biodegradable polymers (e.g., polyglycolic acid (PGA), polycapro- lactone (PCA) or copolymers, such as poly(lactic-co-glycolic acid) (PLGA)) could be alternate substitutes [48,49]. Second, tissue toxicity of biodegradable polymers needs to be taken into account. Although the formation of collaterals did not differ between the BMNCs group and the LA+BMNCs group (Fig. 5B), limb survival significantly improved in the BMNCs alone group but not in the LA+BMNCs group (Fig. 3). Hence, tissue toxicity by LA cannot be ruled out. PLLA has already been applied clinically as a safe biodegradable material [21,50]. However, several reports suggest that the substances formed by degradation of PLLA may have adverse effects on tissue [32,51]. The lack of improvement of limb survival in the LA+BMNCs group may be attributable to such adverse effects. In contrast, NS appears to have no tissue toxicity. When NSs were implanted in the thigh muscle of normal mice, a small number of macrophages were observed around NSs, and there was no severe inflammatory reaction in the implanted area (Fig. S2). Further, at 12 months after implantation, enhanced fibrillization around the NS particles was not observed (Fig. S3). Thus, NS, but not LA, appears to have no tissue toxicity, in agreement with previous our reports [18,20,52]. One possible reason for this result is that the HAp nanoparticles covering the NS surface probably serve as a buffer agent, preventing pH reduction arising from PLLA degradation [18,20,53]. The formation of lactic acid and its oligomer from PLLA degradation reduces pH, and this change is reported to cause inflammatory reactions [32,51]. The product from dissolu- tion of HAp nanocrystals (basic calcium phosphate) has been suggested to serve as a buffer for such reactions [53]. Thus, it appears that coating of NS with HAp nanoparticles resulted not only in cell adhesiveness but also increased safety. However, to ensure further safety, selection of appropriate biodegradable polymers is essential. Third, it is also necessary to estimate the appropriate procedures for treatment, i.e., the appropriate ratio of cells to NS and the preincubation time of cells and NS prior to implantation. Regarding preincubation time, our preliminary data suggested that efficacy of cell-based therapeutic angiogenesis was not diminished even when the step of preincubation of cells with NS was omitted. This result makes this procedure much simpler for clinical use. Meanwhile, there might be scope for improving the conditions for preincubation of cells and NS. For example, additional therapeutic efficacy may be achieved if hypoxic preconditioning [54] is used in combination with the current preincubation conditions. It is reported that preincubation of progenitor cells under hypoxic condition induces the expression of SDF-1 receptor, CXCR4, increasing the benefit of progenitor cell therapy [54]. SDF-1 plays a critical role in recruitment, tissue retention, and cell survival of hematopoietic cells and bone marrow-derived progenitor cells, which promote angiogenesis and tissue regeneration [42]. CXCR4 is normally expressed in hematopoietic cells and progenitor cells, and is upregulated by hypoxia [42,55,56]. As shown in Fig. 6, high levels of SDF-1 expression are observed in ischemic tissue [42]. Hence, increasing the expression of CXCR4 in implanted cells under hypoxic condition through preincubation of NS and cells may further improve tissue retention and cell survival after cell implantation mediated by the SDF-1/CXCR4 axis. Fourth, care may be needed to prevent embolism due to particles at the time of implantation. However, this event can be easily avoided because excessive injection is unlikely to occur unless direct injection into blood vessels occurs carelessly during intramuscular injection. Even with slight injection into blood Nano-Scaffold for Therapeutic Angiogenesis vessels, the influence of minor microembolism arising from 100 mm diameter particles will be negligibly small. In conclusion, we have developed a novel type of injectable cell scaffold and, for the first time, demonstrated that it is a simple and easy device for use as an enhancer of cell-based therapeutic angiogenesis. This nano-scaffold provides promising local envi- ronment for implanted cells for the effects on angiogenesis and arteriogenesis through cell clustering, augmented expression of proangiogenic factors, and supporting cell survival without gene manipulation or artificial extracellular matrix. Supporting Information Figure S1 The course of degradation of NS followed by 3D-CT. (A) NS (30,000 particles) was implanted in the thigh muscle on one side of each normal mouse (C57BL/6NCrSlc). The course of degradation of NS over 1 year period was followed by 3D-CT of live mice immediately and 6 months and 12 months after implantation. Representative 3D-CT images obtained from the same mouse at each point of time are shown. PLLA microspheres containing magnetite (PLA-Particles-M) are used as the core of NS to be detected by X-ray 3D-CT. The NS detected by 3D-CT were visualised as green particles. (B) Quantitative volume analysis of NSs was performed using 3D-CT data obtained from 3 mice immediately and 6 months and 12 months after implantation. Residual NS volume was expressed as a percentage of the NS volume immediately after implantation in each mouse. Data are shown as means (SD). Abbreviations: NS, nano-scaffolds; 3D-CT, three-dimensional computed tomography. (TIF) Figure S2 Inflammatory reactions around NS. NS (3,000 particles) was implanted in the thigh muscle on one side of each normal mouse (C57BL/6NCrSlc). Tissues around NS were examined 7 days, 3 months and 12 months after implantation for inflammatory reactions. Tissue sections were counterstained with DAPI (blue), and immunofluorescence was detected by staining with anti-CD45 antibody (red) and anti-Mac3 antibody (green). Scale bars: 100 mm. Abbreviations: NS, nano-scaffolds. (TIF) Figure S3 Collagen accumulation around NS 12 months after implantation. NS (3,000 particles) was implanted in the thigh muscle on one side of each normal mouse (C57BL/ 6NCrSlc). Tissues around NS were examined 12 months after implantation for enhanced fibrillization. Tissue sections were counterstained with DAPI (blue), and immunofluorescence was observed with anti-fibroblast antibody (red) and anti-collagen I antibody (green), or anti- collagen III antibody (green). The upper panels of each group show normal muscle tissue (normal) and the lower panels show the area around NS (NS). Arrowheads indicate slightly accumulated type I collagen, and arrows indicate slightly accumulated type III collagen. Scale bars: 100 mm. Abbreviations: NS, nano-scaffolds. (TIF) Methods S1 (DOCX) Acknowledgments Supplemental methods. We are grateful to Ms. Masayo Monden, Ms. Chiaki Murakami and Ms. Hitomi Yokoyama, Osaka City University Graduate School of Medicine, for technical assistance. PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35199