## 4 Discussion  
New vessels could be formed via the sprouting of existing vessels (angiogenesis) or the development of de novo vessels from progenitor cells (vasculogenesis), and during these complex processes, the suitable microenvironment which consists of cells, growth factors, and extracellular matrix (ECM) components could regulate both a nascent tube formation and vessel maturation [30; 31]. The 3D scaffold encapsulated with cells and dual growth factors delivery system could mimic the natural microenvironment for vascularization and new bone formation in bone tissue engineering. Traditional growth factor administration caused high toxicity because of short half-lives of growth factor and slow penetration into tissue. Recently, the polymer microspheres have been widely used for growth factor storage and delivery efficiency. Our previous works showed that the linear poly(D,_l_-lactic-_co_-glycolic acid)-_b_-methoxy poly(ethylene glycol) (PLGA-mPEG) block copolymer microspheres had practical advantages on loading growth factor. For instance, the encapsulation efficiency could be regulated by the size of the microspheres, and release profile of growth factor could be adjusted by [LA]/[GA] ratio [19; 20; 21]. In this work, two linear PLGA-mPEG block copolymers with a different [LA]/[GA] ratio (CP-1, CP-2) were used to prepare microspheres, the uniform growth factor loaded microspheres were obtained by the double emulsion method. The encapsulation efficiency of MCP-1 showed a higher value into CP-1 and CP-2 microspheres than that of VEGF (Figure S2). A higher initial loading with respect to the microspheres compared to the previous report (MCP-1, 1200 ng/mg; VEGF, 1200 ng/mg) was operated in this study, thus the release of both growth factors could remain for 4 weeks at least. While the growth factor loaded microspheres were composited into the hydrogel, the release of the growth factor was a little delayed from composite hydrogel compared to the bare microspheres [23; 32]. However, the release profile of growth factor was mainly controlled by the degradability rate of the microspheres, thus different delivery systems could be facilely constructed by designing the proper formulation of microspheres co-loaded with VEGF and MCP-1, VEGF loaded microsphere, and MCP-1 loaded microsphere in hydrogels (Figure 1). On the other hand, VECs transplantation was an effective approach for vascularization [4], and our previous works have proved that composite hydrogel encapsulated with VECs could enhance new blood vessel formation [23]. In this paper, both the linear PLGA-mPEG block copolymer for microsphere preparation and star-shaped PLGA-mPEG block copolymer for hydrogel preparation possessed a good cytocompatibility, thus, VECs survived in the composite hydrogel, and the viability of VECs also increased over days (Figure 2 and Figure S3). The growth factors alone could not promote  
Figure 5: The vessels density and mean vessel diameter were analyzed by the ImageJ software quantitatively according to H&E and Masson stained slices after the implantation of the composite hydrogels with different delivery systems into the necrosis site of femur head in rabbits at the 4 th and 8 th weeks. (**a**) Vessels density increased over weeks for three delivery systems; (**b**) mean vessel diameter also increased over weeks for three delivery systems, and delivery system III showed the highest value at the 4 th week. Non-intervention treatment as a control group (*** \(p<0.001\), \({}^{*}p<0.05\)). the formation of the stable vasculature. So, it was a promising strategy to develop vascularization via the incorporation of VECs into composite hydrogel [33]. Cell-based approaches for therapeutic vascularization, including endothelial progenitor cell delivery, the engineered mesenchymal cell-based patches, and macrophage-based therapeutic strategies, have been studied for many years.