Abstract
Critical-sized bone defects arising from extensive trauma, oncological resections, and congenital abnormalities represent a formidable clinical challenge due to the insufficient endogenous regenerative capacity of skeletal tissue. In this study, we developed an advanced extrusion-based 3D bioprinting strategy utilizing a composite bioink formulated from gelatin methacryloyl (GelMA), sodium alginate, and functionalized graphene oxide (GO) nanosheets encapsulated with bone marrow-derived mesenchymal stem cells (BMSCs). The incorporation of GO markedly enhanced the shear-thinning behavior, shape fidelity, and compressive modulus of the bioprinted constructs, establishing a favorable biomechanical microenvironment that recapitulates native trabecular architecture. In vitro evaluations demonstrated that GO-functionalized scaffolds sustained high BMSC viability (>92%), enhanced cellular spreading, and significantly accelerated osteogenic differentiation, as evinced by elevated alkaline phosphatase activity, extracellular matrix mineralization, and upregulated expression of Runx2, Col-I, and Ocn. Furthermore, the nanocomposite scaffolds stimulated robust paracrine secretion of vascular endothelial growth factor (VEGF), inducing prominent capillary network formation in endothelial cell co-cultures. In an in vivo critical-sized (8 mm) rat calvarial defect model, the BMSC-laden GO nanocomposite scaffolds achieved superior bone volume fraction (BV/TV of 46.8 ± 4.2% at 12 weeks post-implantation) compared to pristine hydrogel and acellular controls. Histological and immunohistochemical analyses verified dense de novo mineralized matrix deposition coupled with abundant CD31-positive neovascular lumina throughout the porous architecture. These findings establish that bioprinted GO nanocomposite hydrogel scaffolds synergistically coordinate osteogenesis and angiogenesis, providing a transformative therapeutic paradigm for structural bone tissue engineering.
