The effective transportation of oxygen, nutrients, and metabolic wastes through new blood vessel networks is key to the survival of engineered constructs in large bone defects. Adipose-derived mesenchymal stem cells (ADSCs), which are regarded as excellent candidates for both bone and blood vessel engineering, are the preferred option for the restoration of massive bone defects. Therefore, we propose to induce ADSCs into osteogenic and endothelial cells differently. A modified hierarchical mesoporous bioactive glass (MBG) scaffold with an enhanced compressive strength was constructed and prevascularized by seeding with endothelial-induced ADSCs (EI-ADSCs). The prevascularized scaffolds were combined with osteogenically induced ADSCs (OI-ADSCs) to repair critical-size bone defects. To validate the angiogenesis of the prevascularized MBG scaffolds in vivo, green fluorescent protein (GFP) was used to label EI-ADSCs. The labeled EI-ADSCs were demonstrated to survive and participate in vascularization at day 7 after subcutaneous implantation in nude mice by double immunofluorescence staining of CD31 and GFP. Regarding the restoration of critical size bone defects, early angiogenesis of rat femur plug defects was evaluated by perfusion of Microfil after 3 weeks. Compared to nonvascularized MBG carrying OI-ADSCs (MBG/OI-ADSCs) and non-cell-seeded MBG scaffolds, the prevascularized MBG carrying OI-ADSCs (Pv-MBG/OI-ADSCs) showed enhanced angiogenesis on the surface and interior. Through dynamic bone formation analysis with sequential fluorescent labeling and Van Gieson's picro-fuchsin staining, we found that the Pv-MBG/OI-ADSCs exhibited the highest mineral deposition rate after surgery, which may be contributed by rapid vascular anastomosis facilitating increased survival of the seeded OI-ADSCs and by the recruitment function for bone mesenchymal stem cells. Therefore, the strategy of time-phase sequential utilization of ADSCs on MBG scaffolds is a practical design for the repair of massive bone defects.
Keywords: adipose-derived stem cells; bone regeneration; critical size bone defect; mesoporous bioactive glass; neovascularization.