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

The aim of the current work is to create 2D silk fibroin and 3D calcium phosphate temporal cellular scaffolds with specifically enhanced porous topographical design by means of femtosecond laser-induced nano- and micro-scale hybrid surface structuring for application in muscle and bone tissue engineering. The created cellular matrices were ablated in a multistep manner using a Solstice Ace system, delivering laser pulses with pulse duration of 70 fs, at λ=800 nm and a focus spot of 25μm. The samples were positioned on a motorized XY translation stage perpendicular or in a variable angle in respect to the laser beam. Based on the previously performed comparative experimental study, optimal laser structuring conditions and raster patterns were chosen for further enhancement of muscular and Schwann cells environment. The most valuable contribution of the study presented is related to the optimization of the bioactivity properties of the studied cell matrices and their subsequent practical application in tissue engineering.


Introduction
The concept of tissue engineering relies on transient cellular matrices, based on biomaterials that simulate the particular tissue extracellular stable matrix (ECM) environment thus ensuring the natural growth of the recipient's own tissue [1].A functional tissue can be created to replace the damaged one by means of the incorporation of "intelligent implants".Such artificial bioscaffolds are usually implanted in the recipient and come into straight contact with cells, the direct adhesion of which is of main importance for the intrabody acceptance of biomedical implants [2,3].Silk fibroin (SF), a protein extracted from Bombyx mori cocoons [4][5][6][7], possesses mechanical stability, controllable biodegradability, biocompatibility and high elasticity, qualities that give the SF priority as a successful substrate for the creation of a temporary muscle cell matrix [8][9][10][11].On the other hand, calcium phosphates are gaining popularity as one of the most preferable materials for temporal bone cellular scaffolds due to their compositional and structural similarity with the bone tissue, non-toxicity, biocompatibility, stability and mechanical resistance [12].About 70% of human bones consist of calcium phosphate (CaP) apatite.That is the reason why CaP substances can be used both as a bone scaffold base material [13], and, at the same time, as a contact biomaterial interface between the incorporated metal-based implant and the natural bone tissue [14,15].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].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.The incident angle of laser irradiation was varied in order to obtain diverse topographical design.Studies of the influence over the chemical composition after every energy delivery were performed.Optimization of the obtained structures in relation to sequenced energy delivery and irradiation in diverse angles of incidence was performed.The most valuable contribution of the study presented is related to the optimization of the bioactive properties of the studied cell matrices and their subsequent practical application in tissue engineering.

3D CaP tablets and 2D SF thin films samples preparation
Tablets of β-TCP were prepared by calcination of a precipitate obtained by dropwise adding a 0.25 M (NH4)2HPO4 to a 0.56M Ca(NO3)2 aqua solutions, both with pH 8.2, at a molar ratio of Ca/P=1.67.The pH of the solutions and it's maintenance during the synthesis was done with NH4OH.The precipitate matured for 24 h and centrifuged with washing; 2% solution of (NH4)2SO4 was added in volume equal to the final suspension volume.After an additional 4 hours of maturation, the precipitate was dried at 100 o C. The dry precursor with a particle size below 0.1 mm was tableted and heated at 1100 o C for 3 hours.The preparation of monophase β-TCP was proved by X-ray powder diffraction analyses using a Bruker D8 Advance diffractometer (Cu Kα radiation), equipped with LynxEye detector (Bruker AXS Advanced X-ray Solutions GmbH, Billerica, MA, USA).The extraction of SF from Bombyx mori Cocoons and the preparation of 2D samples was described in detail elsewhere [19].As a result, SF based 2D thin films (1x1 cm, 120 µm thickness) were obtained.

Femtosecond laser treatment of the 3D CaP tablets and 2D SF thin films
The surface micro-processing of the CaP disks and 2D silk fibroin thin films was performed by means of 6 W femtosecond mode-locked Ti:sapphire laser system (Solstice ® Ace TM , Spectra Physics), working at a central wavelength of λ=800 nm and repetition rate ν=1kHz, TEM00 mode of operation and laser pulse duration of τ=70 fs.The surface structuring was performed in air, using a lens with a 20-cm focal length -the laser beam was focused to a focal spot with a d≈25 μm (figure 1 (a)).The created cellular matrices were ablated in a continuous mode of operation, in a multistep manner, positioned on a motorized XY translation stage.Diverse scanning velocities and laser energies were applied in a sequence.The samples were processed by rastering the focused laser beam over the sample surface.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=0.8J/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).Optimization of the obtained structures in relation to sequenced energy delivery and irradiation in diverse angles of incidence was performed (90 o ,45 o ,30 o ).Each laser-ablated sample was further structurally and chemically analysed with respect to the control laser-untreated cellular scaffold.

Analyses of the biocompatible temporal scaffolds
Scanning electron microscopy (SEM) and energy dispersive X-Ray analysis (EDX) (TESCAN/LYRA/XMU) were used for evaluation of the surface morphology and elemental composition of the calcium phosphate tablets, after sputtering with 20 nm layer of carbon (C), at 4000x magnification; the elemental composition was estimated in [at.%].The Ca/P ratio with respect to control surface was also monitored.The surface roughness of the enhanced porous topographical design of the SF-based cellular matrices was characterized via 3D Optical profiler, Zeta-20.The roughness parameters Ra and Sa were measured by means of line and area roughness analysis.Free software program ProfilmOnline (https://www.profilmonline.com)was used for better visualization of the 3D profile of the laser scan pattern in the true colour images obtained.

Cell viability and differentiation biological evaluation of 2D SF thin films
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.Cells, cultured for 7 and 14 days on G4, G8 and G11 fs processed SF thin films with respect to the control group were analysed with a Leica DMI 6000b inverted fluorescence microscope (Leica Microsystems GmbH).SEM images at 500x/1000x magnification of preliminary biological evaluation with Schwann cells (primary cells isolated from rat sciatic nerves) and fluorescence microscopy images (at 20x) of viability (IF staining -in green), cultured on the silk-based samples were also taken on 4 th day of cultivation, after performing standard alcohol dehydration procedure.

Results and discussion
Selected SEM images (4000x magnification) of fs laser-structured CaP tablets are given in figure 2. As can be clearly seen, by varying laser parameters and the angle of incidence, the morphology of the structures created can be controllably monitoredhigher fluence leads to deeper and more clearly outlined grooves, while lowering the angle of laser incidence results in wider and flatter structures, especially at the higher fluence applied.The grooves, created by raster laser scanning are characterized by granular structure and material ejection around the zones of interaction.Enchased microroughness can be visualized, but in all observed cases, the laser structuring proceeds with no cracks or melted zones formation, neither damage of the structure of the ceramic material is detected.EDX elemental composition [at.%] analysis does not show (figure 2) uncommon elements for CaP tablets, only variations in concentrations of all measured atoms, most pronounced at O atom.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 the case of G4 SF group, where laser patterning is performed at lower laser fluence, the implied raster pattern is clearly defined and merging of the laser grooves is not observed, unlike the other two fs structured SF scaffold groups (G8 and G11).The dimensions of the grooves created -width (≈50 µm) and depth (≈8 µm) (roughness Ra, Sa parameters, respectively) are consistent with the size and shape of the muscle cells, for which the samples serve as temporal scaffolds.The blue fluorescence staining of the nuclei of the murine C2C12 myoblasts confirmed the viability of the cells on seeded the 2D fibroin matrices samples on days 7, and 14 as no visible differences in the cellular viability between fs fs-treated and control samples were observed (figure 4-left).The myogenic differentiation (evaluated by immunofluorescence staining of the myogenic marker myosin heavy chain (MHC) in green) was clearly demonstrated both on the 7 th day (signs of differentiation, elongation of cells) and on day 14 th (fusion of cells to myotubes) of cellular culturing especially on G11 SF samples, where skeletal muscle-like constructs were observed.Fs laser surface optimized raster patterns influenced the C2C12 morphology and organization in the differentiating cells (days 7 and 14).The cells have a more oblong and fusiform shape when cultured on samples G4, G8 and G11, in respect to the disordered spreading and lower level of myogenic differentiation of the C2C12 cells in the control group.
From the SEM and fluorescence images of Schwann cells cultivated on fs-treated and control SF scaffolds (figure 4-right), taken on day 4 of cultivation, a notable difference with myoblast cells is observed -while C2C12 cells are organized along the laser-generated rasters, a visible orientation of the Schwann cells between the grooves is remarked, which could be used as a next step for the development of a fs surface functionalized SF-based cellular scaffold for a parallel co-culturing of Schwann and nerve cells for tissue engineering applications.

Conclusion
The preliminary experiments on CaP tablets confirm that by varying the laser fluence, scanning velocity and incidence angle, very fine control of the structural features (depth, width, micromorphology) of the ceramic samples can be obtained.Further evaluation of the morphological and chemical characteristics and optimization of the laser structuring conditions could enhance the bioactivity properties of the studied CaP cellular matrices.The main contribution of the current work is related to the optimization of laser structuring conditions and raster patterns, chosen for further enhancement of muscular and Schwann cells SF-based environment, that could find practical application in simultaneous two-tissue regeneration and subsequent practical application in tissue engineering.

Figure 1 .
Figure 1.(a) Scheme of Solstice ® Ace TM laser system and the motorized XYZ translation stage, with a perpendicularly positioned to the fs laser beam sample; 2 types of raster scan patterns (b) dx2=2dx1 in the case of SF scaffolds and (c) only one separation interval dxfor ceramic tablets, where fs structuring in diverse angles of incidence was performed (90 o ,45 o ,30 o ).

Figure 4 .
Figure 4. On the left: fluorescence stained images (at 20x magnification) with DAPI (nuclei in blue) and MHC -muscle-specific marker (in green) of C2C12 myoblasts cell line, cultured for 7 and 14 days on G4, G8 and G11 laser processed SF thin films in respect to control group; On the right: fluorescence microscopy images (20x) of viability (IF staining -in green) and SEM images of Schwann cells on fs treated silk samples in respect to control group (500x/1000x) on the day 4 of cultivation.