Surface functionalization of titanium implants with controlled-release glutathione conjugate for antioxidant and osteogenesis

Oxidative stress induced by the overproduction of reactive oxygen species (ROS) during the interaction between titanium (Ti) surfaces and cells commonly results in delayed or compromised osteogenic differentiation. To counteract intracellular ROS overproduction and the oxidized microenvironment, we constructed vinylphosphonic acid-glutathione (VPA-GSH) conjugates synthesized through a thiol-ene click reaction on Ti substrates, endowing them with good biocompatibility, antioxidant capability, and promotion of osteogenesis. In addition, a substantial amount of GSH was controllably released after transferring the VPA-GSH-loaded Ti substrate from a synthetic solution at pH 3.6 to phosphate-buffered saline at pH 7.4. The release began with 1 h and gradually increased, reaching its peak concentration of 39.6 μM between 96 and 120 h. X-ray photoelectron spectroscopy revealed differences in the chemical composition, accompanied by concurrent sulfur and phosphorus peaks, indicating the synthesis and covalent binding of the VPA-GSH conjugates onto the Ti surfaces. Treating Ti surfaces with improved hydrophilicity containing diverse functional groups effectively enhanced MC3T3-E1 cell attachment. The well-developed cytoskeleton and increased vinculin expression in MC3T3-E1 cells showed the biocompatibility of the VPA-GSH-binding Ti surfaces. Moreover, VPA-GSH-treated Ti surfaces facilitated alkaline phosphatase activity and extracellular matrix (ECM) mineralization. Runt-related transcription factor 2 (RUNX2), osteopontin (OPN) and osteocalcin (OCN) gene expression level were significantly up-regulated on VPA-GSH-treated Ti surfaces. Furthermore, low ROS fluorescence intensity in individual cells indicated their regulatory effects. The oxidative stress microenvironment simulated using hydrogen peroxide (H 2 O 2 ) showed that Ti substrates immobilizing VPA-GSH conjugates efficiently scavenged H 2 O 2 and rescued cell viability owing to the controlled release of GSH. Altogether, the proposed strategy demonstrates the potential application of novel antioxidant Ti biomaterials to promote osteogenesis against oxidative stress. • VPA-GSH conjugates was synthesized by thiol-ene click reaction and TiVG surface was developed to immobilize conjugates by hydrothermal method. • TiVG surface regulated MC3T3-E1 cells initial behaviors and further promoted osteogenic differentiation in MC3T3-E1 cells. • TiVG surface play the antioxidant function by alleviating intracellular ROS production and eliminating extracellular H 2 O 2 .