskeleton (red), and with Hoechst #33258 for the cell nuclei (blue). The cytokeratin 14 in the keratinocytes was stained by immunofluorescence (Alexa 488, green). Dragonfly 503 spinning disk confocal microscope with a Zyla 4.2 PLUS sCMOS camera, objective HC PL APO 20x/0.75 IMM CORR CS2.

Video S2 This video shows the two-layer construct of skin cells, depicted in Figure 10, in dynamic mode. The construct was composed of the fibrin-coated PLLA membrane seeded with human dermal fibroblasts, the collagen hydrogel with fibroblasts migrating inside the gel, and the keratinocyte layer on top of hydrogel. The fibroblasts migrated from the membrane into the collagen hydrogel for a period of 18 days. Keratinocytes proliferated on the hydrogel for 14 days. The PLLA membrane with fibroblasts and the collagen hydrogel with immigrated fibroblasts and keratinocytes on the top of hydrogel were stained separately. Both cell types were stained with phalloidin – TRITC for the cell F-actin cytoskeleton (red), and with Hoechst #33258 for the cell nuclei (blue). The cytokeratin 14 in the keratinocytes and the fibrin mesh on the nanofibrous membrane (at the bottom) were stained by immunofluorescence (Alexa 488, green). Dragonfly 503 spinning disk confocal microscope with a Zyla 4.2 PLUS sCMOS camera, objective HC PL APO 20x/0.75 IMM CORR CS2.