with implantation of BMNCs alone, while co-injection of LA did not. Co-implantation of NSs and BMNCs enhances angiogenesis and arteriogenesis To determine whether NS enhances cell implantation-induced angiogenesis, capillary formation associated with implanted BMNCs derived from EGFP-transgenic mice was determined by immunostaining for the endothelial cell marker CD31 (Fig. 4A). The density of CD31-positive capillaries in mice implanted with NS alone (Fig. 4A) was the same as that in the group injected with vehicle alone (data not shown). In the LA+BMNCs group, most of the GFP-positive BMNCs disappeared from around the LA and were no longer colocalized with LA 7 days after transplantation (Fig. 4A), and capillary density around migrating BMNCs was somewhat increased. In contrast, capillary density was markedly increased when BMNCs were co-implanted with NS. Similar enhanced CD31-positive capillary formation in the NS+BMNCs group compared with the LA+BMNCs group was observed in another severe ischemia model in BALB/cAJcl mice (Fig. 4B-a, b, c). If BMNCs alone are implanted, there will be hardly any BMNCs in the implanted area after 7 days (Fig. 2B), thereby complicating the evaluation of capillary formation in the implanted area. Because BMNCs do not adhere to LA, the BMNCs implanted in the LA+BMNCs group will also migrate from the implanted area, and there will be hardly any BMNCs remaining in the implanted area after 7 days, similar to the BMNCs alone group (Fig. 2, 4A). For this reason, LA, which is evaluated as a target for comparison with an NS particle in this study, additionally serves as a marker of the BMNCs-implanted area. Next, we performed X-ray 3D-CT angiographic analysis to determine collateral vessel formation and blood flow. Blood flow of the left hind limb was confirmed to be completely disrupted just after femoral artery resection (Fig. 5A-a). As our micro-CT equipment has a spatial resolution of 48-mm, NS or LA with a diameter of 100 mm could be readily detected, and were visualised as light green particles on 3D-CT images. No collateral arteries had developed 7 days after the operation in the vehicle (Fig. 5A-b) or NS alone groups (Fig. 5A-c). Collateral arteries were somewhat enhanced in the BMNCs alone (Fig. 5A-d) and LA+BMNCs groups (Fig. 5A-e). Interestingly, collateral arteries were well developed around NS in the NS+BMNCs group (Fig. 5A-f). The vascular volume of the ischemic area was measured with 3D-CT angiogram data (Fig. 5B). For objective evaluation of the development of collateral arteries in the ischemic areas, we measured the vascular volume within the lower part of the thigh, ranging from the center of femur’s major axis to the end of femur, based on the arterial phase angiographic data. Resembling the development of microvessels not more than 10 mm in diameter (Fig. 4), the collateral arteries that were detectable with 3D-CT also showed more marked increase in the NS+BMNCs group as compared to that in the BMNCs alone group and the LA+BMNCs group (Fig. 5B). Co-implantation of NSs augments local production of proangiogenic factors induced by BMNC implantation To investigate which proangiogenic factors are involved in enhanced therapeutic angiogenesis in mice treated with NS and BMNCs, candidate cytokines, including VEGF, FGF-2, IL-1b, MCP-1 and SDF-1 were measured by ELISA in ischemic hind limb muscles of the BALB/c mouse. Intramuscular levels of these proangiogenic factors were unaffected by vehicle injection alone Nano-Scaffold for Therapeutic Angiogenesis (data not shown). Intramuscular levels of VEGF and FGF-2 in the NS+BMNCs group were significantly higher than those in the other groups 3 and 7 days after the operation (Fig. 6-a, -b). Levels of IL-1b and MCP-1 in the NS+BMNCs group were slightly but significantly higher than those in the BMNCs group but not those in the LA+BMNCs group 3 days after the operation (Fig. 6-c, -d). However, the levels of IL-1b and MCP-1 were not significantly different among BMNCs alone, LA+BMNCs, and NS+BMNCs groups 7 days after the operation (Fig. 6-c, -d). The level of SDF-1 in the NS+BMNC group was comparable to those in other groups with ischemia at 3 days after the operation. Although SDF-1 levels in the BMNCs alone, LA+BMNCs, and NS+BMNCs groups were higher 7 days after the operation than those in the other groups treated without BMNCs, there were no differences among the 3 groups treated with BMNCs (Fig. 6-e). Importantly, while VEGF levels were normalized even in the BMNCs and LA+BMNCs groups 7 days after ischemic operation, the NS+BMNCs group showed prolonged elevation of VEGF level (Fig. 6-a). This effect may explain enhanced recovery of blood flow in NS+BMNCs group, since long-term exposure to VEGF was reported to be necessary to produce stable microvasculature that is not resorbed after withdrawal of VEGF stimulation [3]. To further clarify the roles of proangiogenic factors, we investigated the localization of VEGF and FGF-2 expression in the ischemic muscle. VEGF expression was mostly co-localized with implanted GFP-positive BMNCs around NS, with some diffusing out of the clusters of BMNCs and NS (Fig. 7A). In contrast, although FGF-2-expressing cells were detected around and within the clusters, many did not express GFP (Fig. 7B), which could be explained by the previous finding that transplanted progenitor cells stimulate resident cells to produce additional secreted factors [8]. Co-implantation of NSs prevents apoptosis of transplanted BMNCs We investigated the possibility that co-implantation of NS prevents apoptotic cell death of BMNCs, and thus contributes to prolonged cell localization. Large numbers of transplanted BMNCs were positive for TUNEL 10 days after the operation when injected alone, suggesting apoptotic cell death (Fig. 8A- upper half, B). Some TUNEL-positive BMNCs did not necessarily express abundant GFP possibly due to the process of cell death. In marked contrast, TUNEL-positive BMNCs were hardly detected when cells were transplanted with NS (Fig. 8A-lower half, B). Biodegradability and non-biotoxic property of NS Finally, we evaluated biodegradability and non-biotoxic prop- erty of NS. The course of degradation of NS over 1 year period was followed by 3D-CT. NS was implanted in the thigh muscle of normal mice. The main degradation mechanisms of PLLA in vivo are random hydrolytic chain scission, enzymatic degradation and acceleration of the breakdown by free radicals released from activated phagocytic cells such as macrophages [32–34]. Reduc- tion in its molecular weight reduces the strength of NS, resulting in cracking and decomposition [32]. NS volume detected by 3D-CT decreased gradually over 1 year period, although 35.168.0% of NS volume was still detected 12 months after implantation (Fig. S1). Next, histological examination was performed to confirm the biodegradability and non-biotoxic property of NS. At 7 days, 3 months, and 12 months after implantation, a small number of CD45 positive leukocytes were recruited around NS, and most of these cells were Mac3 positive macrophages (Fig. S2). The macrophage count around NS was lesser at 7 days than at 3 and 12 months after implantation. At 12 months after implanta- PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35199