## 2 Results
### The Angiogenic and Osteogenic Effects of DFO-Loaded PLGA-PEG-PLGA NPs on hBMSCs in Vitro  
The Western blot results showed that DFO significantly enhanced HIF-1\(\alpha\) expression of hBMSCs (Figure 5A). According to the quantitative real-time polymerase chain reaction (qRT-PCR) results, DFO-loaded NPs significantly upregulated the genetic transcription level of VEGF, the most important downstream angiogenic factor of HIF-1\(\alpha\) pathway, in a time-dependent manner over the course of 48h (Figure 5B), while the expression levels of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-\(\beta\)1 in the DFO-Loaded NPs group were similar to that of the control group or the blank NPs-treated group (Figure 5C,D). Furthermore, the ELISA results (Figure 5E-G) indicated that DFO-loaded NPs significantly promoted the protein secretion of VEGF, which has a direct stimulatory effect on angiogenesis. For bFGF and TGF-\(\beta\)1, although their secretions were increased in the DFO-loaded NPs group, the results had no significant difference in comparison with the other two groups, which were in accordance with the qRT-PCR results.  
With regard to osteogenesis, as shown in Figure 6A, the ALP activity boosted with time in all three groups. When compared with the control group or the blank NPs group, the DFO-loaded NPs group showed significantly higher ALP activity at each predetermined time point, indicating better osteogenic differentiation. Besides, significantly enhanced osteocalcin and run-related transcription factor-2 (Runx-2) genetic transcription levels assessed by qRT-PCR (Figure 6B,C) further demonstrated that the released DFO exerted a stimulative effect on hBMSCs osteogenic phenotype, during the experiment period.  
Figure 4: Cytotoxicity of the blank and DFO-loaded NPs on hBMSCs. (**A**) CCK-8 assay demonstrated acceptable cell toxicity of the NPs and the released DFO for up to 72h; (**B**) Representative images of the Live/Dead staining after cells were exposed to either NPs for 72 h. Live cells were stained green and dead cells were stained red; (**C**) Quantitation of cell death ratio in the representative images of the Live/Dead staining. Bars represent mean \(\pm\) SD (\(n=6\)).  
Figure 5: The angiogenic effect of DFO-loaded NPs on hBMSCs. (**A**) Representative Western blot (above) and semi-quantitative data (below) of HIF-1\(\alpha\) expression at protein level revealed that the released DFO from NPs activated the HIF-1\(\alpha\) signaling pathway; (**B–D**) Released DFO significantly increased the mRNA expression level of VEGF, both at 12 h and 48 h. While for bFGF and TGF-\(\beta\)1, although their transcriptional levels were upregulated in the DFO group, there was no statistical significance compared with the other two groups. Expression levels of target genes were determined by qRT-PCR, normalized using the respective expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH); (**E–G**) Protein secretion levels of VEGF (**E**), bFGF (**F**) and TGF-\(\beta\)1 (**G**) were measured by ELISA. The VEGF protein level increased significantly after DFO treatment. For bFGF and TGF-\(\beta\)1, although their secretions were promoted by the released DFO, there was no significant difference due to the high variations. Bars represent mean \(\pm\) SD (\(n\) = 3). * \(p\) < 0.05 compared to the control group; % \(p\) < 0.05 compared to the blank NPs group.  
Figure 6: The osteogenic effect of DFO-loaded NPs on hBMSCs. (**A**) DFO released from the NPs significantly enhanced the ALP activity after cells were cultured in osteogenic differentiation medium for either 7 or 14 days; (**B,C**) The released DFO significantly increased the mRNA expression levels of osteoclastin and Runx-2, normalized using the respective expression levels of GAPDH. Bars represent mean \(\pm\) SD (\(n\) = 3). * \(p\) < 0.05 compared to the control group; % \(p\) < 0.05 compared to the the blank NPs group.