Artificial Skin – Culturing of Different Skin Cell Lines for Generating an Artificial Skin Substitute on Cross-Weaved Spider Silk Fibres

Background: In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. Methodology/Principal Findings: Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross-sections were stained in Haematoxylin & Eosin (H&E) for microscopic analyses. Conclusion/Significance: Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration

HaCaT cells, cultivated on spidersilk alone.

<p>1^st (A) and 4^th (B) day after seeding. From the corners of the meshwork, cells spread into the meshes and reach confluence within 1 week. bar = 100 µm.</p

Force testing.

<p>The diagrams show graphs describing the generated force of the particular test samples of the control group (n = 5) (A), after a 1 day culture period (n = 4) (B), after a 5 days culture period (n = 8) (C) and after a 21 days culture period (n = 5) (D).</p

3-dimensional cell culturing.

<p>Skin equivalents, cultivated under different medium conditions: Addition of choleratoxin (A), Hydrocortisone (B) or A-2-P (B). bar = 100 µm.</p

Frame design.

<p>Stainless steel straight wires with a diameter of 0,7 mm were bended to frames with side lengths of 1–1,5 cm and wound with spider dragline silk (A). Spider silk was woven in cross pattern to reach a mesh size between 10–100 µm (B). bar = 100 µm.</p

SEM of a confluent MEF cell layer on a frame with spider silk.

<p>The brink of the meshwork, where spider silk fibres do not cross each other is just rarely colonized, whereas a confluent cell-layer can be observed in the central meshwork. Culturing period took 5 days. bar = 1 mm.</p

Immunofluorescence microscopy of 3 dimensional, bilayered Cocultures.

<p>DAPI staining of cell nuclei in blue, Involucrin as specific marker for HaCaT cells in red. bar = 100 µm.</p

MEF cells, cultivated on spidersilk alone.

<p>1^st (A) and 4^th (B) day after seeding. By comparison, MEF cells reach confluence earlier than HaCaT cells. bar = 100 µm.</p

Cultivation at the air liquid interface.

<p>Frames with spider silk, seeded with MEF and HaCaT cells, are lifted on a silicone scale which is filled up with polymer fibres. The surface of the nutrient media is below the altitude of the frame. Media and frame are connected by the polymer fibres, which allow unilateral nutrient supply and metabolite evacuation by diffusion.</p