Figure 1.
SEM image of NS (A) and marked cell adhesiveness to NS in vitro (B).
(A) NS are microspheres approximately 100 µm in diameter (a). The NS surface uniformly coated with nano-scale hydroxyapatite (HAp) crystals was observed at different magnifications (low and high magnification in b and c, respectively). SEM image of an NS cross-section indicating a single layer of nano-scale HAp particles on the NS surface (d). (B) Murine BMNCs were incubated with LA (a) or NS (b, c) at 37°C for 8 h. Large numbers of BMNCs adhered to NS (b, c) but not to LA (a). Scale bars: 100 µm (A-a, B-a, B-b), 5 µm (B-c), 1 µm (A-b), 100 nm (A-c, A-d). Abbreviations: SEM, scanning electron microscopy; NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells.
Figure 2.
Prolonged localization of implanted BMNCs in ischemic tissues by NS.
(A) Colocalization of BMNCs with NS and LA in vivo. Murine BMNCs derived from EGFP-transgenic mice were transplanted together with LA or NS into the thighs in the hind limb ischemic model. Cores of NS and LA containing rhodamine B (orange) were used to indicate localisation of the injected microspheres in ischemic tissues. Tissue sections 7 days after transplantation of LA+BMNCs (a) or NS+BMNCs (b) were counterstained with DAPI (blue), and merged images of DAPI, GFP and rhodamine B are shown. BMNCs (green) were observed as densely clustered around NS (b) but not LA (a). Scale bars: 100 µm. (B) Quantitative evaluation of implanted cells existing in ischemic tissues. Quantitative analysis of intramuscular GFP was performed 3, 7 and 14 days after transplantation. BMNCs were derived from EGFP-transgenic mice. BMNCs were transplanted alone or together with LA or NS into ischemic thigh muscles. Intramuscular GFP values of whole thigh muscles were corrected for total protein and expressed in arbitrary units (n = 6 in each group). *P<0.05 for the NS+BMNCs group compared to the BMNCs alone and LA+BMNCs groups. GFP concentration in normal murine muscle was measured as background (BG). Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells.
Figure 3.
NS enhance limb salvage by BMNC transplantation.
(A) Representative photographs of mice with limb necrosis (left) and limb salvage (right). Limb necrosis was evaluated every day after the ischemic operation in a blinded manner. (B) The survival curve for limb necrosis of hind limb ischemic mice (BALB/cAJcl) after ischemic induction and simultaneous intramuscular implantation of NS+BMNCs (n = 32), BMNCs alone (n = 33), LA+BMNCs (n = 7), NS alone (n = 11) or vehicle alone (n = 17). The curve was obtained using the Kaplan-Mayer method and the difference between the 2 groups was compared using the log-rank test. *: P<0.05. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells.
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 µm. (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.
Figure 5.
Collateral vessel formations in hind limb ischemic model.
(A) 3D-CT angiography of mice (BALB/cAJcl) was performed immediately after the operation (a) and 7 days after implantation of vehicle alone (b), NS alone (c), BMNCs alone (d), LA+BMNCs (e) or NS+BMNCs (f). Representative 3D-CT angiograms are presented. PLLA microspheres containing magnetite (PLA-Particles-M) were used as core NS and LA to be detected by X-ray 3D-CT. Detected NS and LA by 3D-CT were visualised as green particles. (B) Quantitative volume analysis of collateral vessels in the ischemic area used the arterial phase 3D-CT angiogram data (n = 3 in each group). Data are shown as means (SD). *P<0.05 for the NS+BMNCs group compared to the vehicle, NS alone, BMNCs alone, and LA+BMNCs groups. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells.
Figure 6.
Expression of proangiogenic factors in ischemic hind limb muscles treated with transplantation.
Intramuscular levels of proangiogenic factors in whole thigh muscles were quantified in hind limb ischemic model (BALB/cAJcl mice) 3 and 7 days after ischemic induction. Vehicle alone, NS alone, BMNCs alone, LA+BMNCs or NS+BMNCs were injected into ischemic thigh muscles simultaneously with ischemic induction. Intramuscular levels of proangiogenic factors were corrected for total protein and expressed in arbitrary units (n = 6 in each group). Data are shown as means (SD). *, P<0.05 for the NS+BMNCs group compared to the non-ischemic muscle (sham), vehicle, NS, BMNCs and LA+BMNCs groups. **, P<0.05 for the NS+BMNCs group compared to the non-ischemic muscle (sham), vehicle, NS and BMNCs groups. ***, P<0.05 for the NS+BMNCs group compared to the non-ischemic muscle (sham), vehicle and NS groups. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells.
Figure 7.
Colocalization of transplanted BMNCs and VEGF (A) or FGF-2 (B).
Tissue sections from hind limb ischemic mice (C57BL/6NCrSlc) 7 days after transplantation of NS+BMNCs were immunofluorescently stained using anti-mouse VEGF antibody (A) or anti-mouse FGF-2 antibody (B) and DAPI. BMNCs were derived from EGFP-transgenic mice. Scale bars, 100 µm. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells.
Figure 8.
NS prevents apoptotic cell death of implanted cells.
(A) BMNCs (green) were derived from EGFP-transgenic mice. Tissue sections from hind limb ischemic mice (C57BL/6NCrSlc) 10 days after transplantation were counterstained with DAPI (blue). Apoptotic nuclei were stained with tetramethylrhodamine (TMR) (red) by the TUNEL method. Arrowheads indicate cells double-positive for GFP and TUNEL. Scale bars, 100 µm. The upper panels of each group show low magnification (Low) and the lower panels show high magnification (High). (B) The percentage of TUNEL-positive cells out of GFP-positive ones was assessed in 4 low-power fields in each mouse (n = 3 in each group). Data are shown as means (SD). *P<0.05 for the NS+BMNCs group compared to the BMNCs group. Abbreviations: NS, nano-scaffolds; BMNCs, bone marrow mononuclear cells; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling.