Twenty Wistar rats (4 weeks old) were provided by the Laboratory Animal Center of China Medical University (Shengyang, Liaoning, China). All experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China. The study was approved by the ethics committee of The First Affiliated Hospital of China Medical University, Shenyang, China. M199 medium and fetal bovine serum were obtained from Gibco-BRL (Carlsbad, CA, USA). Lymphocyte separation medium was purchased from Tianjin Haoyang Biologicals Technology Co., Ltd (Tianjin, China), 17β-estradiol was obtained from Sigma-Aldrich (St. Louis, MO, USA), CD133, CD34 and CD31-labeled antibodies from Wuhan Boster Biological Technology Co., Ltd (Wuhan, Hubei, China) and Transwell chambers from Corning, Inc. (NY, USA).

The tibia and femur were separated under sterile conditions and the marrow cavity was flushed with M199 medium. The irrigation fluid was collected and mixed fully prior to centrifugation with lymphocyte separation medium (density gradient, 1.077 g/cm³; volume ratio, irrigation liquid to separated liquid was 2:1). Ficoll-Histopaque gradient centrifugation was conducted at 20°C at 384 × g for 20 min. The mononuclear cell layer in the middle was removed and washed in PBS prior to inoculation into a 100 ml culture flask at a density of 1×10⁶ cells/ml. Cells were cultured in M199 medium containing 20% fetal bovine serum in a 5% CO₂ saturated humidity incubator at 37°C for 48 h prior to re-vaccination of non-adherent cells. Half of the medium was changed on the third day with whole replacement on the fifth day. Cells were digested with 0.25% trypsin (containing 1% EDTA) when cell fusion reached >80%. On the seventh day, CD133 and CD34 phenotypes were identified with a flow cytometer.

Adherent cells were collected and counted. EPC suspension (200 μl) was inoculated in a 96-well plate for 48 h with various concentrations of 17β-estradiol (0, 10, 100 nmol/l) following 24 h culture in M199 culture medium without fetal bovine serum. Each concentration was repeated in 5 wells. After 24 h, 20 μl MTT (5 g/l) was added and incubated for 4 h, followed by replacement with DMSO (150 μl/well) and agitation for 10 min. The plate was read at 560 nm on the microplate reader. The average optical density (OD) values of five wells were used to obtain the optimal concentration for cells. Incubation time was used as the abscissa and the OD value as the ordinate for cell growth curve.

In vitro migration assay was performed in a 24-well Transwell chamber (pore size, 8 μm). EPC suspension (200 μl; 1×10⁴ cells/ml) was added into the upper chamber with M199 medium containing various concentrations of 17β-estradiol (0, 10 and 100 nmol/l) in the lower chamber. Cells were cultured for 24 h and cells attached to the upper chamber were removed with a wet cotton swab. The cells were fixed and stained using the Giemsa method. The experiment was repeated three times.

Fibrin gel was used for in vitro angiogenesis experiments. Artificial fibrinogen (30 μl) and 20 μl thrombin were added in sequence to 96-well plates and agitated prior to incubation at 37°C for gel formation. Cells (5×10³) were added and cultured overnight. Next, the culture medium was removed and 30 μl artificial fibrinogen and 20 μl thrombin was added in sequence. Culture medium containing various concentrations of 17β-estradiol (0, 10 and 100 nmol/l) was added and incubated for an additional 24 h. Angiogenesis was observed and three visual fields (magnification, ×200) were selected for blood vessel counting.

Differentiation was assessed on day 4 of primary cell culture. The culture medium was changed to M199 medium containing various concentrations of 17β-estradiol (10 and 100 nmol/l) for 24 h and flow cytometry was performed. Adherent cells were digested with trypsin for single cell suspension solution and centrifuged at 384 × g for 5 min. Cells were resuspended in 50 μl PBS, a CD31-labeling antibody was added and incubated at 4°C for 60 min. Flow cytometry was performed in the dark immediately following PBS washing and re-suspension. PBS was used as the isotype control. WinMDI2.9 software was used to calculate the percentage of CD31⁺ cells.

Data are presented as the mean ± SD and SPSS 10.0 software was used (SPSS, Inc., Chicago, IL, USA) for statistical analysis. Univariate, multifactorial analysis of variance was performed and t-tests were performed to compare between groups. The LSD method was used for the multiple group comparison. P<0.05 was considered to indicate a statistically significant difference.

Freshly isolated mononuclear cells were small, round and evenly distributed in the suspension. Cells adhered to the wells 24 h post-inoculation and gradually grow into a slender spindle or irregular polygon morphology. One week following the removal of unattached cells from the suspension, a small colony was observed and at two weeks, a large colony had formed. At three weeks, colonies had connected and the shape of adherent cells had changed from slender to short, representative of a typical paving stone-like appearance. Vacuoles were observed in the cytoplasm, indicative of aging.

Cells that adhered to the wells after being replated were purified on day 7 of culture. The CD133-positive rate of primary cells was 69.44% and the CD34-positive rate was 81.05% (Fig. 1). These results indicate that the extracted, separated and purified cells are endothelial progenitor cells.

Average OD values of the 0, 10 and 100 nmol/l groups were 0.3490±0.0332, 0.6278±0.0796 and 0.2758±0.0125, respectively and OD was found to be significantly different in the 10 and 100 nmol/l groups compared with the 0 nmol/l group (P<0.05). 17β-estradiol was found to have a significant effect on EPC proliferation, improving the proliferation of EPCs; however, as the concentration of 17β-estradiol increased, EPC proliferation weakened. The 0 and 10 nmol/l groups were selected to generate a cell growth curve (Fig. 2) revealing the following observations: the first three days post-inoculation is the incubation period, in which cells begin to grow adherent to the well with low levels of cell proliferation; following three days, cell proliferation accelerates and proliferation in the 10 nmol/l group was markedly higher than that of 0 nmol/l. This trend continued to the end of primary culture. The rate of proliferation in the 0 nmol/l group began to reduce at day 13 where it entered the platform stage; however, marked levels of proliferation were maintained in the 10 nmol/l group.

Migration of EPCs was analyzed under various concentrations of 17β-estradiol. Migration was enhanced with increases in 17β-estradiol concentration. The number of cells migrating through the pores in the 10 (18.7±4.5) and 100 nmol/l (37.4±9.4) groups was significantly higher than that of the 0 nmol/l group (5.8±1.2) and migration in the 100 nmol/l group was found to be significantly higher than that of the 10 nmol/l group (Fig. 3; P<0.05).

EPCs revealed various angiogenic abilities in the fibrin gel following treatment with different concentrations of 17β-estradiol. In the 0 nmol/l group, a large number of isolated cells were observed in a scattered distribution and few connections between cells were noted. In the 10 nmol/l group, vascular lumen formation was not found; however, the number of cells was higher than that in the 0 nmol/l group and connections between cells were observed. In the 100 nmol/l group, several cells are arranged in a circle and cells exhibited ring connections, constituting a blood vessel lumen. In addition, the formed lumen was rounder than that of the 10 nmol/l group (Fig. 3D–F). The total number of blood vessels in three randomly selected fields was 4.7±1.1, 16.6±2.3 and 27.8±4.2 in the 0, 10 and 100 nmol/l groups, respectively. The number of blood vessels in the 100 nmol/l group was significantly higher than that of the other groups (P<0.05), indicating that the 100 nmol/l group markedly promoted EPC vessel formation.

The specific surface marker of mature vascular endothelial cells, CD31, was analyzed to determine EPC differentiation. EPCs were cultured with various concentrations of 17β-estradiol (10 and 100 nmol/l) and 0 nmol/l was used as a blank control to test the CD31-positive rate by flow cytometry. The CD31-positive rate was 6.65 and 23.30% in the 10 and 100 nmol/l groups, respectively (Fig. 4), indicating that 17β-estradiol induces EPC differentiation into mature endothelial cells. Compared with the 10 nmol/l group, the CD31-positive rate in 100 nmol/l was higher, indicating that EPC differentiation is accelerated by increasing 17β-estradiol concentrations.