Enhanced regenerative potential of polycaprolactone bone substitutes using copper oxide nanoparticles synthesized with walnut husk extract

Copper oxide nanoparticles (CuO NPs) possess suitable properties for bone tissue engineering; however, chemical synthesis limits their biomedical application. This study aimed to produce a bone substitute composed of green-synthesized CuO NPs and 3D-printed poly(α-caprolactone) (PCL). CuO NPs were synthesized using walnut husk extract and characterized according to their size, morphology, and elemental composition. Synthesized NPs were applied onto the 3D-printed scaffolds at concentrations of 2.5 and 5 µg/mL. Cytocompatibility and osteogenic potential of CuO-coated 3D scaffolds were evaluated in both in vitro and in vivo models. Surface coating significantly reduced water contact angles from 114.83 ± 0.2° in uncoated scaffolds to 84.66 ± 2.46° and 42.36 ± 6.28° for the PCL-2.5 and PCL-5 µg/mL CuO groups, respectively. Scaffold swelling after 7 days increased from 14.66 ± 0.57 % in PCL scaffolds to 29 ± 2 % in PCL-5 µg/mL CuO scaffolds. In vitro studies demonstrated that CuO NP-coated scaffolds showed no cytotoxicity and enhanced alkaline phosphatase (ALP) activity in mesenchymal stromal cells, indicating improved osteogenic differentiation. In vivo implantation into the femur bone of Wistar rats showed significant cartilage-to-immature bone transition and over 60 % mature collagen deposition. Micro-computed tomography confirmed higher bone formation in the PCL-CuO group (26.83 ± 3.09 %) compared to blank (11.78 ± 0.09 %) and PCL (14.76 ± 1.16 %) scaffolds. PCL-CuO implantation also led to upregulation of the COL1a1 gene without significant changes in IL-1β and TNFα expression. The findings of the current study present the green-synthesized PCL-CuO scaffold as a multifunctional and bioactive option for enhancing bone regeneration. • Green synthesis of copper oxide nanoparticles (CuO NPs) using walnut green husk extract. • CuO NP coating enhanced 3D-printed PCL scaffold hydrophilicity and biodegradability. • In vitro studies showed enhanced osteogenic differentiation of bone mesenchymal stem cells. • Intra-body implantation promoted bone regeneration with mature bone and collagen formation. • Demonstrates bioactivity and biosafety of CuO-PCL scaffolds for bone healing.