Data - The impact of ultrashort pulse laser structuring of metals on in-vitro cell adhesion of keratinocytes

Description

Methods

Cell Culture

In-vitro studies focused on cell adhesion, morphology, and growth on laser-structured surfaces, human keratinocytes (HaCaT, CLS, Cell Lines Service GmbH, Eppelheim, Germany #330493) were used. 

HaCaTs were cultured in Dulbecco’s modified eagle’s medium (DMEM; high glucose, GlutaMAX-; Thermo Fisher Scientific, Gibco, Paisley, UK) containing 10% fetal bovine serum (FBS; Biochrom FBS Superior, EU-approved, Merck KgaG, Darmstadt, Germany) and 1% penicillin/streptomycin (Pen Strep; Thermo Fisher Scientific, Gibco, Paisley, UK) at 37 °C and in a 5% CO₂ atmosphere (incubator, Sanyo CO₂ incubator MCO-18AIC-UV, Panasonic Biomedical, Osaka, Japan). To detach the cells, for specific analyses, trypsin/ethylenediaminetetraacetic acid (0.25% trypsin/0.38% EDTA; Invitrogen, Gibco, Paisley, UK) was incubated at 37 °C for 7 min.

Methods

Morphology analysis of HaCaT

The morphology of HaCaTs (7 × 10⁵ cells for 2 h; 5 × 10⁵ for 1 d; 3 × 10⁵ cells for 7 d) was assessed using a field emission scanning electron microscope (FE-SEM, Merlin VP compact, Carl Zeiss, Oberkochen, Germany). For this, cells were washed after cultivation with N-(2-hydroxyethyl)-piperazine-N′-(2-ethane sulfonic acid) buffer (HEPES, Sigma-Aldrich, Munich, Germany), fixed with 2.5% glutardialdehyde (GA, Merck, Darmstadt, Germany) and dehydrated with an ascending ethanol concentration series (30% 5 min, 50% 5 min, 75% 10 min, 90% 15 min, 100% twice 10 min). Samples were dried in a critical point dryer (K850, Emitech, Taunusstein, Germany) and finally evaporated with carbon “C” under vacuum conditions (EM SCD 500, Co. Leica, Bensheim, Germany). An HE/SE (high-efficiency secondary electron detector) and an InlensDuo detector were used to image the cells (5 kV).

Methods

Actin cytoskeleton organization

The actin cytoskeleton organization of cells was determined using confocal laser scanning microscopy (cLSM; LSM780, Carl Zeiss Microscopy GmbH, Jena, Germany). Therefore, 5 × 10⁵ cells were cultured on the specimens for 24 h in a 48-well plate. After the 1-day incubation period, HaCaTs were washed three times with phosphate-buffered saline solution (PBS without Ca/Mg; Sigma-Aldrich, Munich, Germany). For fixation, the cells were stored in 4% paraformaldehyde (PFA; Sigma Aldrich) for 10 min, followed by washing steps with PBS (3×) and the permeabilization of the cell membrane with 0.1% 4-(1,1,3,3-Tetramethylbutyl) phenyl-polyethylene glycol (TRITON X-100; Merck KGaA) for 10 min. Finally, the cells were rewashed (3× PBS rinse). For actin staining, the cells were incubated with phalloidin-tetramethyl-rhodamine (TRITC, Sigma Aldrich; 1:15 in PBS) at RT in the dark for 1 h and embedded with Fluoroshield™ DAPI (Sigma Aldrich) after a wash step. Images of the actin cytoskeleton were taken using the LSM780 with software ZEN 2.3 (black edition) (Carl Zeiss) with the ZEISS oil immersion 63× objective (C-apochromat). To analyze the keratinocytes in the dimples, focused confocal images were acquired from multiple focal planes (Z-stack), which were merged into a 3D overlay for analysis.

Methods

Microstructuring using Ultrashort Laser Pulses