Mechanisms of Osteoblast-Like Cells and Bacterial Responses to Copper in Titanium-Copper Alloys

Abstract

Titanium-copper (Ti-Cu) alloys are gaining attention for their dual functionality in promoting osteogenesis while providing antimicrobial protection, making them ideal candidates for dental and orthopedic implants. Copper's ability to enhance bone cell activity and inhibit bacterial growth could help address two critical challenges: successful osseointegration and the prevention of peri-implant infections. This study investigated the cellular and molecular mechanisms by which copper, when incorporated into titanium alloys, stimulates both pro-osteogenic behavior and inhibits bacterial viability. MG-63 osteoblast-like cells were cultured on Ti-5Cu alloy surfaces, and osteogenic activity was assessed through alkaline phosphatase (ALP) activity, collagen deposition, and mineralization assays. Gene expression analysis using qPCR and protein expression via band densitometry provided insights into key pathways, including copper homeostasis and bone matrix formation. The antimicrobial effects of Ti-5Cu were evaluated against common pathogens such as Escherichia coli and Staphylococcus aureus, as well as oral bacteria such as Streptococcus oralis and Fusobacterium nucleatum. Bacterial gene expression was analyzed using qPCR and RNA sequencing. Osteoblast-like cells cultured on Ti-5Cu surfaces showed enhanced ALP activity, increased collagen production, and significant gene upregulation of RUNX2, Osteonectin, Alkaline phosphatase, and BMP-2, driving bone matrix formation. Copper homeostasis proteins, such as CTR1 and ATP7A/ATP7B, were modulated to prevent cytotoxicity while supporting osteogenesis. Ti-5Cu alloys also exhibited broad-spectrum antimicrobial effects, significantly reducing bacterial viability. In S. oralis, stress response genes, CsoR and SOD, were upregulated in response to copper exposure, indicating oxidative stress and disruption of copper homeostasis. Transcriptome analysis found that the alloys induce oxidative stress and disrupt metal homeostasis in commensal bacteria such as S. oralis and Actinomyces naeslundii. The study demonstrates that Ti-5Cu alloys effectively promote osteoblast differentiation and mineralization while preventing bacterial colonization through copper-induced stress responses. These findings support the potential of Ti-5Cu alloys for clinical applications, particularly in dental implants, where both regenerative bone formation and infection prevention are critical for long-term success.