BIOACTIVE CHITOSAN/SILICA COATINGS BY ELECTROPHORETIC DEPOSITION TECHNIQUE FOR ENHANCING STAINLESS STEEL IMPLANTS.

MARIA ROSA KATUNAR; ), FLORENCIA DÍAZ ; ALDO BOCACCINI; JOSEFINA BALLARRE

Mar del Plata

Congreso; 20° CONGRESO INTERNACIONAL DE MATERIALES SAN-CONAMET 2022; 2022

At the present time, the use of stainless steel removable screws for bone or plate fixation is considered, especially in developing countries due to a cost difference with premium alloys. The improvement of implant surfaces is a key issue to make the surgical procedure and the patient recovery, a success. Silica based bioactive glass in nanoparticles shape can be used as apatite (bone) precursors. Sol gel method, contrary to fused generated glasses, can generate porous and homogeneous systems, capable of enhance dissolution rates. Stoeber modified method to generate silica based nanoparticles is suitable for monodisperse nano-particle size ones. The aim of this project is to enhance the bioactivity of stainless steel implants by a chitosan electrophoretic deposited coating containing silica-calcia nanoparticles.Monodispersed spherical bioactive glass nanoparticles (nG) were prepared by a modified Stoeber method (1). Briefly, a solution containing ethanol and TEOS, was added to another solution containing distilled water, ethanol and ammonia solution (28%), under magnetic stirring. After 30 min of reaction, Ca(NO3)2•4H2O was added to the suspension and stirred for another 1.5 h. The obtained suspension was centrifuge, and washed two times with destillated water and two times with ethanol. The wet powder was dried at 60°C in electric furnace and then calcinated at 700 °C for 2h. The obtained particles were milled and characterized by size. Coatings were made by electrophoretic deposition (EPD) method. The solution-suspension used was made with destillated water and acetic acid with 1 g/L of chitosan, and kept under vigorous stirring for 24h at room temperature, then storage in 4 °C. Silica-calcia particles were dispersed in ethanol (0.5 (0.5nG) or 0.75 (0.75nG) g/L) with stirring for an hour. All EPD coatings were obtained by applying a direct current (DC) with a power supplier (2). Planar sheets of AISI 316L stainless steel were used as counter electrodes as well as substrates. The process was conducted by applying a constant voltage of 30V for 3 minutes at room temperature, with a separation between electrodes of 10 mm. Surface characterization of the generated EPD coatings was done by SEM and optical images, roughness, adhesive behaviour and FTIR spectroscopy essays. Also degradation of the coatings and apatite deposition at 7 days of immersion in simulated body fluid at 37 °C was analyzed.To assess cell viability, proliferation, and morphology on the different sample surfaces, murine stromal cells ST-2 were used. For this, 12600 cells cm-2 were seeded on each material and incubated. After 1, 4, and 10 days of cell seeding, WST-8 assay (2-(2-methoxy-4-nitrophenyl)-3-(4- nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) was evaluated. The morphology of ST-2 cells adhered on the different sample surfaces at the same time points was characterized by fluorescence. Calcein solution (4μL/mL) and DAPI solution (1 μL/mL) were used for the staining following standard protocols. All samples were characterized by fluorescence microscopy.The generated particles were analysed in size and tendency of agglomeration: it was found a main diameter of 450nm and with some clusters presence when dispersed in water. The generated EPD chitosan/nanoparticles coatings are homogeneous but with rough surfaces. The Ra parameter of the 0.5 and 0.75nG coatings were 0.49 and 0.695 respectively, compared with the polished stainless steel substrate (0.091). But the adhesion of the layers was good, following the ASTM D3359-14 norm for testing. The thickness of the generated coatings was around 20 microns, being the samples of 0.75nG slightly thicker (25 microns). Regarding the hydrophobicity of the coatings, a comparison between the 0.5nG and the stainless steel substrate was analysed and found that the generated coated surface was more hydrophilic (59.6° vs 86.9°). Different researchers mentioned that the optimum contact angle for cell adhesion is between 50 and 80° (3). Immersion test in SBF demonstrated the presence of phosphates, related with hydroxyl carbonate apatite at the surface of 0.75nG, and in less proportion, in 0.5nG. From the WST-8 assay, it can be deduced that after 1 day, a few amount of cells adhered and proliferated over 0.5nG, 0.75nG and SS316L samples. However, by day 4 the number of cells over all the surfaces increased compared to the first time point. In addition, cells continue growing on every samples evaluated up till 10 days, even though, a delayed could be observed over 0.5nG and 0.75nG conditions Cell morphology on the different samples was also investigated by staining the cell cytoskeleton and nuclei (Figure 1). After one day of seeding, cells over 0.5nG and 0.75nG appeared to be not well spread through all surfaces but they could recover along the days of seeding. By day 10, increased number of cells on all surfaces could be observed; however, in particular over 0.5nG and 0.75nG samples cells adhesion was not homogenous. Nevertheless, cell number over 0.75nG could be higher compared to 0.5nG.2. REFERENCIAS 1. El-Rashidy, A. A., Waly, G., Gad, A., Hashem, A. A., Balasubramanian, P., Kaya, S., Sami, I. (2018). Preparation and in vitro characterization of silver-doped bioactive glass nanoparticles fabricated using a sol-gel process and modified Stöber method. Journal of Non-Crystalline Solids, 483, 26–36.2. Mahlooji, E., Atapour, M., & Labbaf, S. (2019). Electrophoretic deposition of Bioactive glass – Chitosan nanocomposite coatings on Ti-6Al-4V for orthopedic applications. Carbohydrate Polymers, 115299.3. J.I. Rosales-Leal, M.A. Rodríguez-Valverde, G. Mazzaglia, P.J. Ramón-Torregrosa, L. Díaz-Rodríguez, O. García-Martínez, M. Vallecillo-Capilla, C. Ruiz, M.A. Cabrerizo-Vílchez, Effect of roughness, wettability and morphology of engineered titanium surfaces on osteoblast-like cell adhesion, Colloids and Surfaces A: Physicochemical and Engineering Aspects, Volume 365, Issues 1–3, 2010, Pages 222-229, ISSN 0927-7757.3. TOPICO:SAM: 10. Biomateriales4. TIPO DE PRESENTACIÓN SOLICITADA (oral o póster): P (poster)