Antibiotic loading on titanium implants and evaluation of their bioactivities

Surgical intervention is needed for the treatment of some spinal diseases. Using spinal implants, the fusion of two vertebrae can be facilitated, the stability of the spine is by this way ensured, thus the spinal cord strengthened and deformities corrected. Titanium and its alloys are the most widely used materials for spinal implants, due to their suitable properties of biocompatibility, strength-to-weight ratio, and their capacity to prone osseointegration. However, the successful performances of spinal implants also depend on the absence of infections near the implant site. To treat them, gentamicin sulphate (GS), is commonly used for local treatment thanks to its efficiency on both gram positive and gram negative bacteria. For that purpose, Ti6Al4V implant will be coated with a hydrogel based on agarose (Agr), a biocompatible natural polysaccharide, known to improve cell growth and tannic acid (TA), a natural catechol, able to induce crosslink. By loading this hydrogel with gentamicin, the Ti6Al4V surfaces are expected to promote osseointegration whilst presenting antibacterial activity. Sand blasted Ti6Al4V discs, 14 mm diameter, were cleaned before surface activation with 2.5 M NaOH solution. Then, they were let react overnight with dopamine solution (DOPA), dissolved in 10 mM Tris buffer (2 mg/mL) at pH 8.7. Thereafter, gentamicin loading was done as follow: 1 % Agr in water was added to an aqueous solution made of 0.1 % TA, 0.1 % CaCl2 and 1 mg/mL GS. This mixture was then coated on the Ti6Al4V-DOPA surface and let react under UV irradiation for 2h. The efficiency of surface modifications was assessed by X-ray photoelectron spectroscopy XPS analyses, contact angle (CA) measurements, profilometry analyses, and hydrogel characterization was done by FTIR. XPS analyses, allowing to determine the surface atomic concentration clearly exhibit the successful surface grafting of DOPA and Agr. Indeed, carbon percentage is significantly higher when compared to clean Ti6Al4V sample, whereas Ti is no more detected on coated ones. Further, the grafting of GS loaded Agr on Ti6Al4V-DOPA surface is evidenced by the lack of nitrogen from DOPA. Contact angle values also demonstrate the coating presence on surfaces. Profilometry analyses have shown that Ti6Al4V-DOPA and Ti6Al4V-Agr were homogeneously grafted and FTIR analyses have evidenced that GS was efficiency entrapped in the Agr hydrogel coated on Ti6Al4V surfaces. GS release was performed under pseudo-physiological conditions for 28 days and the amount quantified by using ELISA tests. At 6th h, a large amount of gentamicin was released by burst release. But the amount of GS that more than the MIC value of GS on S. aureus bacteria continued to release from TA-containing coatings until day 21. The antibacterial activity of the coatings in antibacterial tests was tested up to day 7 and the GS loaded coatings proved an antibacterial effect on S. aureus bacteria. Bioactivities of discs with Agr coated in different contents were evaluated in cell culture studies. As a result of indirect tests that were performed on Saos-2 cell line, it was determined that the discs showed proliferation effect on cells. In addition, direct testing was performed on the same cell line and the highest number of viable cells was detected on the surface of Agr coated discs containing GS, TA, CaCl2.