Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway

Clinical evidence indicates that insulin therapy improves implant survival rates in diabetic patients; however, the mechanisms responsible for this effect are unknown. Here, we test if insulin exerts anti-oxidative effects, thereby improving diabetes-associated impaired osteoblast behavior on titanium implants. To test this hypothesis, we cultured primary rabbit osteoblasts in the presence of titanium implants and studied the impact of treatment with normal serum (NS), diabetic serum (DS), DS + insulin, DS + tempol (a superoxide dismutase mimetic), DS + insulin + tempol, and DS + insulin + wortmannin. We analyzed cell function, apoptosis, and reactive oxygen species (ROS) production in osteoblasts following the various treatments. Treatment with DS induced osteoblast dysfunction, evidenced by impaired cell attachment and morphology, decreased cell proliferation and ALP activity, and decreased expression of osteogenesis-related genes. We also observed a significant increase in apoptosis. Importantly, treatment with DS resulted in increased production of ROS in osteoblasts. In contrast, treatment with insulin inhibited ROS production, alleviated cell dysfunction, and decreased apoptosis of osteoblasts on the implants. Scavenging ROS with tempol also attenuated cell dysfunction. Compared to insulin treatment alone, the combination of insulin and tempol failed to further improve osteoblast functional recovery. Moreover, the anti-oxidative and pro-osteogenic effects afforded by insulin were almost completely abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrate, for the first time, that insulin treatment alleviates the impaired osteogenesis of titanium implants under diabetic conditions by inhibiting ROS overproduction via a PI3K/Akt-dependent mechanism. Both the anti-oxidative and metabolic properties of insulin should make it a viable therapeutic option to combat diabetic implant failure.