Abstract
Osteoarthritis, a major global cause of pain and disability, is driven by the irreversible degradation of hyaline cartilage in joints. Cartilage tissue engineering presents a promising therapeutic avenue, but success hinges on replicating the native physiological environment to guide cellular behavior and generate tissue constructs that mimic natural cartilage. Although electrical stimulation has been shown to enhance chondrogenesis and extracellular matrix production in 2D cultures, the mechanisms underlying these effects remain poorly understood, particularly in 3D models. Here, we report that direct scaffold-coupled electrical stimulation applied to 3D graphene foam bioscaffolds significantly enhances the mechanical properties of the resulting graphene foam - cell constructs. Using custom 3D-printed electrical stimulus chambers, we applied biphasic square impulses (20, 40, 60 mVpp at 1 kHz) for 5 minutes daily over 7 days. Stimulation at 60 mVpp increased the steady-state energy dissipation and equilibrium modulus by approximately 65% and 25%, respectively, compared to unstimulated controls, while also yielding the highest cell density among stimulated samples. In addition, our custom chambers facilitated full submersion of the hydrophobic graphene foam in media, leading to enhanced cell attachment and integration across the scaffold surface and within its hollow branches. To assess this cellular integration, we employed co-localized confocal fluorescence microscopy and X-ray microCT imaging enabled by colloidal gold nanoparticle and fluorophore staining, which allowed visualization of cell distribution within the opaque scaffold's internal structure. These findings highlight the potential of direct scaffold-coupled electrical stimulus to modulate the mechanical properties of engineered tissues and offer new insights into the emergent behavior of cells within conductive 3D bioscaffolds.