DOI: 10.5281/zenodo.10148654
title: Data - The impact of ultrashort pulse laser structuring of metals on in-vitro cell adhesion of keratinocytes

description: The in-vitro data of the study - The impact of ultrashort pulse laser structuring of metals on in-vitro cell adhesion of keratinocytes - are published here.

Background: Laser patterning is a suitable method for modifying the surface of biomaterials to improve cell adhesion, i.e., designing a new surface topography for external bone fixation pins or implants. 

Aim: The principle of this study was to observe how bio-inspired (deer antler) laser-induced nano microstructures affect skin cell adhesion and growth. 

Objective: The aim was to create pins that enable bacteria-tight skin adhesion to the biomaterial surface. 

Methods: For this purpose, typical fixator metals, steel, and titanium alloys were patterned with ultrashort laser pulses, resulting in periodic nano- and microstructures. In vitro studies with human HaCaT keratinocytes focused on cell adhesion, morphology, actin formation, and growth within 7 days. 

Results: The study showed that surface functionalization influences cell adhesion, spreading, and proliferation. Microdimple clusters on polished bulk metals (DC20) do not affect cell viability. However, they must promote the initial adhesion and proliferation of HaCaTs. In contrast, additional nanostructuring with laser-induced periodic surface structures (LIPSS) promotes cell behavior.

DC20 + LIPSS resulted in improved cell adhesion with a well-distributed cell morphology. 

Conclusion: The bioinspired structures thus showed an advantage in initial cell adhesion. Laser surface functionalization opens up new possibilities for patterning.
description: 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.
description: 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).
description: 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.
description: Microstructuring using Ultrashort Laser Pulses







For the process development and laser structuring of the samples, a femtosecond laser of the type TruMicro 6020 (Trumpf GmbH & Co. KG, Ditzingen, Germany) with a wavelength of λ = 1030 nm, a repetition rate of 1 MHz, and a pulse duration of 530 fs was used [34]. The laser system was integrated into a precise 5-axis micromachining system of GL.evo (GFH GmbH, Deggendorf, Germany). Accurate beam guidance on the planar specimen geometries was carried out using an ExcelliSCAN 14 scanner system (Scanlab GmbH, Puchheim, Germany) with variable f-Theta lenses and corresponding different focal lengths. The laser-induced periodic surface structures (LIPSS) were nanostructured using surface hatching with linearly polarized laser pulses. In contrast, low fluences between 2.8 and 5.6 J/cm2 were used for the fine and highly quantitative structuring of various micro-dimple clusters (DC) without throw-ups and ridges. The fast structuring of the dimples was achieved using on-the-fly processes.

The task was to develop various combinations of dimple patterns and LIPSS in order to assess the deer antler’s porous structure as a bionic specification. For this study, we focused on micro-dimple clusters with a dimple dimeter of 20 µm (DC20), periodic nanostructures (LIPSS), and a combination of both micro-dimples and nanostructures (DC20 + LIPSS), which were compared to polished metals (Ref).