Single point incremental forming of Ti-6Al-4V titanium alloy for biomedical applications: Process optimization and in vitro biocompatibility assessment

Titanium and its alloys are widely utilized in biomedical applications due to their excellent mechanical properties, corrosion resistance, and biocompatibility. However, the relationship between manufacturing process parameters, resulting surface characteristics and biological performance remains poorly understood, limiting the optimization of patient-specific implants. This study investigates the integrated effects of Single Point Incremental Forming (SPIF) process parameters on both mechanical properties and bioperformance of Ti-6Al-4V ELI devices with systematically varied surface roughness. Biological performance is comprehensively defined to include biocompatibility, absence of mutagenic effects, and the stimulation of osteogenic differentiation. Ti specimens were manufactured using SPIF with different combinations of tool diameter, wall angle, and step depth at 450 °C. The SPIFed Ti implants were comprehensively evaluated through surface characterization and chemical composition, microstructural analysis, hardness measurements, wettability, cytotoxicity and genotoxicity tests. Surface roughness varied significantly among SPIF specimens and all of them demonstrated excellent biocompatibility at all time points in both direct and indirect assays. Surface roughness significantly influenced cell behavior, indeed the test characterized by the lowest roughness exhibited the highest direct cell proliferation rate, which was also supported by the results obtained from the chemical surface composition and contact angle measurements. No mutagenic potential was detected in any specimen. Furthermore, gene expression analysis revealed significant upregulation of osteogenic markers across all SPIF surfaces, with specimen having lowest roughness achieving maximal BMP-2 and ALP expression. This study demonstrates that SPIF process parameters critically influence both mechanical and biological performance of Ti-6Al-4V implants through their effects on surface topography and microstructure, highlighting that the optimization of surface roughness through controlled SPIF processing can significantly improve the bioperformance of titanium implants.