Raman spectra (between 1100–1800 cm^-1) of the fresh <i>(A)</i> and aged <i>(B)</i> C₆₀/Ti composites with various Ti concentrations (low: 25%, medium: 45%, high: 70%).

<p>For comparison, a spectrum from the C₆₀ standard is shown.</p

Raman spectrum of thin films formed in Petri dishes by evaporating water solutions after incubation of C₆₀/Ti composites with a low <i>(A)</i>, medium <i>(B)</i> and high <i>(C)</i> content of Ti for 24 hours and then for another 48 hours.

<p>No A_g(2) vibration mode (i.e., no presence of C₆₀) was confirmed.</p

Metabolic activity measured by the XTT test per culture of human osteoblast-like MG-63 cells on day 7 after seeding on fresh and aged C₆₀/Ti composites with various Ti concentrations (low: 25%, medium: 45%, high: 70%).

<p>GS: microscopic glass coverslips, a reference material. No significant differences among the experimental groups were found.</p

Growth and Potential Damage of Human Bone-Derived Cells Cultured on Fresh and Aged C_{60 - Table 1}/Ti Films

<p>Main characteristics of the surface morphology of the prominences for fresh and aged (1-year-old) C₆₀/Ti thin films with various Ti concentrations (low: 25%, medium: 45%, high: 70%). TP/TG—ratio between the thickness of the prominences and the thickness of the grooves, S—particle size, D—particle area density. Scanning areas: 5 x 5 μm².</p><p>Growth and Potential Damage of Human Bone-Derived Cells Cultured on Fresh and Aged C_{60 - Table 1}/Ti Films</p

Numbers of human osteoblast-like MG-63 cells on fresh or aged C₆₀/Ti composites with various Ti concentrations (low: 25%, medium: 45%, high: 70%) on day 1 <i>(A)</i>, 3 <i>(B)</i> and 7 <i>(C)</i> after seeding.

<p>GS: microscopic glass coverslips, a reference material. No significant differences among the experimental groups were found.</p

Flow cytometry of the marker of DNA damage response: gamma-H2AX in human osteoblast-like U-2 OS cells on fresh (A) and aged (B) C₆₀/Ti composites with various Ti concentrations (low: 25%, medium: 45%, high: 70%).

<p>GS: microscopic glass coverslips, reference material; GS+NCS: positive control to phosphorylation of histon H2AX (gamma-H2AX), induced by 1 hour incubation of U-2 OS in neocarzinostatin (NCS; 700ng/mL). M19 and M1 define the percentage of cells with no increase in DNA damage (obtained from cells growing on the reference material, GS); M20 and M2 define the percentage of cells with an increased DNA damage response represented by enhanced phosphorylation of histon H2AX (obtained from cells incubated with NCS).</p

Morphology of human osteoblast-like MG-63 cells on day 3 after seeding on fresh and aged C₆₀/Ti composites with various concentrations of Ti (low: 25%, medium: 45%, high: 70%).

<p>GS: microscopic glass coverslips, reference material.</p

Scheme of the preparation of hybrid fullerene C₆₀/metal composites.

<p>Deposition rates: DR(M) = DR(C₆₀) ~ 1 nm/min. Temperatures during deposition: RT.</p

Immunofluorescence staining of markers of a DNA damage response: 53BP1 (green) and gamma-H2AX (red) in human osteoblast-like U-2 OS cells on fresh and aged C₆₀/Ti composites with various Ti concentrations (low: 25%, medium: 45%, high: 70%).

<p>GS: microscopic glass coverslips, reference material; GS+NCS: positive control of DNA damage response induced by 1 hour incubation of U-2 OS in neocarzinostatin (NCS; 700ng/mL).</p

Growth and Potential Damage of Human Bone-Derived Cells Cultured on Fresh and Aged C₆₀/Ti Films

<div><p>Thin films of binary C₆₀/Ti composites, with various concentrations of Ti ranging from ~ 25% to ~ 70%, were deposited on microscopic glass coverslips and were tested for their potential use in bone tissue engineering as substrates for the adhesion and growth of bone cells. The novelty of this approach lies in the combination of Ti atoms (i.e., widely used biocompatible material for the construction of stomatological and orthopedic implants) with atoms of fullerene C₆₀, which can act as very efficient radical scavengers. However, fullerenes and their derivatives are able to generate harmful reactive oxygen species and to have cytotoxic effects. In order to stabilize C₆₀ molecules and to prevent their possible cytotoxic effects, deposition in the compact form of Ti/C₆₀ composites (with various Ti concentrations) was chosen. The reactivity of C₆₀/Ti composites may change in time due to the physicochemical changes of molecules in an air atmosphere. In this study, we therefore tested the dependence between the age of C₆₀/Ti films (from one week to one year) and the adhesion, morphology, proliferation, viability, metabolic activity and potential DNA damage to human osteosarcoma cells (lines MG-63 and U-2 OS). After 7 days of cultivation, we did not observe any negative influence of fresh or aged C₆₀/Ti layers on cell behavior, including the DNA damage response. The presence of Ti atoms resulted in improved properties of the C₆₀ layers, which became more suitable for cell cultivation.</p></div