A multi-modal approach for correlative evaluation of osteogenesis and osteoclastogenesis on laser-treated titanium, TiAl6V4, and stainless steel

Long-term osseointegration of orthopedic implants mandates a successful interaction between implant surface and resident bone tissue cells, including osteoblasts and osteoclasts. Titanium, TiAl6V4, and Stainless steel are widely used implant biomaterials due to their high biocompatibility and mechanical strength. In this study, we systematically investigated the effects of polishing and laser-induced periodic surface structures (LIPSS) on osteogenic and osteoclastic differentiation across these materials. A correlative multi-modal approach was employed to capture complementary aspects of bone formation and resorption. Scanning electron microscopy (SEM) assessed cell morphology and extracellular matrix deposition, while energy-dispersive X-ray spectroscopy (EDX) quantified elemental composition associated with mineral deposition. Raman spectroscopy enabled molecular characterization of both mineral (phosphate/carbonate) and organic matrix components. These analyses were complemented by alkaline phosphatase (ALP) activity and osteogenic gene expression to evaluate early and late stages of osteoblast differentiation. In parallel, osteoclast responses were characterized using SEM and F-actin imaging for cytoskeletal organization and fusion, together with tartrate-resistant acid phosphatase (TRAP) activity and osteoclast-related gene expression to quantify osteoclast differentiation. Our results reveal a reciprocal relationship between osteoblast and osteoclast activity for higher laser fluence LIPSS. We demonstrate that laser-modified surfaces support better osseointegration by enhancing bone formation while mitigating bone resorption, with corroborating results from various analytical techniques. These findings suggest that LIPSS is a viable method to optimize implant surfaces for improved osseointegration.