(25 mL/site) using a 22-gauge needle as described elsewhere [27]. Ischemic operation, cell transplantation and evaluation of limb necrosis were performed by separate operators in a blinded manner. Measurement of GFP and proangiogenic factors in ischemic tissue We collected whole thigh muscle tissues 3, 7 and 14 days after induction of limb ischemia. The tissue lysate was prepared using a modification of the technique described by Abbott et al. [28]. Briefly, tissue extracts from thigh muscle were prepared by homogenization and lysis with 50 mM Tris-HCl, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.5 mM Na3VO4, 10 mM b-GP, 0.5% NP-40 and protease inhibitor cocktail (Nacalai Tesque, Inc. Japan). The homogenate was centrifuged for 5 min at 10,0006g. The clear supernatant was collected and used for quantification of GFP, proangiogenic factors (VEGF, FGF-2, MCP-1, SDF-1 and IL-1b) and total protein content. Total protein content of each sample was determined by bicinchoninic protein assay. Proangio- genic factors and GFP were quantified using the respective ELISA kits in accordance with the manufacturer’s instructions. Immunohistochemistry and analysis of vessel numbers Thigh tissue sections were prepared using a modification of the technique described by Clausen et al. [29]. Briefly, mice were anaesthetized with pentobarbital (i.p.) and perfused through the left ventricle using 20 ml of saline followed by 100 ml of cold 4% paraformaldehyde (PFA) in phosphate buffer (PB). The thigh tissue was post-fixed in 4% PFA for 2 h. The tissue was then embedded in paraffin or immersed in 20% sucrose in PB overnight followed by freezing in OCT compound (Sakura Finetek, Co. Japan). Paraffin-embedded sections were used for immunohistochemical staining of CD31 as described [30]. Following deparaffinization and rehydration of the sections, endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide before application of blocking serum. Sections were then incubated with anti-CD31 antibody. Immunohistochemical staining was performed by the avidin-biotin complex method (ABC Kit). Colour was developed with DAB. Sections were counterstained with hematoxylin (Sigma Diagnostics, MO). CD31-positive capillary numbers were counted in 4 fields of the injection site. The injection site landmarks were the microspheres (NS or LA). The frozen sections were incubated with anti-mouse VEGF antibody or anti-mouse FGF-2 antibody, followed by incubation with fluorescent secondary antibodies and counterstaining with DAPI. Immunofluorescence was observed using a fluorescence microscope (BZ-8000; Keyence, Osaka, Japan). CT analysis, 3D-CT angiography, and vascular volume measurement We used PLLA microspheres containing magnetite (PLA- Particles-M) as control LA and the core of NS to be detected by X-ray CT scan. Angiography was performed using the technique described by Zhuang et al. [31] with slight modifications. Briefly, BALB/cAJcl mice were anaesthetised with sodium pentobarbital (50 mg/g, i.p.) and injected with 5 ml/g barium sulphate suspension (90 w/v%) directly into the left ventricle. CT images were obtained using a micro-CT scanner (La Theta LCT200; Aloka, Tokyo, Japan) according to the manufacturer’s protocol. The 3D data were constructed from sliced CT images by summing those images along the Z-axis, and vascular volume analysis was performed with an image analyzer (VGStudio MAX software; Nano-Scaffold for Therapeutic Angiogenesis Volume Graphics, Heidelberg, Germany) according to the manufacturer’s protocol. Terminal deoxynucleotidyl transferase-mediated biotin- 16-dUTP nick-end labelling (TUNEL) Assay Apoptotic transplanted BMNCs and host cells were evaluated 10 days after limb ischemia by TUNEL assay in frozen sections with an In Situ Cell Death Detection Kit, TMR Red (Roche Laboratories) in accordance with the instructions provided by the manufacturer. Statistical analysis Data are presented as means (SD). Statistical significance was evaluated by ANOVA and Scheffe´’s test for comparison and contrast between multiple groups. Plots of the estimated limb survival ratio after the operation were constructed by the Kaplan- Meier method and were compared using the log-rank test. In all analyses, P,0.05 was taken to indicate significance. Additional descriptions of experimental procedures can be found in Methods S1. Results Characteristic of nano-scaffold (NS) with enhanced adhesiveness of bone marrow mononuclear cells (BMNCs) in vitro NS are microspheres approximately 100 mm in diameter (Fig. 1A-a), the surfaces of which are coated with a monolayer of HAp nanoparticles 50 nm in diameter (Fig. 1A-b, -c, -d). To assess the cell adhesiveness of NS, SEM was performed after incubation of NS and bare PLLA microspheres (LA) as controls with murine BMNCs at 37uC for 8 h in vitro. The number of cells adhering to NS was much greater than that to LA (Fig. 1B-a and b). High-magnification SEM images showed active cell adhesion to NS (Fig. 1B-c). NS prolonged localization of implanted BMNCs in ischemic tissue To determine the colocalization of implanted cells with injected microspheres, BMNCs from EGFP-transgenic mice and rhoda- mine B-containing PLLA microspheres (orange) as a scaffold core or control microspheres were implanted into the ischemic hind limbs of C57BL/6NCrSlc mice (Fig. 2A). Few implanted BMNCs were observed around LA (Fig. 2A-a), while markedly larger numbers of cells were seen with NS (Fig. 2A-b) in ischemic thigh tissue 7 days after transplantation. Intramuscular levels of GFP derived from transplanted BMNCs were consistently and signif- icantly higher in the group injected with NS than that injected with LA or BMNCs alone at 3, 7 and 14 days after implantation, while GFP levels were not significantly different between BMNCs alone and LA+BMNCs groups (Fig. 2B). Co-implantation of NSs enhances limb salvage by BMC implantation Hind limb ischemia in BALB/c mice was used as an intractable ischemia model as these mice show little spontaneous collateral vessel formation in response to ischemia with ischemic hind limb necrosis [1] (Fig. 3A). The limb survival ratios after the operation in each group were compared using Kaplan-Meier analysis and log-rank statistics (Fig. 3B). In this limb ischemic model, approximately 90% of mice treated with vehicle alone developed hind limb necrosis within 5 days after the operation. Injection of NS alone did not improve limb survival, while BMNC PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35199