Femtosecond Laser Fabrication of Engineered Functional Surfaces Based on Biodegradable Polymer and Biopolymer/Ceramic Composite Thin Films

Surface functionalization introduced by precisely-defined surface structures depended on the surface texture and quality. Laser treatment is an advanced, non-contact technique for improving the biomaterials surface characteristics. In this study, femtosecond laser modification was applied to fabricate diverse structures on biodegradable polymer thin films and their ceramic blends. The influences of key laser processing parameters like laser energy and a number of applied laser pulses (N) over laser-treated surfaces were investigated. The modification of surface roughness was determined by atomic force microscopy (AFM). The surface roughness (Rrms) increased from approximately 0.5 to nearly 3 µm. The roughness changed with increasing laser energy and a number of applied laser pulses (N). The induced morphologies with different laser parameters were compared via Scanning electron microscopy (SEM) and confocal microscopy analysis. The chemical composition of exposed surfaces was examined by FTIR, X-ray photoelectron spectroscopy (XPS), and XRD analysis. This work illustrates the capacity of the laser microstructuring method for surface functionalization with possible applications in improvement of cellular attachment and orientation. Cells exhibited an extended shape along laser-modified surface zones compared to non-structured areas and demonstrated parallel alignment to the created structures. We examined laser-material interaction, microstructural outgrowth, and surface-treatment effect. By comparing the experimental results, it can be summarized that considerable processing quality can be obtained with femtosecond laser structuring.


Introduction
Regenerative medicine and tissue engineering now expands to practically all areas of healthcare. According to a new report by Grand View Research, Inc. (San Francisco, CA, USA) [1], the global tissue engineering market is expected to reach USD 11.53 billion by 2022. Reconstruction of bone tissue defects is a major challenge facing orthopedics, as treatment of skeletal defects has remained a demanding part of many reconstructive surgeries [2,3]. When the gold standard for bone grafting (autogenous bone) is inapplicable, bone tissue engineering is emerging as a possible solution. Recently, temporary "platforms" of various materials for seeding different types of cell cultures and improving study, we focused our attention towards topography of the materials in order to mimic the surface properties similar to natural body tissues.

Sample Preparation
Chitosan (medium molecular weight), hydroxyapatite powder (with particle size 10 µm, ≥ 100 m 2 /g), and nanoZrO 2 (particle size < 100 nm) were purchased from Sigma-Aldrich ® (Munich, Germany). The chitosan powder was dissolved in a 0.5 mol/L CH 3 COOH solution and dH 2 O and heated to 50 • C under continuous stirring for 2.5 h. HAp was added to deionized water and stirred for approximately 2 h without heating. The two solutions were mixed together in diverse ratios of Ch/HAp. To the prepared ratios between Ch and HAp, 1% ZrO 2 (w/v) was added and stirred without heating for 12 h before dripping. A solution of silver nanoparticles AgNps-10% Np (v/v) was added to the prepared ratios between Ch and HAp and stirred without heating for 1 h before dripping.
The biopolymer/ceramic composites were spread over 20 mm × 20 mm glass slides and allowed to dry at room temperature before irradiation by femtosecond laser pulses.
The samples were divided into two groups and treated with femtosecond laser radiation: (1) Pure chitosan thin films.

Experimental Setup
A femtosecond Ti:sapphire laser (Integra-C, Quantronix, Hamden, CT, USA) emitting a central wavelength of 800 nm was used. The pulse duration was 130 fs at 1 kHz repetition rate. The laser system possessed a possibility for variation of the repetition rate ν (from 25 Hz to 1 KHz) via delay generator. The number of laser shots (N) was controlled by computer-driven fast mechanical shutter synchronized from controlling software. The laser beam was focused using a lens with a focal length of 20 cm at normal incidence on the sample surface. It was estimated that the laser spot diameter on the surface of the sample was 50 µm.
A complete set of parameters was selected to deduce the key variables (laser energy, pulse number, overlapping between separate pulses), which effected the morphology, average roughness, and chemistry of the treated areas. Translation was achieved between separate sets of fired laser pulses so that the laser irradiated zone received a defined number of shots. The number of laser pulses (N) was varied from 1 to 100 to examine their influence on morphological change. A schematic of the experimental setup is shown in Figure 1. The microstructured thin films were investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM), fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and X-Ray Diffraction (XRD) analyses.

Sample Preparation
Chitosan (medium molecular weight), hydroxyapatite powder (with particle size 10 µm, ≥ 100 m2/g), and nanoZrO2 (particle size < 100 nm) were purchased from Sigma-Aldrich ® (Munich, Germany). The chitosan powder was dissolved in a 0.5 mol/L CH3COOH solution and dH2O and heated to 50 °С under continuous stirring for 2.5 h. HAp was added to deionized water and stirred for approximately 2 h without heating. The two solutions were mixed together in diverse ratios of Ch/HAp. To the prepared ratios between Ch and HAp, 1% ZrO2 (w/v) was added and stirred without heating for 12 h before dripping. A solution of silver nanoparticles AgNps-10% Np (v/v) was added to the prepared ratios between Ch and HAp and stirred without heating for 1 h before dripping.
The biopolymer/ceramic composites were spread over 20 mm × 20 mm glass slides and allowed to dry at room temperature before irradiation by femtosecond laser pulses.
The samples were divided into two groups and treated with femtosecond laser radiation: (1) Pure chitosan thin films.

Experimental Setup
A femtosecond Ti:sapphire laser (Integra-C, Quantronix, Hamden, CT, USA) emitting a central wavelength of 800 nm was used. The pulse duration was 130 fs at 1 kHz repetition rate. The laser system possessed a possibility for variation of the repetition rate ν (from 25 Hz to 1 KHz) via delay generator. The number of laser shots (N) was controlled by computer-driven fast mechanical shutter synchronized from controlling software. The laser beam was focused using a lens with a focal length of 20 cm at normal incidence on the sample surface. It was estimated that the laser spot diameter on the surface of the sample was 50 µm.
A complete set of parameters was selected to deduce the key variables (laser energy, pulse number, overlapping between separate pulses), which effected the morphology, average roughness, and chemistry of the treated areas. Translation was achieved between separate sets of fired laser pulses so that the laser irradiated zone received a defined number of shots. The number of laser pulses (N) was varied from 1 to 100 to examine their influence on morphological change. A schematic of the experimental setup is shown in Figure 1. The sample was positioned on a motorized xyz translation stage where the sample surface was placed perpendicular to the propagation direction of the incident laser radiation. The sample was positioned on a motorized xyz translation stage where the sample surface was placed perpendicular to the propagation direction of the incident laser radiation.

Morphological and Topography Examination of Textured Biofilms via SEM, Energy-Dispersive X-ray Spectroscopy (EDX), and Atomic Force Microscopy (AFM)
The morphology of the pure chitosan biofilms and their ceramic composites was analyzed using SEM-TESCAN/LYRA/XMU (TESCAN ORSAY HOLDING, a.s., Brno, Czech Republic). The samples were graphene sputtered. Several images were taken at laser treated and non-treated zones for each sample. Energy dispersive X-ray spectroscopy (EDX) was used to analyze the elemental composition of composite thin film surfaces before and after laser irradiation with an operational voltage of 20 kV. Each sample was measured at two positions inside the modified zone.
Roughness was measured by means of an AFM-Bruker Dimension Icon with Scan-Asystin several areas of the treated zones; average values of surface roughness were obtained to characterize the surface after the laser irradiation. In the case of surfaces treated by laser, the AFM tip ran the samples in the direction of the textures.

FTIR Analysis
In order to gain detailed information about the phase transformations and chemical bonds alterations in the samples, IR spectra of the prepared biofilms before and after laser treatment were obtained using FTIR spectrophotometer (IR Affinity-1, Shimadzu, Kyoto, Japan). The measurements were taken in transmittance mode, and the range recorded was from 4500 to 500 cm −1 .

XPS Analysis
X-ray photoelectron spectroscopy (XPS) was performed by means of an AXIS Supra electron spectrometer (Kratos Analytical Ltd., Manchester, UK). XPS method enables high-resolution chemical analysis of polymer films. An evaluation of the surface chemistry of chitosan films after laser processing with a diverse number of applied laser pulses was obtained.

XRD Analysis
The surfaces of the samples after laser treatment were examined by XRD using PANalytical-Empyrean system (Malvern Panalytical, Malvern, UK) equipped with a Cu tube and a PIXcel3D detector. The experimental condition was: 40 kV, 40 mA, exposition 100 s, step 0.03 degree. Software High Score Plus (Malvern Panalytical, Malvern, UK) was used to analyze the obtained X-ray diffraction data.

Cell Seeding, Culture, and Staining on Laser-Textured Samples
The arrangement of living cells on the patterned substrates was achieved via femtosecond laser texturing. The main groups of material used for cellular studies are classified in Table 1. To sterilize the samples, they were placed under a UV lamp of 15 W (Sylvania, London, UK; 254 nm wavelength) at a distance of 45 cm and irradiated for 30 min. Mouse calvaria osteoblasts (MC3T3) and human adipose derived mesenchymal stem cells (ADSCs) (CryoSave, The Cell Factory, Niel, Belgium) were cultured in DMEM Glutamax (Gibco™; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin in a humidified atmosphere at 37 • C and 5% CO 2 . MC3T3 and ADSCs were seeded on laser-textured samples or on controls (non-textured samples or tissue culture plastic) in a 24-well plate. Densities of 100,000 cells and 40,000 cells per sample were used for MC3T3 and ADSCs, respectively. Cell adhesion and proliferation were evaluated after 1 and 6 days.
Live/dead staining with calcein AM/propidium iodide was performed to evaluate cell viability, cell attachment, and cell morphology. After rinsing the samples, the supernatant was replaced with 1 mL phosphate buffered saline (PBS) supplemented with 2 µL (1 mg/mL) calcein AM (Anaspec, Fremont, CA, USA; 89201) and 2 µL (1 mg/mL) propidium iodide (Sigma-Aldrich; P4170). After 10 min incubation in the dark at room temperature, the samples were washed with PBS and evaluated with a fluorescence microscope (Olympus IX 81, Olympus, Bridgeport, CT, USA). humidified atmosphere at 37 °C and 5% CO2. MC3T3 and ADSCs were seeded on laser-textured samples or on controls (non-textured samples or tissue culture plastic) in a 24-well plate. Densities of 100,000 cells and 40,000 cells per sample were used for MC3T3 and ADSCs, respectively. Cell adhesion and proliferation were evaluated after 1 and 6 days. Live/dead staining with calcein AM/propidium iodide was performed to evaluate cell viability, cell attachment, and cell morphology. After rinsing the samples, the supernatant was replaced with 1 mL phosphate buffered saline (PBS) supplemented with 2 µL (1 mg/mL) calcein AM (Anaspec, Fremont, CA, USA; 89201) and 2 µL (1 mg/mL) propidium iodide (Sigma-Aldrich; P4170). After 10 min incubation in the dark at room temperature, the samples were washed with PBS and evaluated with a fluorescence microscope (Olympus IX 81, Olympus, Bridgeport, CT, USA).  The spots presented in Figure 2 were created in a series of irradiations with an increasing number of laser pulses, N = 2 and 5. For N = 2, there was a clearly detectable formation of sponge-like creation (Figure 2a) over the surface baseline. As N increased further to N = 5, the material from the surface began to ablate (Figure 2d). The optimal conditions to obtain the best quality of the fabricated "microfoam-like" structures were determined for a low number of applied laser pulses and low and The spots presented in Figure 2 were created in a series of irradiations with an increasing number of laser pulses, N = 2 and 5. For N = 2, there was a clearly detectable formation of sponge-like creation (Figure 2a) over the surface baseline. As N increased further to N = 5, the material from the surface began to ablate (Figure 2d). The optimal conditions to obtain the best quality of the fabricated "microfoam-like" structures were determined for a low number of applied laser pulses and low and intermediate laser intensities. At an increasing number of applied laser pulses (N), the structures porosity started to deviate, and removal of material was initiated, leading to crater formation.

Femtosecond Laser Fabrication of Chitosan Biofilm Microporous Structures
These results demonstrated that microporous structures were fabricated by femtosecond laser irradiation for specifically selected exposition parameters (N = 2, I = 7.9 × 10 14 W/cm 2 , Figure 2a). A clear difference in the morphological patterns of the chitosan surfaces was observed. Figure 2b,c) shows confocal microscopy images of chitosan films irradiated with increased laser intensity at two selected numbers of applied laser pulses (N = 2 and N = 5), which corresponded to the surface treatment that had the most suitable application in clinical use of the implant's surface. In the case of treatment with N > 2, a formation of cavities through the removal of material by laser was observed, and destruction of microfoam started to take place. It was observed that a surface with uniform roughness and a creation of self-induced porosity due to the nature of non-contact interaction with fs laser radiation and the thin biopolymer films could be obtained under irradiation with a careful selection of a number of applied laser pulses (N~1 or 2) and a laser intensity (I) not exceeding I = 3.1 × 10 15 W/cm 2 [35]. Figure 3 shows the topography image of irradiated chitosan thin film at a diverse number of applied laser pulses and at two energy settings obtained over an area of 20 × 20 µm 2 . The root mean-square roughness (R rms ) at the irradiated zones deviated in relation to an increasing number of applied laser pulses and applied energy, with root mean-square roughness (R rms ) increasing from 0.780 to 2.480 µm. These results demonstrated that microporous structures were fabricated by femtosecond laser irradiation for specifically selected exposition parameters (N = 2, I = 7.9 × 10 14 W/cm 2 , Figure 2a). A clear difference in the morphological patterns of the chitosan surfaces was observed. Figure 2b,c) shows confocal microscopy images of chitosan films irradiated with increased laser intensity at two selected numbers of applied laser pulses (N = 2 and N = 5), which corresponded to the surface treatment that had the most suitable application in clinical use of the implant's surface. In the case of treatment with N > 2, a formation of cavities through the removal of material by laser was observed, and destruction of microfoam started to take place. It was observed that a surface with uniform roughness and a creation of self-induced porosity due to the nature of non-contact interaction with fs laser radiation and the thin biopolymer films could be obtained under irradiation with a careful selection of a number of applied laser pulses (N ~ 1 or 2) and a laser intensity (I) not exceeding I = 3.1 × 10 15 W/cm 2 [35]. Figure 3 shows the topography image of irradiated chitosan thin film at a diverse number of applied laser pulses and at two energy settings obtained over an area of 20 × 20 µm 2 . The root meansquare roughness (Rrms) at the irradiated zones deviated in relation to an increasing number of applied laser pulses and applied energy, with root mean-square roughness (Rrms) increasing from 0.780 to 2.480 µm. The images in Figure 3 show that pure chitosan thin films had distinct surface topography structures produced by different laser expositions. AFM images of chitosan biofilms revealed porous structures with a formation of craters on chitosan layers at higher intensities, which influenced the morphology via producing a rougher surface. The surface roughness deviation was estimated and related to changes due to the laser energy increase and increment of applied N. The images in Figure 3 show that pure chitosan thin films had distinct surface topography structures produced by different laser expositions. AFM images of chitosan biofilms revealed porous structures with a formation of craters on chitosan layers at higher intensities, which influenced the morphology via producing a rougher surface. The surface roughness deviation was estimated and related to changes due to the laser energy increase and increment of applied N.

XPS Chracterization of Pure Chitosan Thin Films
The results of the XPS spectra analysis for pure chitosan films are shown in Figure 4. To compare the effect of laser treatment on the bonding strength, the examined material was separated in relation to applied laser conditions. All spectra were compared to non-irradiated chitosan surfaces.

XPS Chracterization of Pure Chitosan Thin Films
The results of the XPS spectra analysis for pure chitosan films are shown in Figure 4. To compare the effect of laser treatment on the bonding strength, the examined material was separated in relation to applied laser conditions. All spectra were compared to non-irradiated chitosan surfaces. . X-ray photoelectron spectroscopy (XPS) spectra of pure chitosan thin films (a) non-treated and laser irradiated at I = 7.9 × 10 14 W/cm 2 for N = 1, 2, 5, 10; (b)-(g) resolved peaks of C1s, C1s-peak fitting, Ca2p, N1s, O1s, Na1s.
As seen from the XPS analysis in Figure 4, all six spectra exhibited peaks, showing binding energies at C1s, Ca2p, N1s, O1s, and Na1s.
The C1s peak was divided into four peaks 281.7, 283.3, 284.7, and 285.45 eV corresponding to C-C/C-H bond and C-NH/C-NH2 (Figure 4b,c). After femtosecond laser treatment with an increasing number of pulses (N = 1-5), the C-C bond remained almost unchanged, and C-NH/C-NH2 appeared more pronounced when compared to the chitosan control. At 347.4 eV, a Ca3(PO4)2 was present. The N1s spectrum exhibited a peak at 396.2 eV (Figure 4d), and the nitrogen signal arose from the chitosan amino groups and indicated a presence of chitosan in the surface layer. Another pronounced peak appeared at 529.4 eV, which could be assigned to hydroxyl groups (-ОН − ) of O1s line ( Figure 4e). The intensity of the XPS peaks for chitosan did not change when increasing the number of applied laser pulses. These results indicated that the amount of characteristic components of chitosan did not decrease upon increasing N.

Surface Analysis via SEM of Chitosan/Ceramic Composite Films
SEM images of the surface morphology for diverse surface treatments are presented in Figures 5, 6, and 7. Pure chitosan thin films possess a smooth surface. When blended with Hydroxyapatite, the surface morphology changed and showed a topography composed of granules ( Figure 5a). There was a clear difference in the micropatterns of the Ch/HAp biocomposite layers after laser treatment with the formation of a porous area in the interaction zone with grain structures. In Figures 5b and Figure 5c are SEM micrographs of the samples before and after laser treatment. Figure 4. X-ray photoelectron spectroscopy (XPS) spectra of pure chitosan thin films (a) non-treated and laser irradiated at I = 7.9 × 10 14 W/cm 2 for N = 1, 2, 5, 10; (b)-(g) resolved peaks of C 1s , C 1s-peak fitting, Ca 2p , N 1s , O 1s , Na 1s .
As seen from the XPS analysis in Figure 4, all six spectra exhibited peaks, showing binding energies at C 1s , Ca 2p , N 1s , O 1s , and Na 1s .
The C 1s peak was divided into four peaks 281.7, 283.3, 284.7, and 285.45 eV corresponding to C-C/C-H bond and C-NH/C-NH 2 (Figure 4b,c). After femtosecond laser treatment with an increasing number of pulses (N = 1-5), the C-C bond remained almost unchanged, and C-NH/C-NH 2 appeared more pronounced when compared to the chitosan control. At 347.4 eV, a Ca 3 (PO 4 ) 2 was present. The N 1s spectrum exhibited a peak at 396.2 eV (Figure 4d), and the nitrogen signal arose from the chitosan amino groups and indicated a presence of chitosan in the surface layer. Another pronounced peak appeared at 529.4 eV, which could be assigned to hydroxyl groups (-OH − ) of O 1s line ( Figure 4e). The intensity of the XPS peaks for chitosan did not change when increasing the number of applied laser pulses. These results indicated that the amount of characteristic components of chitosan did not decrease upon increasing N.

Surface Analysis via SEM of Chitosan/Ceramic Composite Films
SEM images of the surface morphology for diverse surface treatments are presented in Figure 5, Figure 6, and Figure 7. Pure chitosan thin films possess a smooth surface. When blended with Hydroxyapatite, the surface morphology changed and showed a topography composed of granules ( Figure 5a). There was a clear difference in the micropatterns of the Ch/HAp biocomposite layers after laser treatment with the formation of a porous area in the interaction zone with grain structures. In Figure 5b,c are SEM micrographs of the samples before and after laser treatment.    SEM micrographs of composite Ch/HAp layers reinforced with 1% ZrO 2 revealed the presence of porous granular with nanoscale protrusion architecture Figure 6d. SEM analysis showed the development of granular morphology with porous formation on the sample surface at both laser conditions.
Comparing the different modifications and morphology obtained via laser treatment allowed a higher degree of roughness on the surface. Closer analysis of laser treated surfaces on the middle part of the crater (Figure 6b,d) showed granular structures, which are crucial for cellular interconnectivity, expansion, and proliferation.
higher degree of roughness on the surface. Closer analysis of laser treated surfaces on the middle part of the crater (Figures 6b,d) showed granular structures, which are crucial for cellular interconnectivity, expansion, and proliferation.
Increasing the laser intensity triggers the formation of a deep crater, and at N = 10, I = 3.1 × 10 15 W/cm 2 , glass substrate was reached, creating laser induced periodic surface structures LIPSS on the glass slide, as seen in Figures 7a,b. The period of LIPSS on glass substrate (Figure 7b) is Λ = 900 nm.  Increasing the laser intensity triggers the formation of a deep crater, and at N = 10, I = 3.1 × 10 15 W/cm 2 , glass substrate was reached, creating laser induced periodic surface structures LIPSS on the glass slide, as seen in Figure 7a Comparing the different modifications and morphology obtained via laser treatment allowed a higher degree of roughness on the surface. Closer analysis of laser treated surfaces on the middle part of the crater (Figures 6b,d) showed granular structures, which are crucial for cellular interconnectivity, expansion, and proliferation.
Increasing the laser intensity triggers the formation of a deep crater, and at N = 10, I = 3.1 × 10 15 W/cm 2 , glass substrate was reached, creating laser induced periodic surface structures LIPSS on the glass slide, as seen in Figures 7a,b. The period of LIPSS on glass substrate (Figure 7b) is Λ = 900 nm.  In contrast to the observation of the morphological changes discussed above, we observed a limited interval where the creation of LIPSS in the thin composite layer was observed before the onset of full material ablation (Figure 9) when the number of laser pulses increased up to 10 and the laser intensity was 7.9 × 10 15 W/cm 2 .
Polymers 2018, 10, x FOR PEER REVIEW 11 of 21 Figure 8. EDX spectrum of microstructure fabricated by femtosecond laser irradiation with N = 1, I = 1.5 × 10 15 W/cm 2 of thepolymer/ceramic composite-70% Ch/30% HAp/ZrO2. In contrast to the observation of the morphological changes discussed above, we observed a limited interval where the creation of LIPSS in the thin composite layer was observed before the onset of full material ablation (Figure 9) when the number of laser pulses increased up to 10 and the laser intensity was 7.9 × 10 15 W/cm 2 . The LIPSS on Figure 9 were formed on the composite thin films, which was evidenced also by the acquired EDX analysis where a clear presence of Zr-14.28 wt.%, C-4.64 wt.%, and O-41.95 wt.% components from 70% Ch/30% HAp/ZrO2 thin films were detected. However, the formation of LIPSS was seen only at the edges of the spot formation close to the glass substrate with a periodicity close to the laser irradiation wavelength (Λ = 950 nm), as seen in Figure 9. Evidently, the glass substrate was contributing to the efficient feedback mechanism for LIPSS formation.
It was found that the periodic surface structures described as "ripples" formed preferentially for N = 5 to 10. They were parallel to the laser polarization direction. The formation mechanism of LIPSS was described with the optical interference between the incident wave and a surface scattered electromagnetic wave. LIPSS were fabricated for materials with low linear absorption coefficients. LIPSS were formed at a certain number of laser pulses, inducing nano-roughness. This affected the surface morphology by creating a path for the optimization of scaffold surface properties.

Fourier Transform Infrared Spectra for Laser Modified Chitosan Composite Films
The spectra of Ch/HAp/ZrO2 composites showed a mixture of characteristic transmittance due to the groups listed in Table 2.  The LIPSS on Figure 9 were formed on the composite thin films, which was evidenced also by the acquired EDX analysis where a clear presence of Zr-14.28 wt.%, C-4.64 wt.%, and O-41.95 wt.% components from 70% Ch/30% HAp/ZrO 2 thin films were detected. However, the formation of LIPSS was seen only at the edges of the spot formation close to the glass substrate with a periodicity close to the laser irradiation wavelength (Λ = 950 nm), as seen in Figure 9. Evidently, the glass substrate was contributing to the efficient feedback mechanism for LIPSS formation.
It was found that the periodic surface structures described as "ripples" formed preferentially for N = 5 to 10. They were parallel to the laser polarization direction. The formation mechanism of LIPSS was described with the optical interference between the incident wave and a surface scattered electromagnetic wave. LIPSS were fabricated for materials with low linear absorption coefficients. LIPSS were formed at a certain number of laser pulses, inducing nano-roughness. This affected the surface morphology by creating a path for the optimization of scaffold surface properties.

Fourier Transform Infrared Spectra for Laser Modified Chitosan Composite Films
The spectra of Ch/HAp/ZrO 2 composites showed a mixture of characteristic transmittance due to the groups listed in Table 2. The results illustrated significant differences in the spectra measured from the non-irradiated and laser-processed surfaces of the composite substrates. First, the FTIR spectra exhibited equal numbers and positions of the various transmittance peaks but with a major difference in the intensity of the peaks from the laser treated zones. The shapes of the spectra from both processed areas (N = 5 and N = 10 pulses) were similar ( Figure 10). In the four cases, spectrum from the non-treated surface of chitosan blends exhibited one characteristic group between 3400 and 3000 cm −1 . It corresponded to hydroxyl groups stretching in chitosan. The band between 2990-2880 cm −1 was attributed to -CH "backbone" vibrations, while the peaks near 1650 cm −1 corresponded to C=O stretching and =N-H vibrations of amide structures I and II, which were observed in all samples (laser treated and untreated). Another peak noticed at about 1267 cm −1 from the non-irradiated material surface was characteristic of the =N-H amide III structure. A maximum at about 1400 cm −1 was assigned to -CH 3 and -CH 2 groups in plane deformation vibrations [40,41]. The groups identified at about 1030 to 1020 cm −1 are commonly associated with the stretching of glucosamine in the chitosan structure. The transmittance maxima observed in the range of about 1000 cm −1 and 600-500 cm −1 were attributed to the stretching and flexing vibrations of the PO 4 3− group. These peaks were much more pronounced in the non-irradiated samples in all of the chitosan blends examined, and from the irradiated ones, it was with higher intensity in the case of N = 5 and I = 1.5 × 10 15 W/cm 2 . The area between 1130-1150 cm −1 was identified by P=O stretching vibrations [38]. The results illustrated significant differences in the spectra measured from the non-irradiated and laser-processed surfaces of the composite substrates. First, the FTIR spectra exhibited equal numbers and positions of the various transmittance peaks but with a major difference in the intensity of the peaks from the laser treated zones. The shapes of the spectra from both processed areas (N = 5 and N = 10 pulses) were similar ( Figure 10). In the four cases, spectrum from the non-treated surface of chitosan blends exhibited one characteristic group between 3400 and 3000 cm −1 . It corresponded to hydroxyl groups stretching in chitosan. The band between 2990-2880 cm −1 was attributed to -CH "backbone" vibrations, while the peaks near 1650 cm −1 corresponded to C=O stretching and =N-H vibrations of amide structures I and II, which were observed in all samples (laser treated and untreated). Another peak noticed at about 1267 cm −1 from the non-irradiated material surface was characteristic of the =N-H amide III structure. A maximum at about 1400 cm −1 was assigned to -CH3 and -CH2 groups in plane deformation vibrations [40,41]. The groups identified at about 1030 to 1020 cm −1 are commonly associated with the stretching of glucosamine in the chitosan structure. The transmittance maxima observed in the range of about 1000 cm −1 and 600-500 cm −1 were attributed to the stretching and flexing vibrations of the PO4 3− group. These peaks were much more pronounced in the non-irradiated samples in all of the chitosan blends examined, and from the irradiated ones, it was with higher intensity in the case of N = 5 and I = 1.5 × 10 15 W/cm 2 . The area between 1130-1150 cm −1 was identified by P=O stretching vibrations [38].
The presence of nanoparticles was not detected. The observed FTIR peak in the region of 498-502 cm −1 was attributed to the vibration modes of ZrO3 2− groups, which confirmed the formation of the ZrO2 structure. One band at 600 cm −1 was assigned to the Zr-O-Zr bendings [42,43]. In general, the profile of all peaks for the case of femtosecond irradiated surfaces for all presented The presence of nanoparticles was not detected. The observed FTIR peak in the region of 498-502 cm −1 was attributed to the vibration modes of ZrO 3 2− groups, which confirmed the formation of the ZrO 2 structure. One band at 600 cm −1 was assigned to the Zr-O-Zr bendings [42,43]. In general, the profile of all peaks for the case of femtosecond irradiated surfaces for all presented chitosan/hydroxyapatite concentrations coincided, as the only difference was in the intensity of the lines with a tendency to decrease with the increment of laser intensity and a number of the applied pulses. The main effect of the interaction of laser radiation was characterized by a significant reduction in the maxima associated with the saccharide ring of chitosan, which almost disappeared due to the increase in the laser intensity and the number of laser pulses. This was explained by the greater accumulation of energy in the interaction zone, which was related to the change of the polymer structure in composite biopolymer/ceramics, which in turn led to a change in the structure of the biopolymer. In a similar type of experiment from our earlier studies, we found that the threshold modification for a chitosan specimen was much lower compared to chitosan/hydroxyapatite composites [33], as was seen here for the higher concentration of HAp Figure 10d.

XRD Analysis of Composite Chitosan/Ceramic Films
The XRD of patterns for laser treated Ch/HAp/ZrO 2 blends were analyzed in the 2θ angle.
In order to obtain basic information on the phase variations of the mineral components (hydroxyapatite and zirconia) before and after laser processing of synthesized composites, a series of XRD measurements were performed (Figure 11). The detected base lines of diffractogram occurred at angles 2θ = 14 • , 17 • , 19 • , 25 • , 28 • , 31 • , 34 • , 40 • , and 50 • for each represented sample. The observed XRD peaks around 14-20 • were attributed to the intercalated layers of the chitosan components [44]. The increased sharpness, intensities, and decreased broadness of mentioned peaks could be explained by the evolution in crystal sizes of the hydroxyapatite component [44,45]. Generally, the main differences in the XRD patterns depend on the constituent elements of the scaffold, and they are related to the maximum intensity variations in correspondance to the laser intensity and increase in the number of applied pulses, respectively. The main constituents for all scaffold variations must be taken into consideration. It was observed that the peak assigned to chitosan disappeared, and peaks attributed to the hydroxyapatite appeared more distinguished. These results indicated that HAp addition decreased the crystallinity of chitosan (because it dissolved in acetic acid), while chitosan had no influence on the hydroxyapatite crystallinity structure [46]. On XRD diagrams of composite films, the presence of chitosan peak was notable. However, due to the overlap with the spectrum of HAp, its identification was dificult but was determined by the other performed analysis (FTIR). Less pronounced HAp peaks occurred in the matrices with compositions of 70:30 Ch/HAp + 10% AgNps (Figure 11c),which could be explained by the minor amount of the HAp element present in the matrix, and was the expected result. pulses. The main effect of the interaction of laser radiation was characterized by a significant reduction in the maxima associated with the saccharide ring of chitosan, which almost disappeared due to the increase in the laser intensity and the number of laser pulses. This was explained by the greater accumulation of energy in the interaction zone, which was related to the change of the polymer structure in composite biopolymer/ceramics, which in turn led to a change in the structure of the biopolymer. In a similar type of experiment from our earlier studies, we found that the threshold modification for a chitosan specimen was much lower compared to chitosan/hydroxyapatite composites [33], as was seen here for the higher concentration of HAp Figure 10d.

XRD Analysis of Composite Chitosan/Ceramic Films
The XRD of patterns for laser treated Ch/HAp/ZrO2 blends were analyzed in the 2θ angle. In order to obtain basic information on the phase variations of the mineral components (hydroxyapatite and zirconia) before and after laser processing of synthesized composites, a series of XRD measurements were performed (Figure 11). The detected base lines of diffractogram occurred at angles 2θ = 14°, 17°, 19°, 25°, 28°, 31°, 34°, 40°, and 50° for each represented sample. The observed

Cellular Affinity of Chitosan Ch/Hydroxyapatite (HAp)/ZrO 2 Composite Films
Cellular interaction with surfaces is a main issue that must be considered in the choice of biomaterials. The regulation of cell adhesion on the extracellular matrix (ECM) is governed by molecular mechanisms that provide a connection between the matrix and cell cytoskeleton. ECM topography is an important factor that influences the orientation of cell migration. When cells interact with designed matrices from diverse biomaterials, they gain a certain extracellular physicochemical signal from the topography and chemistry of the implant surface and consequently react. In this study, surface properties of chitosan/ZrO 2 /HAp composite films, specifically surface morphology and chemistry, were examined. The fabricated grooves on the composite thin layers onto which both types of cells were seeded were created in specific conditions that did not initiate material ablation, namely, N = 1, I = 3.1 × 10 15 W/cm 2 (Figure 6a,b). The grooves we obtained as a result of laser assisted modification were with fixed parameters. The estimated width and depth of the produced patterns, measured with confocal microscopy, were d x ≈ 40 µm and d y ≈ 20 µm, respectively ( Figure 12). chitosan had no influence on the hydroxyapatite crystallinity structure [46]. On XRD diagrams of composite films, the presence of chitosan peak was notable. However, due to the overlap with the spectrum of HAp, its identification was dificult but was determined by the other performed analysis (FTIR). Less pronounced HАp peaks occurred in the matrices with compositions of 70:30 Ch/HAp + 10% AgNps (Figure 11c),which could be explained by the minor amount of the HAp element present in the matrix, and was the expected result.

Cellular Affinity of Chitosan Ch/Hydroxyapatite (HAp)/ZrO2 Composite Films
Cellular interaction with surfaces is a main issue that must be considered in the choice of biomaterials. The regulation of cell adhesion on the extracellular matrix (ECM) is governed by molecular mechanisms that provide a connection between the matrix and cell cytoskeleton. ECM topography is an important factor that influences the orientation of cell migration. When cells interact with designed matrices from diverse biomaterials, they gain a certain extracellular physicochemical signal from the topography and chemistry of the implant surface and consequently react. In this study, surface properties of chitosan/ZrO2/HAp composite films, specifically surface morphology and chemistry, were examined. The fabricated grooves on the composite thin layers onto which both types of cells were seeded were created in specific conditions that did not initiate material ablation, namely, N = 1, I = 3.1 × 10 15 W/cm 2 (Figure 6a,b). The grooves we obtained as a result of laser assisted modification were with fixed parameters. The estimated width and depth of the produced patterns, measured with confocal microscopy, were dx ≈ 40 µm and dy ≈ 20 µm, respectively ( Figure 12). Their influence on MC3T3 osteoblasts and adipose derived stem cells (ADSC stem cells) adhesion, proliferation, and orientation were also estimated. It was found that both types of cells strongly responded to microscaled surface topographic structures ( Figure 6) of thin composite chitosan/HAp/ZrO2 composite films with diverse percentages of the main constituents.
On all types of laser modified thin composite films (Ch/1% HAp/1% ZrO2; 70% Ch/30% Hap + 1% ZrO2; 70% Ch/30% Hap + 10% Np; 30% Ch/70% Hap + 10% Np, 70%/30% Ch/HAp), the MC3T3 osteoblasts and ADSC stem cells attached to material surfaces exhibited significant orientation toward laser created stripes when compared to the non-modified parts of the surface. The micrographs of MC3T3 cells shown in Figure 13 were cultured on composite thin films and monitored after the first day. Their influence on MC3T3 osteoblasts and adipose derived stem cells (ADSC stem cells) adhesion, proliferation, and orientation were also estimated. It was found that both types of cells strongly responded to microscaled surface topographic structures ( Figure 6) of thin composite chitosan/HAp/ZrO 2 composite films with diverse percentages of the main constituents.
On the surface of the composite Ch/HAp/ZrO 2 thin films control (Figure 14a), the MC3T3 osteoblast cells exhibited a bipolar shape and began to congregate in the form of clusters. On the laser treated part of the samples surface, the MC3T3 cells exhibited an extended shape directed towards the created microgrooves ( Figure 13).
For all five groups (30% Ch/70% HAp, 70% Ch/30% HAp, 70% Ch/30% Hap + 1% ZrO 2 , 70% Ch/30% Hap + 10% Np, 30% Ch/70% Hap + 10% Np) of modified composite thin films, ADSC stem cells exhibited an elongated shape and grew as an adherent spreading monolayer along the laser formed grooves (Figure 15). Monitoring of ADSC stem cells on all substrates was performed after six days culture time. The enhanced orientation could be observed on most of the composite thin modified films in relation to the control (Figure 14b). On the surface of the composite Ch/HAp/ZrO2 thin films control (Figure 14a), the MC3T3 osteoblast cells exhibited a bipolar shape and began to congregate in the form of clusters. On the laser treated part of the samples surface, the MC3T3 cells exhibited an extended shape directed towards the created microgrooves ( Figure 13).
For all five groups (30% Ch/70% HAp, 70% Ch/30% HAp, 70% Ch/30% Hap + 1% ZrO2, 70% Ch/30% Hap + 10% Np, 30% Ch/70% Hap + 10% Np) of modified composite thin films, ADSC stem cells exhibited an elongated shape and grew as an adherent spreading monolayer along the laser formed grooves (Figure 15). Monitoring of ADSC stem cells on all substrates was performed after six days culture time. The enhanced orientation could be observed on most of the composite thin modified films in relation to the control (Figure 14b). The cellular attachment to the ECM is a major factor in controlling important cellular processes such as adhesion, migration, proliferation, and differentiation. The laser created micropatterns of all samples induces the reorganization of cells movement that affects the cells cytoskeleton, consequently elongating its shape. Terakawa [47] made an extended review in which cell adhesion and behavior on the laser-irradiated surface of biodegradable polymers is described in detail.  On the surface of the composite Ch/HAp/ZrO2 thin films control (Figure 14a), the MC3T3 osteoblast cells exhibited a bipolar shape and began to congregate in the form of clusters. On the laser treated part of the samples surface, the MC3T3 cells exhibited an extended shape directed towards the created microgrooves ( Figure 13).
For all five groups (30% Ch/70% HAp, 70% Ch/30% HAp, 70% Ch/30% Hap + 1% ZrO2, 70% Ch/30% Hap + 10% Np, 30% Ch/70% Hap + 10% Np) of modified composite thin films, ADSC stem cells exhibited an elongated shape and grew as an adherent spreading monolayer along the laser formed grooves (Figure 15). Monitoring of ADSC stem cells on all substrates was performed after six days culture time. The enhanced orientation could be observed on most of the composite thin modified films in relation to the control (Figure 14b). The cellular attachment to the ECM is a major factor in controlling important cellular processes such as adhesion, migration, proliferation, and differentiation. The laser created micropatterns of all samples induces the reorganization of cells movement that affects the cells cytoskeleton, consequently elongating its shape. Terakawa [47] made an extended review in which cell adhesion and behavior on the laser-irradiated surface of biodegradable polymers is described in detail. All cells on the samples showed a high viability (Figures 13 and 15) and were comparable to the cells seeded on the control tissue culture polystyrene ( Figure 14). The results from the live/dead cell staining (Figures 13 and 15) showed that the viable cells (green) were much more than the dead (red stained) ones and was indicative of the influence of the laser modification on the cellular properties. The cellular attachment to the ECM is a major factor in controlling important cellular processes such as adhesion, migration, proliferation, and differentiation. The laser created micropatterns of all samples induces the reorganization of cells movement that affects the cells cytoskeleton, consequently elongating its shape. Terakawa [47] made an extended review in which cell adhesion and behavior on the laser-irradiated surface of biodegradable polymers is described in detail.
All cells on the samples showed a high viability (Figures 13 and 15) and were comparable to the cells seeded on the control tissue culture polystyrene ( Figure 14). The results from the live/dead cell staining (Figures 13 and 15) showed that the viable cells (green) were much more than the dead (red stained) ones and was indicative of the influence of the laser modification on the cellular properties. Both types of cells seeded onto the groove-like patterns demonstrated very high cell viability. Almost no dead cells were present. After a further culture period of six days (Figure 15a-j), the ADSC stem cells proliferated well on the groove-like patterns. This confirmed that the laser treated part of the samples surface created even better conditions for cells proliferation on the "bone mimic medium". This further supports and develops previous research of the group [33,34]. The results obtained are of great importance in tissue engineering due to the requirement of a suitable microstructured matrix, which triggers the development of a large number of oriented cells for bone tissue repairing.

Discussion
It was found that implant surface properties affect primary stability and influence mechanical bonding with the tissue environment. Different studies have reported on the major role of tissue-toimplant adhesion for achieving primary stability. This property can be improved through implant surface processing [47].
Previous studies have been performed on surface processing by utilizing other laser sources and chemical methods, which have drawbacks related to collateral damage and side effects affecting the biocompatibility of the biomaterial [6,17]. The applicability of lasers working in different nanosecond or microsecond pulse durations for material processing is associated with the development of heat affected zones, melting, and evaporation of the material [48,49].
In contrast, the femtosecond laser modification method has significant advantages related to material treatment and ablation with high precision without the development of thermal damage or microcracks formation. Malinauskas et al. successfully applied direct laser writing (DLW) ablation to modify the surfaces of the PLA structures by fabrication of 3D microporous structures with functionalization properties. In their studies, a three-dimensional printing (3DP) approach was applied for the fabrication of macrostructures with internal 3D microstructure. The DLW via ablation was employed to modify the surfaces, hole drilling, and outer geometry shaping of the whole objects and create surface textures on micro-and nano-scales [50].
Interaction in the femtosecond time domain is based on the nonlinear processes of light absorption and ionization [51,54].
In this research, a series of composite thin films was prepared by blending chitosan with hydroxyapatite, Zirconia (ZrO 2 ), and Ag nanoparticles in various proportions, and the surface morphology and chemistry of these composite films were systematically examined.
After the incorporation of a ceramic component with the main constituent, the surfaces of composite films showed a granule-like topography. These observations in surface topography could be assigned to the different crystallization process between chitosan and ceramic particles. The chitosan matrix possesses intermolecular and intramolecular hydrogen bonds. These bonds are involved in the crystallization process by forming particle crystals [55].
The laser treatment at 800 nm laser radiation produced a clean surface modification at a lower intensity range without the formation of melting and cracks. It was noted that the surface treatment of chitosan composite samples by fs laser irradiation produced adequate structuring from the point of view of surface topography. For most types of materials, melting occurred at very high intensities near the peak of the Gaussian beam. Laser modification of biopolymer/ceramic blends initiated a process of morphological surface changes in very low levels of laser intensities. The damage induced by femtosecond laser pulses at intensities exceeding 3.1 × 10 15 W/cm 2 resulted in composite material ablation. Increment of intensity and number of pulses triggered the development of microscale rough structures. As intensity increased further, the formation of deeper craters on the biopolymer and the destruction of polymer/ceramic matrices were observed. In the case of biopolymer/ceramic blends, signs of open porosity were detectable, which suggested the initiation of a bubble formation mechanism attributed to liquid-vapor instability.
From the AFM results of pure chitosan film treatment with diverse expositions to selected number of applied laser pulses (N), it was estimated that by increasing N and laser intensity, the roughness parameter (R rms ) increased proportionally. The overall roughness was a consequence of the superposition of the created patterns (depending on the set condition for overlapping between separate laser shots) and additional side effects like cracks or pores formation. It was found that the laser parameters interplay (intensity and number of pulses) affected surface roughness [54]. The tunability of laser processing via variation of laser intensity and number of pulses had a joint effect on the morphology of the modified material. In order to obtain porous patterns, both the intensity and the number of pulses had to be kept (in the case of pure chitosan and also for its ceramic blends) in its low range. SEM, FTIR, and XPS analysis proved that this regime led to defined structuring without drastic alteration of chemical composition. Ideal porous modifications could be achieved by irradiation with a low number of pulses (N = 1 and 2) for deeper patterns and an increase of I and N led to the disruption in the quality of the structures.
The examination of the influence of laser created surface patterns on composite films on two types of cells (MC3T3 osteoblasts and ADSC stem cells) provides a preliminary model for cell-interface studies. The monitoring of cellular adhesion and proliferation on all six groups of composite films was achieved. The results from cellular studies on fs laser modified films showed indicative proliferation and orientation of MC3T3 osteoblasts and ADSC stem cells compared to the non-treated control. One possible explanation for these discoveries was that the substrate surfaces had increased roughness that mimicked, in part, the microstructure of the native extra-cellular matrix (ECM). In the human body, the ECM serves as a support to cells and guides their mobility [60].
Moreover, the influence of surface patterning on protein adsorption [47] must also be explored. It has been reported that protein absorption plays a main role in cellular adhesion, which is explained in detail in the review of Khalili and Ahmad [60].
Another property that has been found to drastically affect cell adhesion is the presence of microporosity in biocomposite scaffolds [61], which is an important factor in allowing cellular infiltration and proliferation inside a volume. The natural hierarchical architecture of bone ranges from pores of around 1 µm (interaction with proteins) to 1 to 20 µm (cellular in-growth), 100 µm and up to more than 500 µm (for cellular growth and bone in-growth). With laser-assisted technology, control of porosity via the selection of a number of applied laser pulses and intensity can be achieved and adapted for implant materials.

Conclusions
In summary, femtosecond laser material processing represents a fast and precise method for initiating controlled morphological changes at the micrometric scale on the surface of chitosan and its ceramic blends. Enhancement of physicochemical and morphological properties of the chitosan and its ceramic composite blends were studied. The interplay between the applied laser parameters and the treatment conditions gives diverse structural alterations. These studies show that the laser surface processing technique is an alternative method to conventional surface treatment techniques like sandblasting and acid etching because it permits easy control of the roughness without additional chemical contamination, which in turn helps to enhance cellular adhesion. Tuning laser parameters permits close control over pattern type and properties like roughness and depth.