Results
Phase I (01/04/2022 - 31/12/2022)
The MAPLE experiments conducted for the deposition of thin films were carried out in a stainless steel chamber, with the help of a KrF* excimer laser source (λ = 248 nm, τFWHM = 25 ns), model COMPexPro 205. The morphology of the surfaces of the BG57+VD3 films obtained through MAPLE was investigated using SEM. The morphology of the film surface changed with the addition of VD3, becoming smoother. EDS spectra highlighted the constituent elements of the Ti substrate and confirmed the presence of BG on the substrate. FTIR revealed efficient transfer of the composite material and preservation of the chemical integrity of all elements in the film. BG57+Mel samples were immersed in SBF and investigated after various intervals. FTIR spectroscopy was used to assess chemical changes, and SEM to evaluate the morphological modification of surfaces, after various immersion times in SBF. Regardless of the immersion time, the morphology of the surfaces did not visibly change. EDS analysis of the BG57+Mel films, after various immersion times, highlighted the spots associated with Ti, and the peaks associated with Ca, and P, as well as Cl and Na (characteristic of salts deposited from SBF on the film,) during immersion in SBF.
The cytotoxicity of Mel and VD3 solutions was evaluated on NCTC L929 cells, using MTT and LDH tests 24 hours after incubation. The biocompatibility of the MAPLE films was evaluated on HDF cells, and the degree of biocompatibility was assessed through MTT and LDH tests. According to the LDH test, all tested materials had higher values compared to the control sample. Cellular morphology evaluation was performed using fluorescence microscopy following the cultivation of human cells on the tested materials for 24 hours. Cultivating human cells on the tested materials did not induce major cellular changes. After testing the inflammatory profile of human cells cultivated on the thin films obtained by MAPLE, it was observed that the thin films did not induce an inflammatory process at the cellular level, the cytokine profile being similar to that of the control sample. The evaluation of the antimicrobial activity of Mel and VD3 solutions, before incorporation into thin films, was carried out by the microdilution method in liquid medium.
The results obtained in this phase thus allow for the continuation of the subsequent stages of the project.
Phase II (01/01/2023 - 31/12/2023)
In the release studies of AMP and VD3, as well as the surface transformations of the coatings, complex experiments were conducted to evaluate the performance of biomaterials in various contexts.
In the first part of the studies, the BG57+VD3 samples were analyzed under static and dynamic conditions to simulate interaction with human body fluids. A constant release of VD3 was observed over time, and the surface transformations were characterized by FTIR spectroscopy, highlighting the formation of carbonated apatite. Subsequently, transformations at the surface levels of the BG57+VD3 and BAV coatings were explored, identifying various changes through FTIR spectroscopy.
The process of carbonated apatite formation was highlighted, consolidating the coatings' efficiency in the simulated physiological environment.
To verify the biocompatibility of the thin films, tests were carried out on two different cell lines. Tests such as MTT, LDH, Live-Dead, and ELISA were conducted to evaluate cellular viability and the reaction to the tested materials. The results indicate the biocompatibility of the BAV thin film.
Furthermore, the antimicrobial activity of the thin films against Gram-positive and Gram-negative bacteria was tested. The thin films show anti-biofilm activity against standard and clinical isolates of bacteria, such as S. aureus, P. aeruginosa, E. coli, E. faecalis. The BAV-based material inhibits biofilm in some cases. In addition to these aspects, further analyses were conducted, such as the wettability properties of surfaces coated with BG57 and BAV, highlighting changes in hydrophilicity depending on the applied thin film.
Phase III (01/01/2024 - 31/03/2024)
During the third phase of the project, the focus was on the detailed analysis of BAV thin films, to evaluate their performance and biocompatibility.
The studies encompassed a series of experiments designed to mimic the real conditions of the human body and to investigate the films' capability to release active substances like AMP and VD3 in a controlled and sustainable manner. A constant release of active substances was noted, indicating the films' effectiveness in maintaining long-term therapeutic action. This aspect is crucial for the potential use of the films in medical applications, where controlled drug release can significantly improve treatment and healing.
Surface analysis of the films through FTIR spectroscopy highlighted the formation of carbonated apatite, a marker of bioactivity and successful material integration in the physiological environment. This underscores the potential of BAV thin films to support not only healing but also tissue integration, crucial aspects in their application as coatings for implants or in tissue engineering.
Biocompatibility tests conducted on various cell lines, including assessments such as MTT, LDH, Live-Dead, and ELISA, confirmed the excellent compatibility of the BG57+VD3+Mel films with the cellular environment. These results highlight the material's ability to support cell growth and viability, making it a promising option for various biomedical applications.
Furthermore, the thin films demonstrated remarkable antimicrobial activity against a wide range of pathogens, including Gram-positive and Gram-negative bacteria and fungi. This anti-biofilm activity emphasizes the films' utility in preventing infections associated with medical devices, a significant challenge in the health sector.
Additional analyses, such as studies on the wettability properties of the coated surfaces, indicated improvements in hydrophilicity, beneficial for promoting cell adherence and tissue integration. These findings strengthen the argument that BAV thin films can play a significant role in optimizing the interface between implants and host tissue, enhancing the success of medical interventions and the quality of patients' lives.