Critical-sized bone defects, as well as delayed and non-union fractures, are unable to heal naturally without targeted intervention. Bone graft was the gold method in treating such conditions with potential deleterious disadvantages including immune rejection, persons needed for graft harvesting, increased pain and morbidity, and failure of healing in large bony lesions. Recently, the regenerative therapeutic strategies involving stem cells, microvesicles, and biological scaffolds have shown promising results in bone healing even in large-sized defects in experimental and clinical studies. Mesenchymal stem cells and microvesicles can secrete growth factors that aid vascularization and osteoblast differentiation. This systematic review represents an overview that investigates the advantages, disadvantages, and outcomes of using mesenchymal stem cells, exosomes, and biological scaffolds to manage critical-sized bone defects. In recent years, regenerative therapy for the treatment of critical-size bone defects was considered a major issue in bone tissue engineering and received extensive attention. Stem cells, EVs, and scaffolds played a crucial role in the improvement of the rate and quality of osteogenic differentiation, mechanical strength, and osteogenic conductivity. Studies on EVs and composite biomaterials showed promising results for the future of bone tissue engineering.