Clinical and biochemical profiles of patients.

<p>Continuous variables are summarized as mean (SD), whereas medians [interquartile range] are shown for variables with skewed distributions. Prevalence was reported as a percentage. BMI = body weight (kg)/height (m²). Delta power is the total power of the electroencephalograph signal within the delta frequency bands during REM sleep latency.</p><p>CA-IMT, carotid artery intima-media thickness; HbA1c, glycosylated hemoglobin; non-HDL-C, non-high density lipoprotein cholesterol; eGFR, estimated glomerular filtration rate; REM, rapid eye movement.</p><p>Clinical and biochemical profiles of patients.</p

Correlations between HbA1c, REM sleep latency, and log delta power and their effects on CA-IMT.

<p><b>(a)</b> HbA1c exhibited significant negative correlation with REM sleep latency (ρ = −0.342, p = 0.007). <b>(b)</b> REM sleep latency exhibited significant and positive correlation with log delta power during REM sleep latency (ρ = 0.510, p = 0.001). <b>(c)</b> CA<b>-</b>IMT exhibited significant and negative correlation with REM sleep latency (ρ = −0.274, p = 0.031) and log delta power during REM sleep latency (ρ = −0.300, p = 0.018). HbA1c, glycosylated hemoglobin; REM, rapid eye movement; CA-IMT, carotid artery intima-media thickness.</p

Enhancement of Cell-Based Therapeutic Angiogenesis Using a Novel Type of Injectable Scaffolds of Hydroxyapatite-Polymer Nanocomposite Microspheres

<div><h3>Background</h3><p>Clinical trials demonstrate the effectiveness of cell-based therapeutic angiogenesis in patients with severe ischemic diseases; however, their success remains limited. Maintaining transplanted cells in place are expected to augment the cell-based therapeutic angiogenesis. We have reported that nano-hydroxyapatite (HAp) coating on medical devices shows marked cell adhesiveness. Using this nanotechnology, HAp-coated poly(l-lactic acid) (PLLA) microspheres, named nano-scaffold (NS), were generated as a non-biological, biodegradable and injectable cell scaffold. We investigate the effectiveness of NS on cell-based therapeutic angiogenesis.</p> <h3>Methods and Results</h3><p>Bone marrow mononuclear cells (BMNC) and NS or control PLLA microspheres (LA) were intramuscularly co-implanted into mice ischemic hindlimbs. When BMNC derived from enhanced green fluorescent protein (EGFP)-transgenic mice were injected into ischemic muscle, the muscle GFP level in NS+BMNC group was approximate fivefold higher than that in BMNC or LA+BMNC groups seven days after operation. Kaplan-Meier analysis demonstrated that NS+BMNC markedly prevented hindlimb necrosis (<em>P</em><0.05 vs. BMNC or LA+BMNC). NS+BMNC revealed much higher induction of angiogenesis in ischemic tissues and collateral blood flow confirmed by three-dimensional computed tomography angiography than those of BMNC or LA+BMNC groups. NS-enhanced therapeutic angiogenesis and arteriogenesis showed good correlations with increased intramuscular levels of vascular endothelial growth factor and fibroblast growth factor-2. NS co-implantation also prevented apoptotic cell death of transplanted cells, resulting in prolonged cell retention.</p> <h3>Conclusion</h3><p>A novel and feasible injectable cell scaffold potentiates cell-based therapeutic angiogenesis, which could be extremely useful for the treatment of severe ischemic disorders.</p> </div

SEM image of NS (A) and marked cell adhesiveness to NS <i>in vitro</i> (B).

<p>(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.</p

Collateral vessel formations in hind limb ischemic model.

<p>(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). *<i>P</i><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.</p

Expression of proangiogenic factors in ischemic hind limb muscles treated with transplantation.

<p>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). *, <i>P</i><0.05 for the NS+BMNCs group compared to the non-ischemic muscle (sham), vehicle, NS, BMNCs and LA+BMNCs groups. **, <i>P</i><0.05 for the NS+BMNCs group compared to the non-ischemic muscle (sham), vehicle, NS and BMNCs groups. ***, <i>P</i><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.</p

Angiogenesis in hind limb ischemic model.

<p>(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 (<i>n</i> = 3 in each group) (c). Data are shown as means (SD). *<i>P</i><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.</p

NS prevents apoptotic cell death of implanted cells.

<p>(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). *<i>P</i><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.</p

NS enhance limb salvage by BMNC transplantation.

<p>(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 (<i>n</i> = 32), BMNCs alone (<i>n</i> = 33), LA+BMNCs (<i>n</i> = 7), NS alone (<i>n</i> = 11) or vehicle alone (<i>n</i> = 17). The curve was obtained using the Kaplan-Mayer method and the difference between the 2 groups was compared using the log-rank test. *: <i>P</i><0.05. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells.</p

Prolonged localization of implanted BMNCs in ischemic tissues by NS.

<p>(A) Colocalization of BMNCs with NS and LA <i>in vivo</i>. 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). *<i>P</i><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.</p