Ethanol-driven structural transitions of human serum albumin and surface-assisted 2D network formation on Ti-6Al-4V: A kinetic-morphological map

Résumé fourni par la source

Ti-6Al-4V, a widely used titanium alloy for load-bearing implants, is rapidly conditioned by adsorbed proteins in vivo. Protein conditioning layers on Ti-6Al-4V implants strongly influence subsequent cell and microbial interactions, yet reproducible process parameters for well-defined human serum albumin (HSA) coatings are still lacking. Ethanol–water mixtures drive conformational transitions of HSA, but it remains unclear when β-rich, thioflavin‑T (ThT)–positive HSA solution states translate into laterally connected protein nanofiber (PNF) coatings on Ti‑6Al‑4V. Here, we map ethanol-driven HSA structural transitions and PNF formation across ethanol fraction (20–80 % v/v), HSA concentration (500–1500 µg·mL -1 ) and incubation time (6–72 h), and refine the effects of temperature (65–85 °C) and pH (3/7.4/10). Solution-state readouts comprise time-resolved ThT fluorescence (ThT kinetics) and far-UV circular dichroism (CD) spectroscopy measured on a selected subset of conditions. They are linked to surface outcomes using whole‑disc ThT-positive area fraction, atomic force microscopy (AFM)‑resolved fibrillar coverage/connectivity, and X-ray photoelectron spectroscopy (XPS). We found that β‑rich solution-state signatures of HSA form readily under acidic conditions, yet β‑sheet enrichment is not sufficient for a connected coating: at 60 % ethanol and 65 °C, pH 10 produces a percolating fibrillar mesh with high AFM fiber coverage, whereas pH 3 yields sparse fibers despite substantial ThT‑positive surface area. The endpoint morphology and chemistry data support an operational surface-assisted assembly model in which adsorption/retention and lateral reorganization at the Ti-6Al-4V interface govern whether β-active deposits develop into a connected fibrillar network. The resulting parameter-process map provides design parameters for reproducible HSA-derived PNF coatings on Ti-6Al-4V and a framework for systematic studies of protein-conditioned implant interfaces.

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