| Objective: To study the effect of silymarin on rat tibial fracture at different times through method of tissue morphology and iconography. Observed fibroblast growth factor 2( FGF-2) expression levels in fracture site by immunohistochemistry.Method: Seventy two female S-D rats, aged eight weeks, were enrolled in this study. The open osteotomy model was choosen. The rats were then randomly assigned to three groups, Group A were treated with placebo, Group B were treated with SIL(100mg/Kg/d) after surgery. Group C were treated with SIL(200mg/Kg/d) after surgery. Bone callus and average healing scores were evaluated by radiographs in 2, 4, 6 weeks after operation. At the same time checked with Serum Biochemical Analyses(alkaline phosphatase ALP, Alanine aminotransferase ALT, Aspartate transaminase AST, Serum creatinine SCr, urea nitrogen BUN, calcium Ca), Eight rats of every group were killed at 2, 4, 6 weeks after radiographs measure the isolated rat tibia bone mineral density(BMD), The Histological Examination(hematoxylin and Shandon Instant eosin H&E) were performed to observe the histological changes of fracture healing, and fibroblast growth factor 2( FGF-2) immunohistochemical staining were used for detect the expression in bone callus. All resμlts were analysed with statisical software SPSS 22.0.Result:1. Radiological examination: 2 weeks after operation of fracture showed the bone callus growth obviously between with three groups. 4 weeks after operation, the fracture lines began to blur, and significantly reduce the volume of callus. 6 weeks after operation, some rats have reached the bone healing, and the cavum medullary have recanalization. 2 weeks and 4 weeks, the average fracture healing score of group were shown to be significantly differences between control Groupand other two groups(P<0.05), but there were no significant difference between Group SIL-L and SIL-H(P>0.05). In the 6th week there were no significant difference between three groups(P>0.05);2. Hematoxylin eosin staining: Histological observation showed that at the 2th week after operation, there are a lot of fibrous tissue and cartilage at the fracture ends with in three groups; at the 4th week, the fibrous was gradually replaced by immature bone. The histological scores were significantly different between the two observation groups and the control group in the 2th and 4th weeks(P<0.05), but no significant difference was observed between two observation groups(P>0.05); at the 6th week, the endochondral ossification process of group SIL-H was faster than that of control group and group SIL-L, with fuzzy fracture line in group SIL-H and clear fracture line in control group and group SIL-L; the histological scores were different between the group SIL-H and the other groups(SIL-H VS SIL-L: P<0.05; SIL-H VS Control: P<0.01),but no difference was observed between control group and group SIL-L(P>0.05);3. The level of ALP in serum: The serum ALP showed the group SIL-H was higher than control group(P<0.01) and group SIL-L(P<0.05) in 2th weeks. Between the group SIL-L and control group was no significant difference. But in 4th and 6th weeks, three groups showed no significant difference(P>0.05).4. The isolated rat tibia bone mineral density(BMD):The BMD values of Group SIL-L and Group SIL-H at each time point were higher than that of the control group after operation(P<0.05). The BMD values of Group SIL-H at each time point were higher than that of the Group SIL-H, but no significant difference between two observed groups(P>0.05);5. The level of Ca2+ in serum: The Ca2+ showed the group SIL-H and group SIL-L was lower than control group in 2th weeks(P<0.05), but there were no significant difference in the group SIL-L and group SIL-H(P>0.05). In the 4th and 6th weeks, there were no significant difference between three groups(P>0.05).6. Immunohistochemistry: 2 weeks after operation the three groups showed the FGF-2 expression in fibroblasts, cartilage, osteoblasts and bone matrix. the three groups reached a peak. 2 weeks, two observation groups compared with the control group the expression of FGF-2 was significantly increased(P<0.01), the group SIL-H higher than group SIL-L there were significant difference between this two groups(P<0.01). 4th weeks and 6th weeks, the expression of FGF-2 was declined in three groups. in 4th weeks the observation group compared with the control group the expression of FGF-2 was significantly increased(P<0.01), there was significant difference between the group SIL-L and group SIL-H.(P<0.05).6th weeks, the observation groups FGF-2 expression higher than control group, but three groups was no significant difference(P>0.05).7. Alanine aminotransferase ALT, Aspartate transaminase AST, Serum creatinine SCr and urea nitrogen BUN: 2th, 4th and 6th weeks after operation, the serumal level of ALT, AST, SCr and BUN were no significant difference in three groups.Conclusion:According to the experimental resuts, we can draw the following conclusion:1. Silymarin can promote fracture healing in rats.2. Silymarin can increase the level of BMD. It can increase the level of ALP and decrease the level of Ca in serum in early stage.3. Silymarin promote fracture healing in rats maybe related to the expression of FGF-2.4. No significant toxic effects are found for Silymarin in this study. |
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