matrix scaffolds [12–14]. These strategies were reported to successfully promote long-term cell survival and capillary forma- tion, resulting in functional recovery of perfusion in ischemic organs. However, the cumbersome procedures and techniques required may be an obstacle to the treatment of large numbers of cells in clinical use. Therefore, simple, safe and effective novel approaches to retain transplanted cells in place are urgently required. Hydroxyapatite (HAp) is the main mineral component of bones and teeth, and artificially synthesised HAp has been used extensively as a biomaterial because of its excellent adhesive properties, not only to hard and soft tissues but also to cells of various types [15]. However, due to the mechanical weakness and brittleness of HAp ceramics, they have been confined to applications with low mechanical stress. To overcome its mechanical weakness, we recently developed a high-dispersed and crystallised nano-scale HAp coating for flexible polymer substrates [16–18]. The novel composite retained most of the mechanical properties of the substrate [16] and showed improved cell adhesion and reduced inflammatory reaction due to the surface HAp nanocrystals [17–20]. Poly(L-lactic acid) (PLLA) microspheres were used as a polymer substrate, to generate the biodegradable and injectable cell scaffold. PLLA is one of the most promising biodegradable polymers and has been used in the fields of orthopaedic and reconstructive surgery and tissue engineering [21] as it is not necessary to remove the material after healing. However, PLLA has the disadvantage of low cell adhesion due to its hydrophobic surface [22]. Our HAp nanocrystal coating is expected to overcome the disadvantages of PLLA microspheres [20]. Thus, nano-scale HAp-coated PLLA microspheres, named nano-scaffold (NS), were generated using our nanotechnology as a non-biological, biodegradable and injectable type of cell scaffold. In this study, we investigated the ability of NS to enhance cell- based therapeutic angiogenesis by retention of implanted cells. Methods Animals BALB/cAJcl mice were purchased from Clea Japan, Inc. (Tokyo, Japan). Enhanced green fluorescent protein (EGFP)- transgenic C57/BL6-Tg (CAG-EGFP) mice and wild-type C57BL/6NCrSlc mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). The procedures used in this study were approved by the Animal Care and Use Committee of Osaka City University Graduate School of Medicine, Osaka, Japan (approval number, 09023). Materials The PLLA microspheres (PLA-Particles), PLLA microspheres containing rhodamine B (PLA-Particles-redF) and PLLA micro- spheres containing magnetite (PLA-Particles-M) were purchased from Micromod Partikeltechnologie (Rostock, Germany). Rat anti-CD31 antibody (clone: MEC13.3) was obtained from BD Pharmingen (San Diego, CA). The avidin-biotin complex (ABC) kit and the 3,39-diaminobenzidine (DAB) substrate kit were purchased from Vector Laboratories (Burlingame, CA). Rat monoclonal anti-vascular endothelial growth factor (VEGF) antibody (clone: RM0009-2G02) was purchased from Angio- Proteomie (Boston, MA). Rabbit polyclonal anti-fibroblast growth factor-2 (FGF-2) antibody was obtained from Abcam (Cambridge, UK). Alexa Fluor 594-conjugated goat anti-rat IgG antibody and Alexa Fluor 594-conjugated chicken anti-rabbit IgG antibody were purchased from Invitrogen Life Technologies (Carlsbad, CA). 49,6-Diamino-2-phenylindole dihydrochloride (DAPI) was Nano-Scaffold for Therapeutic Angiogenesis obtained from Pierce Biotechnology (Rockford, IL). Enzyme- linked immunosorbent assay (ELISA) kits for VEGF, FGF-2, interleukin 1-b (IL-1b), monocyte chemotactic protein-1 (MCP-1) and stromal cell-derived factor-1 (SDF-1) were purchased from R&D Systems (Minneapolis, Minn). The GFP ELISA kit was obtained from Cell Biolabs (San Diego, CA). Barium sulphate was purchased from Sakai Chemical Industry (Osaka, Japan) and used as contrast material for three-dimensional computed tomography (3D-CT) angiography. Fabrication of HAp nanocrystals and injectable cell scaffold The dispersed HAp nanocrystals with an average diameter of 50 nm and rod-like shape were prepared with a wet chemical process and used after calcination with an antisintering agent— poly(acrylic acid, calcium salt)—at 800uC for 1 hour, as described in our previous reports [23]. HAp nanocrystal-coated PLLA microspheres were fabricated as described [24]. Briefly, the PLLA microspheres (0.1 g) were treated with alkali (pH 11.0; adjusted with 25% ammonia solution) for 1 h at room temperature to introduce carboxyl groups onto the surfaces, washed with water, and then dried under reduced pressure. The alkali-treated and dried microspheres were washed with ethanol and immersed in a 1.0% HAp ethanol dispersion for 1 h at room temperature with stirring. The HAp-coated PLLA was washed five times with ethanol by sonication for 3 min, dried under reduced pressure, and used as NS. Bare PLLA microspheres (LA) were used as controls. The NS and LA were characterised in terms of size, particle size distribution and morphology using scanning electron microscopy. BMNC isolation Isolation of BMNCs was performed as described [25]. Briefly, BMNCs were harvested from 8-week-old male mice [BALB/cAJcl or C57/BL6-Tg (CAG-EGFP)] by washing of the tibiae and femora with serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) and separated using Ficoll-Paque Plus (GE Healthcare AB, Sweden). BMNC adhesion on microspheres in vitro and scanning electron microscopy observation Murine BMNCs (56106 cells) were incubated with each microsphere preparation (3,000 particles) at 37uC for 8 h. After incubation, microspheres were harvested using a cell strainer (35- mm nylon mesh; BD Falcon, Japan). The samples were fixed with 2.5% glutaraldehyde for 1 h and dehydrated with aqueous ethanol (30%, 50%, 70%, 90%, 99%, 100%) media and 100% n-butanol for 15 min in each step. They were subsequently lyophilized and observed under scanning electron microscopy (SEM) (JSM-6301F; JEOL Ltd., Tokyo, Japan) operated at 5 kV. BMNC transplantation in hind limb ischemia model Eight-week-old male mice (BALB/cAJcl or C57BL/6NCrSlc) were used for the hind limb ischemia model. Unilateral hind limb ischemia was induced by resection of the left femoral arteries, veins and side branches under anaesthesia with sodium pentobarbital (50 mg/g) injected intraperitoneally (i.p.) as described [26]. BMNCs (56106 cells) were suspended in 100 mL of serum-free DMEM with or without 1.5 mg (3,000 particles) of NS or LA, and incubated at 37uC for 3 h. Immediately after operative resection of the artery and vein, 100 mL of the suspension including 56106 cells and/or 3,000 particles of each microsphere preparation was injected intramuscularly into the ischemic thigh muscle at 4 sites PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35199