Concentration-Dependent Human Serum Albumin Layers on Ti-6Al-4V Modulate Mammalian Cells and S. aureus Responses

Orthopaedic Ti-6Al-4V implants face two persistent risks: early infection and limited osseointegration. Human serum albumin (HSA) coatings may influence interactions between titanium interfaces in both mammalian and bacterial cells, but these effects may depend on the concentration used during coating preparation. In this study, Ti-6Al-4V discs were PEI-functionalized and coated with HSA solutions ranging from 200 to 500 µg·mL−1. Surface properties were assessed by AFM, contact-angle goniometry, and XPS, while apparent surface-associated HSA and short-term coating retention were estimated using a BCA depletion approach. The apparent surface-associated HSA, estimated from solution depletion, increased with increasing coating concentration, and all coated samples remained ultrasmooth (Ra < 1 nm), providing a comparable physical baseline. In FBS-free DMEM, at least 70% of the initially estimated HSA remained after 3 days. SaOS-2 metabolic activity was reduced at coating concentrations of 200–300 µg·mL−1, whereas at concentrations of 400–500 µg·mL−1 the values did not differ from polished Ti-6Al-4V after 72 h. LDH release remained unchanged across groups. No significant changes were observed in the expression of osteogenic markers and miRNAs analysed among the HSA-coated samples. Macrophage-like cell (THP-1) attachment was reduced on HSA-coated surfaces, while inflammatory gene expression remained largely unchanged. S. aureus adhesion was quantified by CFU counting after 3 h and 24 h, and SEM analysis after 3 h. When culturing S. aureus on 500 µg·mL−1 coated discs, an approximately 1-log10 reduction in recoverable S. aureus CFU vs polished Ti-6Al-4V was observed. Overall, these findings demonstrate a concentration-dependent response to HSA-modified Ti-6Al-4V, with favourable short-term cytocompatibility at higher coating concentrations and reduced S. aureus adhesion at the highest concentration tested.