Nano-Scaffold for Therapeutic Angiogenesis Figure 8. NS prevents apoptotic cell death of implanted cells. (A) BMNCs (green) were derived from EGFP-transgenic mice. Tissue sections from hind limb ischemic mice (C57BL/6NCrSlc) 10 days after transplantation were counterstained with DAPI (blue). Apoptotic nuclei were stained with tetramethylrhodamine (TMR) (red) by the TUNEL method. Arrowheads indicate cells double-positive for GFP and TUNEL. Scale bars, 100 mm. The upper panels of each group show low magnification (Low) and the lower panels show high magnification (High). (B) The percentage of TUNEL- positive cells out of GFP-positive ones was assessed in 4 low-power fields in each mouse (n = 3 in each group). Data are shown as means (SD). *P,0.05 for the NS+BMNCs group compared to the BMNCs group. Abbreviations: NS, nano-scaffolds; BMNCs, bone marrow mononuclear cells; DAPI, 49,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. doi:10.1371/journal.pone.0035199.g008 additional increase in these 3 cytokines after cell implantation, the change observed was not significant enough to explain why marked improvements in blood flow and limb salvage were observed only in the NS+BMNCs group. Three days after transplantation/ischemic induction, IL-1b and MCP-1 levels tended to be high in the NS+BMNCs group, though there was no significant difference in the levels between the NS+BMNCs group and the LA+BMNCs group. SDF-1 expression was not affected by cell implantation after 3 days. Seven days after transplantation/ischemic induction, tissue IL-1b, MCP-1, and SDF-1 levels were significantly higher in the cell-implanted groups (BMNCs group, LA+BMNCs group, and NS+BMNCs group) than in the vehicle group and the NS alone group (Fig. 6). This is probably because large amounts of VEGF and FGF-2 are mainly secreted from implanted cells [3,7,8,43], while IL-1b, MCP-1, and SDF-1 are additionally secreted from muscle cells, endothelial cells, and stromal cells activated by the implanted cells [41,43–46]. In the present study, neovascularization was not stimulated by the elevation of these cytokines 7 days after implantation in the BMNCs alone group and the LA+BMNCs group. The exact reason for this finding is unknown. One possible explanation is the interaction with other proangiogenic factors. For neovasculariza- tion, coordinated actions of multiple proangiogenic factors appear to be important [3,47]. It appears that in the BMNCs alone group and the LA+BMNCs group, these cytokines failed to function satisfactorily because VFGF levels decreased to that of the non- ischemic group (sham) 7 days after ischemic induction (Fig. 6-a). VEGF has been reported to play an important role for SDF-1 activity [47], which might also be true for IL-1b and MCP-1. There are still several limitations to the use of NS for cell-based therapeutic angiogenesis in clinical and practical use. First, it is necessary to control the absorption time of NS in the ischemic tissues to ensure both therapeutic efficacy and safety. When normal mice were followed by 3D-CT for 1 year after NS implantation, NS decreased gradually over time, but 35.1% of the particle volume remained after 1 year (Fig. S1). Although too rapid absorption can reduce the cell-retaining potential, it appears that modulation of NS is necessary to accelerate its absorption appropriately to avoid residual foreign particle accumulation in the living body. To achieve appropriate absorption, other PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35199