Results
Phase 1: Laser fabrication and testing of ZnO thin films
We obtained ZnO thin films on stainless steel substrates by the PLD method, using a KrF* excimer laser source (λ = 248 nm, τFWHM ≤ 25 ns). The thin films were morphologically, compositionally and structurally characterized. SEM investigations performed on the sample obtained with the highest number of pulses, revealed a relatively uniform deposition, with specific ZnO structures and with a thickness of ~100 nm. The EDS spectra revealed both the substrate constituent elements and the ones characteristic of Zn and O. The crystalline structure of the ZnO thin films was revealed by XRD. By AFM it could be observed that the surface of the films was not smooth and the grains did not have a homogeneous distribution. The deposition of the multifunctional composites (BG57+0.01SIMV, BG57+0.02SIMV, BG57+NS) on stainless steel and Ti substrates was performed by the MAPLE method, using the same laser source. Also, within this phase, we developed a borate BG doped with Ce (BBGi); we deposited thin films of BBGi and BBGi+PVP. The surface morphology of the thin films deposited by MAPLE was examined by SEM. The MAPLE - obtained thin films were tested on the 3T3 osteoblast cell line. The results of the MTT assay demonstrated that the number of cells cultured on the BBGi films increased during the tested experimental period. LDH values obtained from cells grown on all films were not higher than those of the control, indicating no induction of cytotoxicity. BBGi-based films stimulated the production of reactive oxygen species by RAW macrophages and BG57-based films did not induce oxidative stress following co-cultivation with RAW macrophages. After the Live Dead test, it was observed that all MAPLE-obtained thin films are biocompatible, but the BBGi one presented the best degree of biocompatibility.
The results obtained in this phase allow the subsequent stages of the project to be carried out.
Phase 2: Functionalization of SS Substrates Using the MAPLE Technique
In the first part of this report, we used FTIR spectroscopy to analyze the substrates coated using the MAPLE technique in the previous stage. We evaluated the integrity of chemical functions and the stoichiometry of the deposited films, including BG57+SIMV, BG57+NS, BG57+SIMV+NS, and BG57+LOV. The FTIR spectra revealed the distinct characteristics of these thin films, highlighting the presence of NS and SIMV in the composition. We also observed the specific vibrations of functional groups, confirming the successful use of the MAPLE technique in obtaining composite coatings. We detailed our observations regarding the FTIR spectra of the BG57+LOV films, comparing them with drop-cast samples. We highlighted the characteristic peaks of LOV and the involved functional groups. Additionally, we noted the overlap of certain peaks with those of the BG substrate, confirming the homogeneity of the composite coatings.
The surface topography analysis of the AFM samples revealed that, even though the films do not have a uniformly smooth surface, they completely cover the substrates without obvious gaps. We explored the surface transformations of the thin films in interaction with SBF. We immersed the samples in SBF and subjected them to analysis under static and dynamic conditions. The constant release of active substances from the thin films was evidenced by UV-Vis spectroscopy. This result underscores the stability and effectiveness of our composite coatings in the context of interaction with the physiological environment. After each immersion interval, the samples were visualized using SEM.
The films were subjected to electrochemical tests, and it was observed that the addition of SIMV and NS influenced the electrochemical behavior of the system, with some formulations showing better corrosion resistance. The immersion experiments in SBF demonstrated the constant release of active substances from the films over time, regardless of the simulated conditions.
The BG+ST+NS films were transferred onto SS substrates previously coated with ZnO, and this stage was achieved using MAPLE technology. The experimental parameters, such as laser energy and fluence, were presented.
The research team studied the surface topography of the BG+ST+NS/SS samples using AFM microscopy. Tapping mode scans were performed on various areas of the samples.
Subsequently, the biocompatibility of the samples was evaluated on G292 osteoblasts, and the results were obtained through various tests, including the MTT test for cell viability, the LDH test for cytotoxicity, and the Live/Dead test for cell morphology and viability. The oxidative stress and reactive nitrogen species analysis was performed on RAW macrophages. BG57-based materials did not induce oxidative stress or the production of reactive nitrogen species.
The study of antimicrobial activity included testing on different microbial strains. Materials with SIMV and ZnO showed the best antimicrobial and anti-biofilm activity.
The results were disseminated through participation in scientific conferences and the publication of three scientific articles. Additionally, a patent was filed with OSIM.
Phase 3: Functionalization of SS Substrates Using the MAPLE Technique
Thin films of BG+ST+NS were deposited on SS substrates (0.8x0.8 cm²) coated with ZnO. Additional Si (100) wafers were used to facilitate structural, morphological, and compositional analysis. The MAPLE technique was used for deposition, with specific experimental parameters.
The biocompatibility of the samples was tested on three cell lines: fibroblasts, epithelial cells, and monocytes. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, seeded at a density of 10⁴ cells per well, and incubated for 24 hours with the test materials. Biocompatibility was evaluated using MTT and LDH assays. The results showed an increase in cell numbers on LOV and SIMV-based materials, indicated by increased absorbance values at 550 nm. LDH release indicated the absence of necrotic cell death for most thin films, although films on ZnO coatings showed slight cytotoxicity.
The morphology and viability of cells in the presence of materials were evaluated using Live/Dead assays on HDF and epithelial cells. Viable cells were stained with calcein and Hoechst. All tested materials were biocompatible, as evidenced by MTT, LDH, and Live/Dead assays.
Sterilized samples were exposed to microbial suspensions (10⁶ CFU/mL) in a liquid medium. After 24 hours, microbial viability was determined by serial dilution and plating on agar medium. All tested thin films exhibited activity against Gram-negative bacteria (E. coli, P. aeruginosa). Activity was also observed against a clinical isolate of MRSA (methicillin-resistant Staphylococcus aureus). No significant activity was observed against Gram-positive strains (S. aureus, E. faecalis). The films showed activity against C. albicans.
FTIR spectroscopy was used to evaluate the chemical integrity and stoichiometry of the thin films, revealing the presence of functional groups and confirming the successful deposition of composite coatings.
SEM images showed irregular structures associated with the ZnO layer on the SS surface. AFM analysis provided surface roughness parameters, with values between 0.09 and 0.1 µm.
Spectrophotometric measurements showed a gradual release of the therapeutic agent, with different release profiles depending on the coating composition. SEM analysis post-immersion in SBF revealed surface changes, with regular structures indicating ongoing transformations.
The evaluation of the electrochemical behavior of the films showed that SIMV-ZnO exhibited better corrosion resistance compared to LOV-ZnO.
The findings from this stage indicate successful deposition and functionalization of SS substrates using the MAPLE technique, with promising biocompatibility and antimicrobial properties. Further preclinical and clinical studies are recommended to validate these materials for practical medical applications.
(*) During the implementation period of the Project, the PhD student successfully defended her theses (Oana Gherasim, "Multifunctional bionanostructured materials", Summa cum laude qualification)