Effect of Copper and Zinc on the Bioactivity and Cells Viability of Bioactive Glasses

Synthetic biomaterials are considered like a great option to replace bone grafts. Bioactive glasses are able to form a hydroxyapatite layer Ca10(PO4)6(OH)2 on their surface when they are immersed in a simulated body fluid. This formation induces a bone bonding improving the bone growth [1,2]. Copper and zinc are interesting elements for biomedical applications because they present physiological properties. Copper has anti-inflammatory, anti-infectious and anti-bacterial properties. It is necessary for a lot of biological processes as angiogenic response [3]. Zinc introduces in several enzymatic processes, inhibits the bone resorption and promotes the proliferation of osteoblast [4]. The aim of this work is to study the effect of Cu and Zn on the formation of hydroxyapatite.


Introduction
Synthetic biomaterials are considered like a great option to replace bone grafts. Bioactive glasses are able to form a hydroxyapatite layer Ca 10 (PO 4 ) 6 (OH) 2 on their surface when they are immersed in a simulated body fluid. This formation induces a bone bonding improving the bone growth [1,2]. Copper and zinc are interesting elements for biomedical applications because they present physiological properties. Copper has anti-inflammatory, anti-infectious and anti-bacterial properties. It is necessary for a lot of biological processes as angiogenic response [3]. Zinc introduces in several enzymatic processes, inhibits the bone resorption and promotes the proliferation of osteoblast [4]. The aim of this work is to study the effect of Cu and Zn on the formation of hydroxyapatite.

Melting synthesis of bioactive glasses doped with Cu and Zn
For elaboration of bioactive glasses, oxides (SiO 2 , CaO, Na 2 O, P 2 O 5 , CuO, ZnO) weighed and mixed in a polyethylene bottle for 2 hours using a planetary mixer. 0.1wt% of Cu and/or Zn were introduced to have the same quantities as in human bones. Three doped glasses were elaborated: 0.1% Cu in glass matrix named 46S6-0.1Cu, 0.1% Zn in glass matrix named 46S6-0.1Zn and 0.1% Cu + 0.1% Zn in glass matrix named 46S6-0.1CuZn.
The premixed mixtures were melted in platinum crucibles that were placed in an electric furnace. The first rise of temperature rate was 10°C/ min and it was hold at 900°C for 1 hour to achieve the decarbonation of all products. The second rise of temperature rate was 20°C/min and it was hold to 1350°C for 3 hours. The samples were casted in preheated brass cylindrical molds (13 mm*8 mm high) and annealed at 565°C for 4h near the glass transition temperature of each glass. The obtained cylinders were reserved for the "in vitro" evaluations. Several cylinders were crushed using a mechanical crusher Retsch Fisher Block Scientific RM100. The obtained powder was sieved using Retsch Fisher Block Scientific AS200 sieve in order to have a powder presenting a granulometry of 40 μm. Cylinders were used for "in vitro" studies without cells and powder was used for "in vitro" studies with cells.

Physico-chemical characterizations
The bioactive glasses, before and after immersion in the SBF, were characterized by a powder X-ray diffraction analysis (BRUKER AXS D8 ADVANCE diffractometer by using Cu target, Bragg-Brentano geometry); FT-infrared spectrophotometer (Bruker Equinox 55); Scanning Electron Microscopy (Jeol JSM 6301F) and Inductively Coupled Plasma-Optical Emission Spectroscopy (Spectro Ciros Vision Ametek) to evaluate the structure, the morphology and the kinetic of the ionic exchanges respectively.

Statistical analysis:
The quantitative results from the MTT tests were analysed using Statview V, one-way ANOVA followed by PLSD Fisher test to determine the significant differences layout.

Release of metals and pH of the conditioned medium:
Concentrations of metal released in the bioactive glass conditioned medium were assayed using method described for SBF. The pH was measured with an electronic pH meter Mettler Toledo FE20/EL20.

Physico-chemical results
The amorphous structure of all glasses was confirmed by XRD analyses before immersion in the SBF. The SEM micrographs showed that the surfaces of samples are smooth before immersion.
The XRD patterns for pure and doped glasses after 30 days of immersion in the SBF are presented in Figure 1. The formation of apatite is confirmed with three diffraction maxima at 25.8, 31.8 and 53.4 (2θ°) corresponding to the (002), (211) and (004) apatite reflections [6] for 46S6. Concerning doped glasses, the (002) apatite reflection decreases in intensity compared to 46S6. Moreover, the (211) reflection becomes more pronounced compared to XRD pattern before immersion. The disappearance of (004) apatite reflection is observed for doped glasses. Therefore, the crystallization of the apatite layer is slowed down when the doping elements were added.

Biological results
After 24 hours of incubation, tested biomaterials have a proliferative action on osteoblast cells (p<0.0001). This action is more acute with 0.1% Cu compared to 0.1% Zn. After 72 hours of incubation, this trend is reversed (Figure 4).
After 24 hours of incubation, bioactive glass 46S6 shows a cell proliferative effect on its own, whereas cell growth (p<0.0001) is not affected while using metal-enclosed materials. After 72 hours of incubation, cytotoxicity occurs but remains modest using copper as  metal (about 80% viability). Materials enclosing mixed copper/zinc assume a higher toxicity than single metal-enclosed glasses. 46S6 bioactive glass shows a significant proliferative effect (p=0.03) ( Figure 5).
Using ICP-OES, the concentration of metals in the conditioned medium were measured. After 24 hours of incubation, 0.028 ppm of Zn and 1.203 ppm of Cu were released by 46S6-0.1CuZn; 1.123 ppm of Cu was released by 46S6-0.1Cu.
Bone cytotoxicity of the product is evaluated using human osteoblast SaOS2 cell lines. Assuming the biomaterial could be permeable to revascularization, its endothelial cytotoxicity is evaluated using human endothelial EAHY 926 cell line too.
While using bioactive glasses, a moderate cytotoxicity was observed after seventy two hours of incubation. The increased sensitivity of SaOS2 compared to EAHY 926 cells (cell growth induction as well as inhibition) suggests that the effects must be cell-and time dependent. Although metal ions cytotoxicity has not been described on the presently studied cell lines, examples in literature show a cell-and time dependent effect [7,8], which also appear to be dose dependent [8,9] for copper and zinc.
Dose dependent effect may not be imputable to the biomaterial itself since it is not influencing cell growth in its metal-free version (it is actually enhancing it).
Regarding copper, a lethal dose (LD50) has been reported as 46 mg.L -1 after 24 hours of incubation on mice fibroblasts L929 [7] whereas zinc assume cytotoxic concentration variations from 10 to 250 mg.L -1 [8,9]. Besides leached amounts well below the cytotoxic doses our bioactive glasses allows, after an incubation time of 24 hours, a release of 1.12 mg.L -1 for copper and 0.028 mg.L -1 for zinc. On the other hand the increasing toxicity observed after 72 hours must be related to an increase of pH values of the incubation medium while metal ions are added to the materials (pH is of 7.51, 7.6 and 7.52 for Cu, Zn and both respectively versus 7.48 for pure glass).

Conclusions
The physicochemical results show the formation of an hydroxyapatite for pure and doped bioactive glasses. These layers present different morphologies according to the introduced doping element. The cytotoxicity assays do not show toxicity of the biomaterials after 24 hours of incubation on osteoblast or endothelial cells. The mixture of Cu and Zn in the glassy matrix allows improving the cell proliferation. These results confirm the advantage of Cu and Zn for biomedical applications because they present different kinetics of release. These biomaterials offer an alternative for the orthopaedic or maxillo-facial surgery. Pure or doped glasses could be used according to several parameters as age, gender and site of implantation.