Publication: Influence of vitamin c incorporated polycaprolactone towards oxidative stress related response in bone regeneration in vitro
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Date
2025-03
Authors
Abdulhameed, Elaf Akram
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Abstract
Bone regeneration remains a critical challenge in biomaterial science due to reactive oxygen species (ROS) generation induced by biomaterial implantation during the wound healing process. Oxidative stress following biomaterial implantation can lead to chronic inflammation and impair tissue-material integration, thus hindering effective healing. Incorporation of antioxidants into biomaterials may control oxidative stress and enhance bone regeneration. This study investigated the use of polycaprolactone (PCL) membrane incorporated with Vitamin C (Vit C) to mitigate ROS-mediated damage and elucidate the mechanisms that enhance the osteogenic and angiogenic processes required for bone regeneration in-vitro. Two types of PCL membrane were produced, first using 11 wt% PCL membrane incorporated with 25 wt% Vit C (PCL-Vit C) and the second was 11 wt% PCL membrane alone. Both membranes were characterized using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS), Fourier-transform infrared spectroscopy (FTIR) and surface hydrophilicity. Vit C release from PCL-Vit C membrane was quantified colorimetrically. Viability and attachment studies of hFOB 1.19 cells on membranes were carried out using XTT assay, SEM and confocal microscopy. ROS generation was measured and its influence on osteogenic and angiogenic markers for biomineralization was investigated using monoculture hFOB 1.19 cells in phase I, co-culture of osteoblast-osteoclast (OB-OC) in phase II and co-culture of osteoblast+endothelial cells (hFOB+HUVEC) in phase III of the study; through mRNA gene expressions, ELISA protein expressions, mineralization staining studies, western blotting and MAPK signalling pathways. Material characterization revealed smooth, fine, bead-free fibres with FTIR peaks simulating PCL in both membranes, higher hydrophilicity in PCL-Vit C membrane and sustained controlled release of Vit C in the first hour. Results in phase I showed PCL-Vit C had lower ROS levels compared to PCL membrane with improved osteoblast adhesion, proliferation, and differentiation. In phase II OB-OC co-cultures, PCL-Vit C membrane enhanced key osteogenic markers ALP, Col1 and OCN, reduced the RANKL/ OPG ratio, and enhanced mineral deposition, suggesting a favorable impact on osteoblastogenesis while inhibiting osteoclastogenesis. In phase III hFOB+HUVEC co-cultures, PCL-Vit C resulted in reduced Hypoxia-inducible factor 1-alpha (HIF-1α) expression, activation of MAPK pathways and increased VEGF release, promoted angiogenic-osteogenic coupling which is critical for neovascularization. This study underscores the therapeutic potential of antioxidant incorporation into biomaterials to mitigate ROS-induced oxidative stress, thus optimizing cellular microenvironments for bone regeneration. These findings provided a foundational basis for the development of ROS-regulating smart biomaterials that supported osteogenic and angiogenic processes for bone regeneration.