The Influence of Vitamin C-incorporated Polycaprolactone on Osteogenesis in Osteoblast-Osteoclast Co-culture In Vitro.

OBJECTIVES

Implantation of biomaterials generates reactive oxygen species (ROS) in the peri-implant microenvironment. Bone loss occurs when ROS levels exceed the local antioxidant capacity. The aim of this study was to investigate the influence of vitamin C-incorporated polycaprolactone (PCL-Vit C) membrane in scavenging ROS and enhancing biomineralisation in osteoblast-osteoclast (OB-OC) co-culture system.

METHODS

OB-OCs were cultured on polycaprolactone (PCL) and PCL-Vit C membranes under osteogenic conditions to mimic the bone microenvironment. ROS generation was measured using flow cytometry. ALP and the RANKL/OPG ratio were determined by colorimetric and ELISA assays, respectively. Gene expression of ALP, Col1, Runx-2 and OCN and protein expression of Runx-2, BMP-7, Col1 and OCN were determined by real-time PCR and western blotting, respectively. Activation of P38, ERK and JNK and beta-catenin expression was also analysed.

RESULTS

OB-OC grown on PCL membrane generated a higher amount of ROS compared to those on PCL-Vit C. Colorimetric assays revealed a significantly higher ALP activity in OB-OC co-cultures on PCL-Vit C membranes. Furthermore, OB-OC grown on PCL-Vit C membrane showed significant upregulation in mRNA levels of ALP, Col1 and OCN with lower RANKL/OPG ratio and higher amounts of mineralisation nodules. Runx-2 expression was comparable in both membranes. Western blotting showed a significant increase in phosphorylation of P38 MAPK, ERK, JNK and beta-catenin expression in OB-OC maintained on PCL membrane, plausibly due to increased ROS levels, compared to PCL-Vit C.

SIGNIFICANCE

OB-OC grown on PCL-Vit C membrane scavenged ROS and supported a higher osteogenic potential environment for OB-OC co-culture in vitro.