Abstract
Decellularized extracellular matrix (dECM) is a promising bioink because it replicates the biochemical and structural features of native tissues. However, tissue-derived dECM is limited by restricted availability and potential immunogenicity. To overcome these challenges, we developed a macromolecular crowding (MMC)-enhanced cell-derived extracellular matrix (CD-ECM) bioink with improved yield and biofunctionality. MC3T3-E1 pre-osteoblasts were cultured under MMC conditions, followed by decellularization and enzymatic processing to generate a printable CD-ECM bioink. The MMC strategy markedly increased extracellular matrix (ECM) yield, collagen and glycosaminoglycan (GAG) content, and mechanical stability compared to conventional cultures. The optimized CD-ECM bioink exhibited reliable printability in both extrusion-based and digital light processing (DLP) 3D bioprinting, enabling fabrication of constructs with high shape fidelity and cell viability. Incorporation of α-tricalcium phosphate (α-TCP) further enhanced osteogenic performance, resulting in elevated alkaline phosphatase (ALP) activity, increased calcium deposition, and upregulation of osteogenic markers, including runt-related transcription factor 2 (RUNX2), collagen type I alpha 1(COL1A1), ALP, and osteocalcin (OCN). These findings highlight the synergistic interaction between ECM-derived biochemical cues and α-TCP-mediated ionic signaling. Overall, the MMC-enhanced CD-ECM/α-TCP bioink offers a versatile, biologically active, and osteoinductive platform for advanced bone tissue engineering and regenerative applications.