The 3D-printed hybrid biodegradable hydrogels composed of alginate, gelatin, and cellulose nanocrystals (CNCs) were prepared to provide a favorable environment for cell proliferation, adhesion, nutrients exchange, and matrix mineralization for bone tissue engineering (BTE) applications. The hybrid scaffolds exhibited enhanced mechanical strength compared to the pure polymer scaffolds. The biocompatibility, differentiation potential, and bone regeneration potential of the printed scaffolds were evaluated by DAPI staining, live-dead assay, alizarin Red-S (ARS) staining, real-time PCR (qRT-PCR), and μCT analysis, respectively. Enhanced cell proliferation has occurred 1% CNC/Alg/Gel scaffolds compared to the control. The cells were adequately adhered to the scaffold and exhibited the flattened structure. Improved mineralization was observed in the 1% CNC/Alg/Gel scaffolds' presence than the control, showing their mineralization efficiency. A significant enhancement in the expression of osteogenic-specific gene markers (Runx2, ALP, BMP-2, OCN, OPN, BSP, and COL1) has occurred with 1% CNC/Alg/Gel than the control, indicating their osteogenic potential. Furthermore, enhanced bone formation was observed in the scaffolds treated groups than the control in the calvaria critical-sized defects (CCD-1) model, suggesting their improved bone regeneration potential. Therefore, the fabricated scaffolds have the potential to explore as a biomaterial for tissue engineering.
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