In Vitro Biological Evaluation of Titanium Implants Anodized With Aqueous Extract of Psidium guajava and Conventional...

Titanium implants can release ions that damage cells. Anodization creates a protective oxide layer, but conventional acid methods raise environmental and safety concerns. This study evaluates in vitro cytotoxicity and osseointegration with the Psidium guajava aqueous leaf extract (PgE) as an alternative anodization electrolyte for titanium plates. For this purpose, 1-cm² titanium plates were anodized with H₃PO₄, a combination of H₃PO₄+HF or PgE, and each group was assessed for its impact on cell viability, adhesion, and proliferation in osteosarcoma (Saos-2) and fibroblast (NIH-3T3) cells. The plate surfaces were characterized by energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (PXRD), and contact angle measurements using the sessile drop method, demonstrating greater hydrophilicity in plates anodized with PgE. Cytotoxicity was assessed using both indirect assays (extraction media) and direct assays, where cells were seeded directly onto titanium plates; cell viability was determined by MTT and NRU assays after 24 and 96 h. Osseointegration was analyzed by counting nuclei through DAPI staining and adhesion using scanning electron microscopy. In indirect assays, all samples showed no significant difference compared to the control (ANOVA/Tukey's; p < 0.05), while direct assays demonstrated an average increase in cell viability on all anodized plates. The number of cell nuclei increased on H₃PO₄ and PgE plates in both cell lines compared to pure titanium. SEM analysis revealed improved cell morphology and adhesion on surfaces anodized with H₃PO₄ or PgE, whereas cells on H₃PO₄+HF appeared thinner and less spread, suggesting impaired cellular anchorage. The results show that using PgE as an anodization electrolyte for titanium plates caused no cytotoxicity and enhanced cell proliferation and adhesion, outperforming conventional H₃PO₄+HF electrolyte. This makes it a promising and sustainable alternative aligned with green chemistry principles.