Optimizing the Surface Structural and Morphological Properties of Silk Thin Films via Ultra-Short Laser Texturing for Creation of Muscle Cell Matrix Model

Abstract

Temporary scaffolds that mimic the extracellular matrix’s structure and provide a stable substratum for the natural growth of cells are an innovative trend in the field of tissue engineering. The aim of this study is to obtain and design porous 2D fibroin-based cell matrices by femtosecond laser-induced microstructuring for future applications in muscle tissue engineering. Ultra-fast laser treatment is a non-contact method, which generates controlled porosity—the creation of micro/nanostructures on the surface of the biopolymer that can strongly affect cell behavior, while the control over its surface characteristics has the potential of directing the growth of future muscle tissue in the desired direction. The laser structured 2D thin film matrices from silk were characterized by means of SEM, EDX, AFM, FTIR, Micro-Raman, XRD, and 3D-roughness analyses. A WCA evaluation and initial experiments with murine C2C12 myoblasts cells were also performed. The results show that by varying the laser parameters, a different structuring degree can be achieved through the initial lifting and ejection of the material around the area of laser interaction to generate porous channels with varying widths and depths. The proper optimization of the applied laser parameters can significantly improve the bioactive properties of the investigated 2D model of a muscle cell matrix.

Rationally designed ultra-fast laser surface texturation of biocompatible temporal scaffolds

“…Laser irradiation in an air atmosphere changes the surface properties of the material, causing both the formation of calcium hydroxide and carbonate complexes, leading to a change in the concentrations of atoms and the Ca/P ratio on the surface. Based on previously performed comprehensive experimental evaluation of the structural and chemical composition of the structured SF based thin films [19] with the presented laser parameters, in the current work only the 3D profile real colour images of the optimized raster patterns, chosen for further biological evaluation with muscular and Schwann cells are presented (figure 3). Although the grooves in G8 and G11 look very similar to each other, a difference in the depth and width (Ra, Sa respectively) of the fs raster patterns is clearly defined -the increase in the fluence and velocity applied leads to wider grooves and to thinner samples.…”

“…In order to avoid a spatial overlap between the separate laser-created grooves, precisely defined separation intervals (dx) were defined. In the case of the 2D silk fibroin thin films already optimized laser parameters were used (group G4laser fluence F=0.4 J/cm 2 , scanning velocity V=1.7 mm/s, group G8 -F=0.8 J/cm 2 , V=1.7 mm/s, group G11 -F=1.7 J/cm 2 , V=3.8 mm/s) [19], but the created raster scanning design (dx1=25 µm and dx2=50 µm) was specifically optimized according to the muscle cells' dimensions [20], so enhanced seeding conditions to be achieved (figure 1 (b)). In the case of the 3D CaP tablets ablation (dx=50 µm), the incident angle of irradiation was also varied in order to obtain a diverse topographical design (F=0.8 and 2.4 J/cm 2 , V=1.5 mm/s) -figure 1 (c).…”

“…Cellular experiments for biological evaluation of the enhanced topographical design of the laserstructured 2D SF thin films muscle cellular scaffold were also performedthe detailed myoblasts cell line C2C12 seeding protocol was described elsewhere [19]. Cellular viability and muscle differentiation of C2C12 myoblasts cell line (CLS, Eppelheim, Germany) were monitored by means of fluorescence staining (images at 20x magnification) with DAPI (nuclei in blue) and muscle-specific marker (myosin heavy chain in green), respectively.…”

“…Optimal ECM living conditions could be achieved, simply by optimizing the parameters of the applied laser radiation. The results of previously performed by the group ultra-fast laser surface texturing experiments, described elsewhere [18,19], and structural and chemical characterization, 3D roughness analysis, wettability evaluation and preliminary cellular experiments, show that by changing the parameters of the applied femtosecond laser radiation, different degree of surface structuring can be obtained. Based on the performed comparative experimental study for evaluation of different modes of processing of the prepared cellular tissue scaffolds, optimal laser structuring conditions and raster patterns were chosen for further enhancement of muscular and Schwann cells environment.…”

“…Ultrafast laser processing is implemented in tissue engineering for implant high-precision surface functionalization that preserves the quality composition of the matrix, which is own to the ultrashort time of interaction with the material. The method introduces monitored hierarchical porosity and surface roughness to the cellular implants that allows controlled cellular adhesion, differentiation and motion to the desired selective region and, in the same time strengthened interface bond with recipient tissues [16][17][18][19]. Femtosecond laser treatment is a contactless method for creation of controllable porosity on the biomaterial surface, that can strongly influence cellular behaviour and can be used to manufacture a highly ECM imitating implant for the injured tissue that is capable to promote recovery and minimize undesired side effects as immune response and inflammation [16,17].…”