## 3 Discussion  
In this study, we successfully prepared PLGA-PEG-PLGA NPs from PLGA-PEG-PLGA triblock copolymers via a solvent extraction/evaporation method and demonstrated for the first time that this PLGA-PEG-PLGA NPs could support a sustained release of encapsulated DFO, with its angiogenic and osteogenic bioactivities preserved. The drug release assay showed that a total of 89.7% of the initially encapsulated DFO was released from the NPs over the duration of 30 days, in a controlled profile. Furthermore, this vehicle system showed reasonable cytotoxicity. Most importantly, we explored the pre-clinical application potential of this DFO-loaded NPs by systematically evaluating its long-term angiogenic and osteogenic coupling effects on hBMSCs and HUVECs, two most important cell types participate in human bone regeneration process. During bone regeneration, the osteogenesis mediated by BMSCs-derived osteoblasts is of great importance [27]; on the other hand, osteogenesis is highly dependent on angiogenesis. That is to say, angiogenesis is the fundamental requirement for skeletal regeneration. The underlying mechanisms  
Figure 7: The angiogenic effect of DFO-loaded NPs on HUVECs. (**A–D**) The relative mRNA expression levels of VEGF, ANGPT-2, EGF and PDGF-BB following exposure to blank or DFO-loaded NPs solution which was sequestered in the upper chamber of transwell plate. Cells showed significantly increased genetic transcriptions for ANGPT-2, EGF and PDGF-BB at 24, 48, 72 h; for VEGF, statistical significance was found at 48 and 72 h. Bars represent mean \(\pm\) SD (\(n=3\)); (**E**) DFO released from the NPs significantly enhanced the tube formation activity after incubation for 24 h (scale bar = 300 \(\upmu\)m). Bars represent mean \(\pm\) SD (\(n=5\)). * \(p<0.05\) compared to the control group; % \(p<0.05\) compared to the the blank NPs group. are as follows: (1) newly formed blood vessels transport oxygen, nutrients and BMSCs that are indispensable for bone defect repair; (2) the vasculature could serve as a scaffold for bone-forming cells and new bone tissue; and (3) endothelial cells could affect bone formation indirectly via paracrine actions [2; 3; 28; 29; 30]. Thus, to find a therapeutic method that has the dual effects of osteogenesis and angiogenesis is a promising choice to modulate bone defect healing. Recent studies have shown that the HIF-1\(\alpha\) signaling pathway exerts coupling functions in physiological response mechanism, including angiogenesis and osteogenesis [31]. Iron chelators, first used in clinical practice to manage plasma iron levels, have been demonstrated to be a class of hypoxia mimics and activators of HIF-1\(\alpha\) pathway. Accordingly, comprehensive biological and pharmacological activities were achieved by using different class of iron chelators. For example, the application of multimodal iron-chelating compounds M30 and HLA20 showed effective neuroprotective and neurorescue activities in a neural degenerative disease model [32]. Similarly, [PERSON] et al. reported that M30 showed a protective effect for hepatocytes against ethanol-induced injury [33]. VAR10303, another multitarget iron chelator, could improve the motor performance and extended survival rate of amyotrophic lateral sclerosis mouse when co-applied with high calorie/energy-supplemented diet [34]. While after treating pulp cells with iron chelator hinokitiol, the collected conditioned medium showed enhanced angiogenic properties both in vitro and in vivo [35]. However, as far as we know, there are few reports investigating the biological effects of DFO on hBMSCs and HUVECs, cell types that are crucial for bone regeneration. Thus, we hypothesized in this study that the application of DFO, which could activate the HIF-1\(\alpha\) signaling pathway under normoxia conditions, would induce angiogenic factor secretions and osteogenic differentiation of BMSCs, and finally boost bone modeling. However, the lifetime of DFO is always very short, and it is necessary to exploit a local sustained release depot for it.