Nano-Scaffold for Therapeutic Angiogenesis Figure 4. Angiogenesis in hind limb ischemic model. (A) Tissue sections from hind limb ischemic mice (C57BL/6NCrSlc) 7 days after transplantation of NS, BMNCs, LA+BMNCs or NS+BMNCs were immunofluorescently stained using anti-mouse CD31 antibody (red) and counterstained with DAPI (blue). BMNCs (green) were derived from EGFP-transgenic mice. The areas circled with dashed lines indicate the presence of NS or LA. Scale bars, 100 mm. (B) Quantitative evaluation of capillary density was performed by immunohistochemical staining using anti-mouse CD31 antibody in ischemic hind limbs of mice (BALB/cAJcl) 7 days after transplantation of NS, LA or NS+BMNCs. Typical staining of CD31-positive capillaries in high-power field in LA+BMNCs and NS+BMNCs groups are shown in (a) and (b), respectively. Arrowheads indicate representative CD31-positive capillaries. CD31-positive capillary numbers were counted in 4 low-power fields of the injection site, which had microspheres (LA or NS) as landmarks in each mouse (n = 3 in each group) (c). Data are shown as means (SD). *P,0.05 for the NS+BMNCs group compared to the NS and LA+BMNCs groups. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells. doi:10.1371/journal.pone.0035199.g004 tion, invasion by macrophages inside the decomposed particles was noted (Fig. S2). These data suggested that the recruitment of macrophages around NS was due to the mild foreign-body reaction to eliminate foreign matter [35]. In addition, although slight increase in the number of fibroblast and the amount of type I and type III collagens around the particles were noted 12 months after implantation, no intense chronic inflammation, which could promote the strongly enhanced fibrogenesis by fibroblast [35,36], was observed (Fig. S3). Discussion In this study, we presented usefulness of a novel and feasible injectable cell scaffold to enhance cell-based therapeutic angio- genesis. Co-implantation of NS markedly prolonged the localiza- tion of implanted BMNCs in ischemic tissues (Fig. 2). Angiogenesis and collateral formation of arteriogenesis were significantly induced around the BMNC and NS clusters (Fig. 4, 5), resulting in significant limb salvage (Fig. 3). These results further emphasize that the prolonged localization of implanted cells is important for induction of effective neovascularization in ischemic tissues. Moreover, our nano-scaffold could be applied to various cell types, including autologous BMNCs, stem cells, EPCs, PBMNCs, and adipose-derived regenerative cells, for therapeutic angiogen- esis [1–4,6,37], because HAp shows excellent adhesive properties to various types of cells [15]. Peptide hydrogel and collagen-based matrix have both been reported as injectable cell scaffolds in animal models [13,38,39]. These matrix scaffolds also provide an angiogenic environment for successfully promoting long-term cell survival and capillary formation. However, the cumbersome procedures and techniques to prepare matrix scaffold containing cells may be an obstacle to the treatment of large numbers of cells in clinical use. In addition, a large volume of matrix may be required to implant the same number of cells compared with simple cell implantation due to the limited cell retention capacity of the matrix scaffold (,56106 cells/ml) [13,38,39]. This could limit the implantation of a sufficient number of cells for therapy, since the injection space in the myocardium and limb muscles is restricted. In contrast, the procedure for mixing our NS particles into cell solutions is much simpler, and the NS mixture barely affects the volume of the originally prepared cell solutions for implantation. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35199