The objective of this study was to evaluate the effects of mechanical loading scenarios on tissue differentiation and bone formation within trabecular bone defects, by integrating computational simulations with experimental validation. A three-dimensional finite element model with a mechano-regulation tissue differentiation algorithm was employed to predict tissue differentiation under mechanical loading. Experimental studies were performed by using porcine trabecular bone explants with fibrin gel in the defects. The applied mechanical loadings were designed to represent low and high mechanical strains. Computational simulations suggest that lower loading (20 N) had more uniform bone formation within the explant defect, in which mature bone was filling approximately 93% of the defect. In contrast, higher loading (100 N) resulted in less bone formation, in which mature bone occupied approximately 20% of the defect, immature bone occupied approximately 55%, and the rest were cartilage, fibrous tissue, and resorption. These model predictions do not match with experimental outcomes, which showed limited bone formation within the explant defects, with most fibrin gels contracting and not integrating with the explant defects’ edges as anticipated. The discrepancy between model predictions and experimental results highlights the complexities of bone formation within trabecular bone defects and the limitations of the model. This study contributes to the understanding of the mechanobiology of bone regeneration, offering insights into enhancing bone healing in trabecular bone defects.