Combined Treatment with an Anticoagulant and a Vasodilator Prevents Steroid-Associated Osteonecrosis of Rabbit Femoral Heads by Improving Hypercoagulability

Abstract

Steroid-associated osteonecrosis of the femoral head remains a challenging problem in orthopedics worldwide. One pathomechanism is ischemia of the femoral head, as a result of thrombus formation and vasoconstriction. The present study investigates the effects of combination prevention with enoxaparin and EGb 761 on steroid-associated ONFH in rabbits. Rabbits were randomly divided into 5 groups (control group, model group, enoxaparin group, ginkgo group, and combination group). With the exception of the control group, the groups of rabbits were modeled with lipopolysaccharide and methylprednisolone acetate. Starting with modeling, the enoxaparin group and ginkgo group were injected with 1 μg/kg/day enoxaparin subcutaneously and orally given 40 mg/kg/day EGb 761 for 4 weeks, respectively; the combination group received both treatments. After modeling for 6 weeks, the hematology data indicated prolonged PT and APTT in the three prevention groups. The micro-CT examination revealed higher bone density and better structure; histomorphometry revealed significant pathological changes. Immunohistochemistry revealed higher expression of BMP-2 and VEGF, thus revealing better osteogenesis and angiogenesis activities. Among the three prevention groups, the combination group had the most efficient results. In conclusion, the combined prevention with an anticoagulant and a vasodilator has the potential to decrease the incidence of steroid-associated ONFH in rabbits.

Figure 1

Coagulation analysis. Hematological data of PT (a) and APTT (b) in the five groups of rabbits (normal group (●), model group (■), enoxaparin group (▲), ginkgo group (▼), and combination group (◆)). Data are expressed as the mean (n = 6); ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 2

Micro-CT scanning images. A representative micro-CT image from the model group (b, g) showed that the trabecular bone was sparser in the subchondral bone compared with the control group (a, f). In the prevention groups (enoxaparin group (c, h), ginkgo group (d, i), and combination group (e, j)), the trabecular bone was thicker and denser. The circles in the images represented the chosen area to analyze the micro-CT parameters.

Figure 3

Micro-CT data. Parameters analysis of the micro-CT was performed on the differences in CT values (a), BV/TV (b), Tb.Th (c), and Tb.Sp (d) in the 5 groups. Data are presented as the mean ± SD (n = 6). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 4

Histological observation. Representative photomicrographs of the subchondral bone (a, b, c, d), the empty lacunae (e, f, g, h), and ratio of empty lacunae (i) of the femoral heads in the four groups. (a, e) The model group showed sparser and more disordered trabecular bone with more empty lacunae, surrounded by fewer marrow cells and more marrow fat cells. In the (b, f) enoxaparin group and (c, g) the ginkgo group, the trabecular bone ranged more regularly. A decreased number of empty lacunae were observed. (d, h) The combination group showed fewer empty lacunae, and the trabecular bone was surrounded with normal marrow. Stain: hematoxylin and eosin; magnification: ×40 (a, b, c, d), ×200 (e, f, g, h). Data are presented as the mean ± SD. ^∗p < 0.05; ^∗∗p < 0.01.

Figure 5

Immunohistochemical staining. Analysis of BMP-2 and VEGF protein in the femoral heads from each group (100x magnification). The model group (a, e) showed slight BMP-2 immunoreactivity and VEGF immunoreactivity in the bone tissue. In the enoxaparin group (b, f) and ginkgo group (c, g), increased BMP-2 and VEGF expressions were observed. The combination group (d, h) showed intense expression of BMP-2 and VEGF compared with that in the other groups, especially in the bone marrow. Quantitative analysis of BMP-2 (i) and VEGF (j) protein was performed in each group and the mean density was calculated. Data are presented as the mean ± SD. ^∗p < 0.05; ^∗∗p < 0.01.