Vapor-Phase-Deposited Ag/Ir and Ag/Au Film Heterostructures for Implant Materials: Cytotoxic, Antibacterial and Histological Studies
Abstract
:1. Introduction
2. Results
- “Ti” carriers: pristine Ti6Al4V discs; according to atomic force microscopy (AFM) data, the average roughness (Ra) is 30 nm (Figure 1a);
- “Ti*” carriers: surface-polished Ti6Al4V discs; according to AFM data, the ordered grooved altitude differences are up to 300 nm whereas Ra is 60 nm (Figure 1b);
- “CFR-PEEK” carriers: polyetheretherketone discs reinforced with carbon fiber (30%); according to AFM data, Ra is 55 nm.
2.1. Deposition and Characterization of Ir and Au Sublayers
2.2. Deposition and Characterization of Ag/Ir and Ag/Au Heterostructures
2.2.1. Ag/Ir and Ag/Au Heterostructures Obtained via the PVD Method
2.2.2. Ag/Ir and Ag/Au Heterostructures Obtained via MOCVD
2.3. Biological Studies
2.3.1. Antibacterial Activity
2.3.2. Cell Viability
2.3.3. In Vivo Biocompatibility
3. Discussion
3.1. Film Heterostructures: Active Component Formation
3.2. Antibacterial Activity
3.3. In Vivo and In Vitro Biocompatibility
4. Materials and Methods
4.1. Materials
4.2. Preparation of Ir, Au Layers and Ag/Ir, Ag/Au Heterostuctures
4.3. Methods
4.4. Biological Studies
4.4.1. Antibacterial Activity Test
4.4.2. Cytotoxicity via the XTT Test
4.4.3. Subcutaneous Implantation in Animals
4.4.4. Histological Test
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample | Implantation Time (Days) | Packing Density | Microvessels | Macrophages | Lymphocytes | FBCs | Mast Cells |
---|---|---|---|---|---|---|---|
Ag/Ir/Ti | 30 | 1–2 | 1 | 1 | 0 | 0 | 0 |
90 | 1 | 1 | 1 | 0 | 0 | 0 | |
Ag/Ir/Ti* | 30 | 0–1 | 1 | 0 | 0 | 0 | 0 |
90 | 2–3 | 1 | 2–3 | 0 | 0 | 0 | |
Ag/Au/Ti | 30 | 2 | 1 | 0 | 0 | 0 | 0 |
90 | 1–2 | 0 | 0 | 0 | 0 | 0 | |
Ag/Au/CFR-PEEK | 30 | 0–1 | 1 | 0 | 0 | 0 | 0 |
90 | 1–2 | 2 | 1 | 0 | 0 | 0 | |
Ag/Ir/CFR-PEEK | 30 | 1–2 | 1 | 0 | 0 | 0 | 0 |
90 | 1 | 1 | 1 | 0 | 0 | 0 |
Sample | Initial Ag Content (μg/cm2) | Implantation Time (30 Days) | Implantation Time (90 Days) |
---|---|---|---|
Ag/Au/CFR-PEEK | 13.2 ± 1.3 | 1.8 ± 0.1 | 0.89 ± 0.08 |
Ag/Ir/CFR-PEEK | 13.5 ± 1.3 | 1.4 ± 0.1 | 0.89 ± 0.08 |
Ag/Ir/Ti | 15.7 ± 1.5 | 5.2 ± 0.2 | 2.6 ± 0.1 |
Ag/Ir/Ti* | 16.2 ± 1.5 | 3.2 ± 0.2 | 1.4 ± 0.1 |
Ag/Au/Ti | 18 ± 1.5 | 16 ± 1 | 13.2 ± 0.3 |
Substrate | Deposition Conditions | Sample Series | ||||
---|---|---|---|---|---|---|
Precursor | Load (mg) | Temperature (°C) | Details | |||
Tv | Td | |||||
CFR-PEEK | Ag | 5 | 1300 | 150 | PVD UVM.71 installation, U = 400 V, I = 400 mA 8 × 10−7 Torr | Ag/CFR-PEEK |
Ir/CFR-PEEK | Ag/Ir/CFR-PEEK | |||||
Au/CFR-PEEK | Ag/Au/CFR-PEEK | |||||
[Ag(cod)(hfac)]2 | 40 | 120 | 300 | MOCVD hand-made installation, v(Ar) = 9 L/h, v(H2) = 12 L/h, 5.7 Torr | ||
Ir/Ti | Ag/Ir/Ti | |||||
Ir/Ti* | Ag/Ir/Ti* | |||||
Au/Ti | Ag/Au/Ti |
Method | Abbreviation | Equipment and Experimental Details | Interpretation |
---|---|---|---|
X-ray diffraction | XRD | Shimadzu XRD-7000 diffractometer, Shimadzu, Kyoto, Japan, CuKα radiation (2θ = 10–80°). | XRD patterns of Au/Ti and Au/CFR-PEEK samples were indexed according to the RDF-2 database (ICDD, Newtown Square, PA, USA) (version 2022). |
Inductively coupled plasma atomic emission spectroscopy | ICP-OES | High-resolution spectrometer iCAP 6500 (Thermo Fisher Scientific, Waltham, MA USA). The registration of samples was performed at the axial observation of plasma: cooling argon flow was 12 L/min, secondary was 0.5 L/min, registration time was 5 s, power supplied to an ICP inductor was 1150 W. | The nitric acid solutions of as-deposited samples (Table 3, Column 7) and samples after 30 and 90 days of implantation were prepared using the standard procedure described in Ref. [25]. The Ag and Au contents were calculated using the most intense analytical lines: 208.209, 242.795, 267.595 nm for Au, and 328.068, 338.289 nm for Ag. |
X-ray photoelectron spectroscopy | XPS | PHOIBOS-150 analyzer with 1D-DLD detector, FOCUS-500 monochromator (SPECS, Berlin, Germany), Al Kα radiation, hv = 1486.71 eV, 14 kV, 200 W). Calibration of binding energies by the Fermi level of the valence band (0.0 eV). | The spectra of Ag/Au/CFR-PEEK, Ag/Ir/Ti, and Ag/Au/Ti samples were processed in the CASA program version 2.2 software (Tokyo, Japan) using the Voigt function. The background was taken into account using the Shirley method. For Ir, Au 4f peaks fitting, the parameters proposed by Pfeifer et al. were used [73]. |
Atomic force microscopy | AFM | Microscope with a semi-contact mode with an Ntegra Prima II (NTMDT, Moscow, Russia): length was 123 µm, width was 34 µm, force constant was 17 N/m, and resonance frequency was 230 kHz. | The roughness parameters of bare Ti, Ti* substrates and samples (Table 3, Column 7) were calculated using the Nova SPM version 4.0 software 197374, St. Petersburg, Savushkina street, building 83 k3, office 236, Russia |
Scanning Electron Microscopy | SEM | HITACHI UHR FE-SEM SU8200, Hitachi, Ltd., Hitachi, Japan | All samples (Table 3, Column 7) were scanned at the same conditions (3 keV, LA detector). SEM images of the samples (Tiff files) were imported into the program image analysis version 1.54 software (ImageJ) National Institutes of Health, 9000 Rockville Pike, Bethesda, Rockville, MA, USA (version 2.0.0) to study Ag particles size distribution in case of Ag/CFR-PEEK. |
Energy-dispersive X-ray spectroscopy | EDX | EDX-analizator EX-2300BU connected with SEM, JEOL-ISM 6700 F microscope, Tokyo, Japan | EDX spectra of Ag/Ir/CFR-PEEK and Ag/Au/CFR-PEEK were recorded using Quantax 70 Bruker version 1.3 software, Schwazchid st. 12, Berlin, Germany |
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Sergeevichev, D.S.; Dorovskikh, S.I.; Vikulova, E.S.; Chepeleva, E.V.; Vasiliyeva, M.B.; Koretskaya, T.P.; Fedorenko, A.D.; Nasimov, D.A.; Guselnikova, T.Y.; Popovetsky, P.S.; et al. Vapor-Phase-Deposited Ag/Ir and Ag/Au Film Heterostructures for Implant Materials: Cytotoxic, Antibacterial and Histological Studies. Int. J. Mol. Sci. 2024, 25, 1100. https://doi.org/10.3390/ijms25021100
Sergeevichev DS, Dorovskikh SI, Vikulova ES, Chepeleva EV, Vasiliyeva MB, Koretskaya TP, Fedorenko AD, Nasimov DA, Guselnikova TY, Popovetsky PS, et al. Vapor-Phase-Deposited Ag/Ir and Ag/Au Film Heterostructures for Implant Materials: Cytotoxic, Antibacterial and Histological Studies. International Journal of Molecular Sciences. 2024; 25(2):1100. https://doi.org/10.3390/ijms25021100
Chicago/Turabian StyleSergeevichev, David S., Svetlana I. Dorovskikh, Evgeniia S. Vikulova, Elena V. Chepeleva, Maria B. Vasiliyeva, Tatiana P. Koretskaya, Anastasiya D. Fedorenko, Dmitriy A. Nasimov, Tatiana Y. Guselnikova, Pavel S. Popovetsky, and et al. 2024. "Vapor-Phase-Deposited Ag/Ir and Ag/Au Film Heterostructures for Implant Materials: Cytotoxic, Antibacterial and Histological Studies" International Journal of Molecular Sciences 25, no. 2: 1100. https://doi.org/10.3390/ijms25021100