Microfabrication of copper-containing ZnO nanoparticles for enhanced cellular behaviour: preliminary in vitro evidence toward future dental implant-on-chip biomaterial models

This study investigates the synthesis, physicochemical characterisation, and preliminary in vitro biological evaluation of copper-containing zinc oxide (Cu-ZnO) nanoparticles with distinct morphologies, with the long-term aim of informing future dental implant-on-chip biomaterial models. Four nanoparticle formulations were synthesised using a continuous hydrothermal flow system: ZnO_1, ZnO_2, Cu-ZnO_1 (flake-shaped), and Cu-ZnO_4 (sphere-shaped). The physicochemical characterisation showed that Cu-ZnO_1 exhibited the highest BET surface area (11.86 m²/g), a reduced crystallite size (30.11 nm), and a predominantly flake-like morphology, features that may contribute to formulation-dependent cell-material interactions. Biological evaluation with human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) revealed that Cu-ZnO_1 showed the highest resazurin metabolic activity among the tested formulations. ECM analysis via Sirius Red and Alizarin Red staining revealed no significant difference in collagen production, but ZnO_2 induced the highest calcium deposition (0.18 absorbance units at 405 nm). VEGF expression peaked on Day 21, with Cu-ZnO_4 and Cu-ZnO_1 showing the highest levels at absorbance values of 3.4 and 3.0, respectively, translating to estimated VEGF concentrations of 750–1000 pg/mL. These findings suggest that Cu-containing ZnO nanoparticles, particularly Cu-ZnO_1, may support favourable early cell-material interactions and VEGF-associated responses under conventional in vitro culture conditions. However, the present study does not include organ-on-chip fabrication, microfluidic culture, dynamic flow conditions, dental implant-on-chip validation, XPS confirmation of Cu lattice incorporation, or ion-release analysis. Therefore, the findings should be interpreted as preliminary materials-screening evidence that may inform future dental implant-on-chip biomaterial studies. Accordingly, the biological findings are not attributed solely to confirmed Cu lattice doping but are interpreted in relation to formulation-associated differences, including morphology, surface area, particle size, and detectable Cu presence.