7. Treatment of Ischemic HindlimbThe ischemic hindlimb models were randomly divided into the ischemic, bFGF, CM, and bFGF-CM groups (n = 5). In the ischemic group, the animals did not receive any under treatment. In the bFGF group, on average 15��g of bFGF in 3mL of PBS was injected into two sites along the sides of the longitudinal incision, each site was 0.8cm2 in area, and the injection was 1cm in depth. In the CM group, the CM (0.8cm in diameter) was implanted into the same sites as that in bFGF group. In the bFGF-CM group, the complex of bFGF and CM (0.8cm in diameter) was implanted into the sites described previously. The implanted sites were stitched as a marker.2.8. Oxygen Saturation Parameter AnalysisThe hindlimbs of the animals were tested for hemoglobin oxygen saturation to evaluate the ischemic hindlimb perfusion at 1, 2, and 4 weeks.

Oxygen saturation of hemoglobin was checked by the hemoglobin absorption spectrum with a spectrometer (GE Medical Systems Information Technologies) at a wavelength range from 500 to 620nm by using a clamp probe placed on both of the animals’ hind feet, the hair of which had been removed. Measurements were performed in a chamber at room temperature. The data was calculated by the parameter ratio of the right foot/left foot in each animal.2.9. Histological and Immunohistochemical ExaminationThe animals were killed at 4 weeks. Samples from the implanted sites with stitched markers in ischemic muscle were treated with 10% neutralized formalin, embedded in paraffin, and cut into 4��m sections.

The samples in the ischemic group without treatment were harvested from the same sites as the other groups. The sections were stained with hematoxylin and eosin. The specimens were also immunohistochemically stained with an antibody against von Willebrand factor (vWF; Dako, Carpinteria, CA, USA) to determine the quantitative capillary density and with an antismooth muscle ��-actin (SMA) antibody (Dako) to evaluate the quantitative maturation of arteries. Positive staining indicated the presence of capillaries or mature vascular vessels. Vessel densities were calculated as the number of vessels/mm2.2.10. Statistical AnalysisData is expressed as means �� SD. Analysis of variance (ANOVA) followed by Student’s test was used to determine the significant differences among the groups. All data were considered significant at P < 0.

05.3. Results3.1. Characterization of the CM and Dacomitinib the bFGF-CM ComplexBoth the CM and the bFGF-CM complex were white multiporous collagen matrices that look the same from the scanning electron microscope image and the gross view (Figure 1(a)). The average pore size was 100��m (Figure 1(b)) with 80.2% porosity. The pores in the CM are interconnected (Figure 1(b)). The CM had a high rehydration capacity; the rehydration ratio reached 2.94 �� 3.4 (Figure 1(d)).Figure 1Characterization of the CM and the bFGF-CM complex.