Hierarchically Porous Calcium Carbonate Scaffolds for Bone Tissue Engineering

Hierarchically porous CaCO₃ scaffolds comprised of micro- (diameter = 2.0 ± 0.3 μm) and nano-sized (diameter = 50.4 ± 14.4 nm) pores were fabricated on silicon substrates using a supercritical CO₂-based process. Differentiated human THP-1 monocytes exposed to the CaCO₃ scaffolds produced negligible levels of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α), confirming the lack of immunogenicity of the scaffolds. Extracellular matrix (ECM) proteins, vitronectin and fibronectin, displayed enhanced adsorption to the scaffolds compared to the silicon controls. ECM protein-coated CaCO₃ scaffolds promoted adhesion, growth, and proliferation of osteoblast MC3T3 cells. MC3T3 cells grown on the CaCO₃ scaffolds produced substantially higher levels of transforming growth factor-beta and vascular endothelial growth factor A, which regulate osteoblast differentiation, and exhibited markedly increased alkaline phosphatase activity, a marker of early osteoblast differentiation, compared to controls. Moreover, the CaCO₃ scaffolds stimulated matrix mineralization (calcium deposition), an end point of advanced osteoblast differentiation and an important biomarker for bone tissue formation. Taken together, these results demonstrate the significant potential of the hierarchically porous CaCO₃ scaffolds for bone tissue engineering applications.