Gallium-based ASD Antibacterial Coatings For Orthopaedic Implants

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1 Gallium-based ASD Antibacterial Coatings For Orthopaedic Implants Francesco Bucciotti, PhD 1, Cinzia Della Valle, PhD 2, Gabriele Candiani, Phd 2, Daniele Pezzoli, PhD 2, Livia Visai, Phd 3, Lia Rimondini, Prof. 4, Andrea Cochis, Msc 4, Elvira De Giglio, PhD 5, Alberto Cigada, Prof. 2, Roberto Chiesa 2. 1 Eurocoating S.p.A, Cirè di Pergine Valsugana, Italy, 2 Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Milano, Italy, 3 Department of Molecular Medicine, and Center for Tissue Engineering (C.I.T.), University of Pavia, Pavia, Italy, 4 Departments of Health Sciences, University of Piemonte Orientale, A. Avogadro, Novara, Italy, 5 Department of Chemistry, University of Bari "A.Moro", Bari, Italy. Disclosures: F. Bucciotti: None. C. Della Valle: None. G. Candiani: None. D. Pezzoli: None. L. Visai: None. L. Rimondini: None. A. Cochis: None. E. De Giglio: None. A. Cigada: None. R. Chiesa: None. Introduction: Nowadays titanium and titanium alloy represent the maily used materials for hard tissue applications. One of the main complications related to titanium implantable devices is represented by infections which often lead to the implant failure and revision as well as strong antiseptic treatments. Among the different strategies nowadays available, the use of surface modification with specific metal ions represents a promising approach to fight implants related infections. In particular gallium has recently shown efficacy in the treatment of infections [1]: exploiting the chemical similarity of Ga3+ with Fe3+, it can interfere in the Fe metabolism in a wide range of bacteria. The aim of this work is to develop and characterize new biocompatible biomimetic treatments with anodic spark deposition (ASD) technique on titanium characterized by antibacterial properties. Methods: Three different surfaces were prepared using commercially grade II titanium samples (12 mm diam., 0.5 mm thick) and was produced starting from the same electrolytic solution called SiB (0,03M Na2SiO* 2H2O, 0,1 M β-gp, 0,3M C4H6CaO4* H2O and 0,036M NaOH) [2]. Afterwards, Ga-Nitrate and the chelating compounds were added to the SiB solution to produce the final antibacterial treatment. The chelating agents are necessary to avoid the salts precipitation. All the samples were anodised in galvano-static condition with a current density of 10 ma/cm2. The following materials were obtained: i) GaCis: obtained adding Ga-nitrate and L-cysteine in the starting electrolytic solution (final potential: 295 V); ii) GaOss: developed adding Ganitrate and oxalic acid to the starting electrolytic solution (final potential: 325 V);iii) SiB-Na: anodisation using the starting solution (final potential: 295 V). Specimens treated with SiB were subjected to an alkali treatments in 5M NaOH for 2h at 60 ± 2 C. This alkali etching aimed to produce a surface sodium enrichment and to increase the Ca/P (calcium/phosphorous) ratio. The resulting samples called SiB-Na were used as control samples. Ti grade II was used as second control. Surface properties were evaluated with scanning electron microscopy (SEM), electron dispersion spectroscopy (EDS) and X-ray photoelectron Spettroscopy (XPS). The Ga release was assessed up to 21 days of soaking in D-PBS with inductively coupled plasma optical emission spectrometry (ICP/OES). The mechanical characterization of the coating was performed using micro indentation and scratch test. The direct and indirect cytotoxicity was evaluated using Saos 2 (human osteoblastic like-cells line ECACC number ). The Alkaline Phosphatase (ALP) activity was evaluated up to 21 days of cell culture in contact with the antibacterial surfaces using a colorimetric assay kit (ab83369, abcam ). The bacterial adhesion and viability were evaluated against S.mutans, S.epidermidis and E.coli bacterial strains after 3h and 24 h of incubation. The long lasting antibacterial activity was then evaluated after 21 day of samples soaking in D-PBS. S.mutans, S.epidermidis and A.baumannii biofilm formation was also investigated. Results: The treatments studied are characterized by a microporous surface (Fig.1) enriched with silicon, calcium, phosphorus and sodium elements. GaCis and GaOss spectra revealed the gallium presence onto surfaces; particularly, the gallium peak on GaOss resulted higher than those observed in GaCis sample (data not shown). The EDX data were confirmed by the XPS analisys (data not shown). Different morphology is reported for GaOss treatment in which the pores diameter results smaller than that of the other ASD coatings and it is charaterized by a not regular pore size distribution. Both treatments revealed that Ga is mainly released in the first 7 days of soaking in D-PBS, reaching a plateau after 21 days. The mechanical characterization reported an optimal coating adherence without any presence of coating delamination. Already after 24h of culture, the Saos2 osteoblast-like cells attached onto the ASD surface were fully spread and flattened with a completely branched shape. The ALP activity of Saos2 cells onto the investigated treated surface compared to the controls is reported in Fig. 2. The ALP synthesis growth for all the samples between day 1 to day 21. GaOss treatment showed a significative higher ALP activity than on all the other ASD antibacterial treatments, also better than the control SiB-Na (p<0.05). Both treatments revealed that Ga is mainly released in the first 7 days of soaking in D-PBS, reaching a plateau after 21 days. Both the Ga based treatments showed a strongly reduced bacterial proliferation against all the bacterial strain analized even after 21 days of antibacterial agent realeased from samples, indicating a long lasting antibacterial activity (Fig. 2). A strong biofilm inhibition was found against S.mutans, S.epidermidis and A.baumanni bacteria strains (Fig.3). Discussion: The developed antibacterial ASD coatings are characterized by homogeneus and reproducible morphological and

2 chemico-physical properties suitable to favor the osteoblastic cells attachment and proliferation, representing attractive platforms for osteoblastic cell differentiation toward the production of new bone tissue in comparison to the optimum control SiB-Na, already patented and clinically used. The presence of the antibacterial agents together with their kinetic of release are able to confer bacteriostatic and long lasting bactericidal properties to the coatings against the most commonly diffused bacteria strains related to titanium-implant associated infections. Among the developed treatments, the gallium-based treatments, in particular GaOss, showed the best properties in stimulating the osteoblastic activity toward the new bone formation and the highest capability in limit the bacteria adhesion, proliferation and biofilm formation for all of the bacterial strains here considered and represent the best candidate for future studies. Significance: This work demonstrated the possibility of successfully use the Anodic Spark Deposition (ASD) to develop biomimetic treatments on titanium substrates, characterized by long lasting antibacterial properties maintaining high osteointegrative potential. In light of these considerations, the developed treatments represent a promising class of antibacterial and high osteointegrative coatings for dental and orthopedic applications. Acknowledgments: - References: [1] Kaneko Y et al., J Clin Invest Apr;117(4): [2] Della Valle C et al., J Appl Biomater Function Mater (2012)

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4 ORS 2014 Annual Meeting Poster No: 1944

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