Nano-Scaffold for Therapeutic Angiogenesis Figure 1. SEM image of NS (A) and marked cell adhesiveness to NS in vitro (B). (A) NS are microspheres approximately 100 mm in diameter (a). The NS surface uniformly coated with nano-scale hydroxyapatite (HAp) crystals was observed at different magnifications (low and high magnification in b and c, respectively). SEM image of an NS cross-section indicating a single layer of nano-scale HAp particles on the NS surface (d). (B) Murine BMNCs were incubated with LA (a) or NS (b, c) at 37uC for 8 h. Large numbers of BMNCs adhered to NS (b, c) but not to LA (a). Scale bars: 100 mm (A-a, B-a, B-b), 5 mm (B-c), 1 mm (A-b), 100 nm (A-c, A-d). Abbreviations: SEM, scanning electron microscopy; NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells. doi:10.1371/journal.pone.0035199.g001 implantation slightly but significantly improved the limb survival ratio to about 20%. Implantation of BMNCs with NS (NS+BMNCs group) markedly improved limb survival compared Figure 2. Prolonged localization of implanted BMNCs in ischemic tissues by NS. (A) Colocalization of BMNCs with NS and LA in vivo. Murine BMNCs derived from EGFP-transgenic mice were transplanted together with LA or NS into the thighs in the hind limb ischemic model. Cores of NS and LA containing rhodamine B (orange) were used to indicate localisation of the injected microspheres in ischemic tissues. Tissue sections 7 days after transplantation of LA+BMNCs (a) or NS+BMNCs (b) were counterstained with DAPI (blue), and merged images of DAPI, GFP and rhodamine B are shown. BMNCs (green) were observed as densely clustered around NS (b) but not LA (a). Scale bars: 100 mm. (B) Quantitative evaluation of implanted cells existing in ischemic tissues. Quantitative analysis of intramuscular GFP was performed 3, 7 and 14 days after transplantation. BMNCs were derived from EGFP-transgenic mice. BMNCs were transplanted alone or together with LA or NS into ischemic thigh muscles. Intramuscular GFP values of whole thigh muscles were corrected for total protein and expressed in arbitrary units (n = 6 in each group). *P,0.05 for the NS+BMNCs group compared to the BMNCs alone and LA+BMNCs groups. GFP concentration in normal murine muscle was measured as background (BG). Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells. doi:10.1371/journal.pone.0035199.g002 Figure 3. NS enhance limb salvage by BMNC transplantation. (A) Representative photographs of mice with limb necrosis (left) and limb salvage (right). Limb necrosis was evaluated every day after the ischemic operation in a blinded manner. (B) The survival curve for limb necrosis of hind limb ischemic mice (BALB/cAJcl) after ischemic induction and simultaneous intramuscular implantation of NS+BMNCs (n = 32), BMNCs alone (n = 33), LA+BMNCs (n = 7), NS alone (n = 11) or vehicle alone (n = 17). The curve was obtained using the Kaplan-Mayer method and the difference between the 2 groups was compared using the log-rank test. *: P,0.05. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells. doi:10.1371/journal.pone.0035199.g003 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35199