**7. The previous studies on large bone treatment using 3D bio-printing**

Pitaco et al. [145] developed a hypertrophic cartilage graft for the replacement of critical-sized defects. The bio-ink was prepared with Human mesenchymal stem/stromal cells loading in the fibrin-based bio-ink and 3D bio-printing was performed in the polycaprolactone framework. The designed constructs were implanted into nude mice and expressed a high potential for bone remodeling with high efficiency in vascularization and bone formation. In another trial Sun et al. [146] introduced a multicomponent bio-ink for the treatment of critical-sized bone defects in diabetes mellitus patients. The gelatin, gelatin methacryloyl, and 4-arm poly (ethylene glycol) acrylate bio-ink were loaded with BMSCs, RAW264.7 macrophages, and BMP-4-loaded mesoporous silica nanoparticles. Silica nanoparticles could enhance the mechanical stability and control the release of BMP-4. The existence of BMP-4 led to the polarization of RAW264.7 to M2 macrophages to produce BMP-2 and anti-inflammatory factors and the dual presence of BMP-2 and BMP-4 facilitated the osteogenic differentiation of BMSCs. Zhang et al. [147] developed a human mesenchymal stem cell-laden graphene oxide/alginate/gelatin bio-ink with different concentrations of graphene oxide (0.5, 1, and 2 mg/ml) to create bone scaffolds. The results demonstrated that the scaffolds containing 1 mg/ml graphene oxide had higher shape fidelity, osteogenic differentiation, and mineral volume for mimicking critical-sized calvarial bone defects. Furthermore, Dong et al. [148] evaluated the rabbit bone marrow mesenchymal stem cells and bone morphogenetic protein-2 encapsulated chitosan hydrogel in 3D printed polycaprolactone scaffolds with the aim of bone tissue regeneration. The cellular investigation after 2 weeks confirmed high osteogenesis and bone matrix formation of the proposed scaffold. Khoshnood et al. [149] introduced a novel 3D bio-printed composition for bone regeneration. According to their study, printing 1 wt % tragacanth and 3 wt % alginate bio-ink can provide favorable printability and viscosity. The addition of tragacanth to the alginate modulates the biodegradation ratio to 21 days. Furthermore, adding hydroxyapatite improved osteoconductivity. The prepared bio-inks provided high cell differentiation support with the expression of CD105, CD44, and CD90 surface markers. Hao et al. [150] clinically investigated the printing capacity to treat complicated and large acetabular bone defects. The personalized titanium prosthesis was designed based on computed tomography and X-ray and the final model was achieved using Siemens NX software and Magics software and then the printing was performed. After this step, the prosthesis was trimmed, polished, sandblasted, and cleaned. Then, a surgical process was performed. According to the radiographic and software analysis, the 3D-printed prosthesis showed a high Harris hip score recovery rate in all patients. Wang et al. [151] used 3D-printed polylactic acid (PLA) and nano-hydroxyapatite scaffolds for large bone treatment. The polymeric part was used to provide the required mechanical and degradation rate of the bone scaffolds and nano-hydroxyapatite was added to provide osteoconductivity and improve the load-bearing capacity. The prepared scaffolds were tested *in-vivo* in the rabbit model. The high biocompatibility and osteogenic properties proved the high potential of these scaffolds for further future studies. Li et al. [152] introduced bio-active 3D printed scaffolds for the regeneration of large and load-bearing bone

defects. The scaffolds compound was strontium-hardystonite-gahnite and the scaffolds were tested in the large bone-sized defects of the sheep tibia for 3 and 12 months. Mathematical modeling confirmed and highlighted the importance of the surgical process and implant fixation as much as the implant compositions and the *in-vivo* results revealed high induction of bone formation after 12 months.
