A total of 24 healthy young mongrel dogs were randomly divided into PTFE and Dacron groups. In each group, 8 dogs were implanted with prostheses that had been seeded with CD34+ cells, while 4 dogs were implanted with prostheses that had been seeded with autogenous blood only, as a control. The study was approved by the Animal Care and Use Committee of Shandong University (Jinan, China).

Following intraperitoneal anesthesia with 30 mg/kg pentobarbital sodium, 15–20 ml bone marrow was aspirated from the posterior superior iliac spine and mixed with heparin at a dose of 10 IU/ml. The mononuclear cells underwent Ficoll separation (specific gravity, 1.077) and were then diluted with phosphate-buffered saline (PBS) to a concentration of 2×10⁷ cells/ml. Cells were incubated with anti-canine CD34 monoclonal antibodies (BD Biosciences, Franklin Lakes, NJ, USA) and then CD34+ beads. The cell bead mixture was passed through the immunomagnetic separation system (immunomagnetic beads; Miltenyi Biotec, Bergisch Gladbach, Germany) and then washed several times to remove nonspecifically bound cells. The solution was released from the magnet and the positively selected CD34+ cells were eluted from the system. The selected cells were assessed by Trypan blue staining. The CD34 + cells were mixed with 1 ml 1X phosphate buffer saline (PBS) + 2% horse serum buffer and stained by CD34-FITC antibody (130-081-001; Miltenyi Biotec) and then Fc antibody was blocked, finally the CD34+ cells were analyzed by flow cytometry. Finally, the CD34+ cells were maintained at 4°C overnight.

A solution of 0.8 ml plasma, separated from peripheral venous blood, and 0.1 ml CaCl₂ (2.5%) was injected into the graft lumen to form a layer of fibrin coagulum in the graft inner wall. The lumen measured 6 mm in diameter and 3 cm in length. The graft was clipped and rotated for ~5 min to ensure an adequate spread. The solution was then removed from the lumen and a mixture of 0.5 ml CD34+ cells, 0.5 ml plasma and 0.1 ml CaCl₂ (2.5%) was injected into the graft lumen with vascular clamps in place at each end of the graft. The graft was rotated for ~20 min and maintained in a sterile Petri dish at room temperature for implantation. Only 1 ml plasma separated from canine peripheral venous blood was injected into the control grafts with vascular clamps in place at each end of the graft.

In total, 24 mongrel dogs underwent surgery with intraperitoneal anesthesia of 30 mg/kg pentobarbital sodium. A midline laparotomy incision was performed and the infrarenal abdominal aorta was isolated by blunt and sharp dissection. Following proximal clamping below the renal arteries and distal clamping above the aorto-iliac bifurcation, a 2-cm segment of the abdominal aorta was resected. The grafts were then implanted with 5-0 Prolene sutures for end-to-end anastomosis. The same approach was performed on the inferior vena cava (Fig. 1A). Postoperatively, 100 mg/day antiplatelet drug (aspirin) was administered for 2 months.

Venous blood samples were collected during surgery and at days 10, 30 and 60 following surgery. Serum concentrations of 6-keto-PGF1α and TXB₂ were determined by enzyme immunoassays. Kits for 6-keto-PGF1α (515211.1) and TXB₂ (519031.1) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA).

At days 10, 30, 60 and 100 following surgery, 2 dogs from the PTFE experimental group and 2 dogs from the Dacron experimental group were sacrificed by exsanguination following the induction of deep anesthesia. At day 60 and 100, 2 dogs from the PTFE control group and 2 dogs from the Dacron control group were also sacrificed by exsanguination. The specimens were removed, opened longitudinally and photographic images were captured. Next, the tissue samples were evaluated with hematoxylin and eosin (H&E) staining, immunohistochemical analysis of factor VIII (FVIII) and CD34 antigen and scanning and transmission electron microscopy.

The specimens were fixed in 10% formaldehyde solution and embedded in paraffin. Specimens were stained with hematoxylin for 5 min and flushed once with PBS. Hydrochloric acid and ethanol were used for separation and washed once with PBS. Eosin staining was performed for 3 min followed by PBS flushing. Finally specimens were dehydrated with alcohol and sealed with neutral gum seal.

Paraffin sections were placed into 3% H₂O₂-PBS at room temperature for 15 min, then washed with PBS three times every 3 min. The microwave antigen repair was performed as follows: slices were put into the repair of liquid (citrate buffer) for 10 min at 95°C and 20–30 min at the room temperature and washed with PBS three times every 3 min, then blocked by normal sheep serum at wet box of 37°C for 1 h or 4°C overnight. The sealing fluid was absorbed and primary antibodies were added at 4°C for the night. PBS wash was performed three times every 3 min, biotin-labeled secondary antibodies were added and reacted at 37°C for 30 min to 1 h. PBS wash was performed three times every 3 min, 35 to 50 μl horseradish peroxidase-labeled streptavidin was added and reacted at 37°C for 30 min to 1 h. PBS wash three times every 3 min, DAB was added and color development was performed without light (PBS 50 ml +20 mg DAB+7 μl 30% H₂O₂). Finally, slices were counterstained with 10% hematoxylin, washed with 1% hydrochloric acid-ethanol, dehydrated with alcohol and sealed with neutral gum.

Specimens were flushed with heparin saline, fixed by glutaraldehyde and 1% osmic acid, dehydrated by ethanol, embedded with epoxy resin, double stained by uranyl acetate-lead citrate and then photographed with scanning electron microscopy.

All experimental parameters were analyzed for statistical significance using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). The Student’s t-test or one way analysis of variance was used to compare the statistical significance of the differences between the two groups. P<0.05 was considered to indicate a statistically significant difference.

The grafts were patent following surgery (Fig. 1A). All the grafts were observed to adhere tightly to the surrounding tissue. There were no apparent deformations or signs of infection (Fig. 1B). In the experimental groups, the longitudinal section at day 10 showed the neointima covering part of the cavity surface, while at day 30 the neointima completely covered the graft and there was no thrombosis or stenosis present (Fig. 1C). Graft stenosis differed between day 60 and 100 (Table I) and the stenosis rates from the PTFE and Dacron test groups were significantly lower compared with those of the respective control groups at day 60 (P=0.001) and day 100 (P<0.001). All venous grafts were occlusive (Fig. 1D) in the control groups at day 100 following surgery. There was no indication of infarction in the lower limb or other organs in any of the 24 dogs.

Cells isolated by the immunomagnetic bead-based system were identified to be CD34+ cells by flow cytometry (Fig. 2A). The average number of viable CD34+ cells isolated in each group was 2.6±0.3×10⁷ cells/ml, as determined by Trypan blue exclusion prior to seeding.

Immunocytochemical staining with FVIII/von Willebrand factor and CD34 antibodies resulted in staining of endothelial cells (Fig. 2B). On the seeded grafts, there was a layer of neointima consisting of a single layer of endothelial cells on the surface, shown to be positive with H&E staining. There were varying amounts of fibrin coagulum with some blood cells (Fig. 2C). The subintima was largely composed of smooth muscle cells, fibroblasts and collagen. There were no osteocytes, osteoblasts or microcalcification in the seeded grafts (Fig. 2D).

Endometrial thickness measurements are shown in Fig. 3A and B. There were significant differences in neointimal thickness between the experimental and control groups (PTFE, artery P<0.001 and vein P<0.001; Dacron, artery P=0.001 and vein P<0.001; Fig. 3B). However, no significant differences were identified between the two CD34+ cell-seeded groups (P=0.84; Fig. 3A).

Postoperatively, new endothelial cells in the experimental groups synthesized significantly higher levels of PGI₂ compared with the levels in the control groups (PTFE, P=0.001; Dacron, P=0.001; Fig. 3C). In the control groups, vascular damage was not repaired as quickly, the platelet release reaction was enhanced and TX synthesis was significantly increased. By contrast, in each of the experimental groups, due to the protective effect of the endothelial cells, the TXB₂ concentration was significantly lower compared with that in the respective control group (PTFE, P<0.001; Dacron, P=0.001; Fig. 3D).

In the experimental groups at day 10, the density of the endothelial cells was low and the cells exhibited spindle morphology. The transformation of specific endothelial cells into spindles was visible in the images of scanning and transmission electron microscopy (Fig. 4A and B). In the experimental groups at day 30, a large number of endothelial cells appeared at the anastomoses. In addition, the number of endothelial cells gradually decreased from the ends to the middle of the graft (Fig. 4C and D). At day 60, the scanning and transmission electron microscopy images showed a continuous layer of endothelial cells on the CD34+ cell-seeded graft surfaces. Additionally, the closer the anastomoses, the higher the number of endothelial cells (Fig. 5A and B). In the middle of the grafts, endothelial cells were sparse but almost covered the surface (Fig. 5C and D). In the experimental groups at day 100, endothelial cells were arranged tightly and were more mature (Fig. 6A). The endothelial cells were arranged more closely at the anastomoses than elsewhere. At high magnification, it was evident that the cells were spread well with rich pseudopodia (Fig. 6B).

On the control grafts, the majority of the surface was covered with a film of fibrin material mixed with blood cells. In addition, a small amount of necrosis and senescence was present in the endothelial cells at day 60 (Fig. 6C and D).