Cytotoxicity and antibacterial properties associated with the dopant release of Cu-doped Biphasic Calcium Phosphate (BCP) powders, mainly composed of hydroxyapatite mixed with β-tricalcium phosphate powders, were investigated. Twelve BCP ceramics were synthesized at three different sintering temperatures (600 °C, 900 °C and 1200 °C) and four copper doping rates (x = 0.0, 0.05, 0.10 and 0.20, corresponding to the stoichiometric amount of copper in Ca₁₀Cu_x(PO₄)₆(OH)_2-2xO_2x). Cytotoxicity assessments of Cu-doped BCP powders, using MTT assay with human-Mesenchymal Stem Cells (h-MSCs), indicated no cytotoxicity and the release of less than 12 ppm of copper into the biological medium. The antibacterial activity of the powders was determined against both Gram-positive (methicillin-sensitive (MS) and methicillin resistant (MR) Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The Cu-doped biomaterials exhibited a strong antibacterial activity against MSSA, MRSA and E. coli, releasing approximatively 2.5 ppm after 24 h, whereas 10 ppm were required to induce an antibacterial effect against P. aeruginosa. This study also demonstrated that the culture medium used during experiments can directly impact the antibacterial effect observed; only 4 ppm of Cu²⁺ were effective for killing all the bacteria in a 1:500 diluted TS medium, whereas 20 ppm were necessary to achieve the same result in a rich, non-diluted standard marrow cell culture medium.

The results presented in this paper, based on the powder X-ray diffraction technique followed by Rietveld analyses, are devoted to the mechanism of silver incorporation in biphasic calcium phosphates. Results were confirmed by SEM observation. Samples were synthesized via the sol-gel route, followed by heat treatments. Two incorporation sites were highlighted: Ca²⁺ replacement by Ag⁺ into the calcium phosphates (HAp: hydroxyapatite and β-TCP: tricalcium phosphate), and the other as metallic silver Ag° nanoparticles (formed by autogenous reduction). The samples obtained were thus nanocomposites, written Ag°/BCP, composed of closely-mixed Ag° particles of about 100 nm at 400 °C (which became micrometric upon heating) and calcium phosphates, themselves substituted by Ag⁺ cations. Between 400 °C and 700 °C the cationic silver part was mainly located in the HAp phase of the composition Ca_10−xAg_x(PO₄)₆(OH)_2−x (written Ag⁺: HAp). From 600 °C silver cations migrated to β-TCP to form the definite compound Ca₁₀Ag(PO₄)₇ (written Ag⁺: TCP). Due to the melting point of Ag°, the doping element completely left our sample at temperatures above 1000 °C. In order to correctly understand the biological behavior of such material, which is potentially interesting for biomaterial applications, its complex doping mechanism should be taken into consideration for subsequent cytotoxic and bacteriologic studies.