Li Zhengzheng, Zhao Junwei, Yi Zhigang, Luo Wei, Li Kang, Wang Yuliang, Wang Jing, An Liping, Ma Jinglin
Department of Orthopedics, Lanzhou University Second Hospital, Lanzhou Gansu, 730030, P. R. China.
Orthopedics Key Laboratory of Gansu Province, Lanzhou University Second Hospital, Lanzhou Gansu, 730030, P. R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2016 Oct 8;30(10):1225-1232. doi: 10.7507/1002-1892.20160251.
To investigate the mechanism of Semaphorin 3A (Sema3A) in fracture healing after nerve injury by observing the expression of Sema3A in the tibia fracture healing after traumatic brain injury (TBI).
A total of 192 Wistar female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into tibia fracture group (group A, =48), TBI group (group B, =48), TBI with tibia fracture group (group C, =48), and control group (group D, =48). The tibia fracture model was established at the right side of group A; TBI model was made in group B by the improved Feeney method; the TBI and tibia fracture model was made in group C; no treatment was given in group D. The tissue samples were respectively collected at 3, 5, 7, 14, 21, and 28 days after operation; HE staining, immunohistochemistry staining, and Western blot method were used for the location and quantitative detection of Sema3A in callus tissue.
HE staining showed that no obvious changes were observed at each time point in groups B and D. At 3 and 5 days, there was no obvious callus growth at fracture site with inflammatory cells and fibrous tissue filling in groups A and C. At 7 and 14 days, fibrous tissue grew from periosteum to fracture site in groups A and C; the proliferation of chondrocytes in exterior periosteum gradually formed osteoid callus at fracture site in groups A and C. The chondrocyte had bigger size, looser arrangement, and more osteoid in group C than group A. Group B had disorder periosteum, slight subperiosteal bone hyperplasia, and no obvious change of bone trabecula in group B when compared with group D. At 21 and 28 days, cartilage callus was gradually replaced by new bone trabecula in groups A and C. Group C had loose arrange, disorder structure, and low density of bone trabecula, big callus area and few chondrocyte and osteoid when compared with group A; group B was similar to Group D. Immunohistochemistry staining showed that Sema3A expression in chondrocytes in group C was higher than that in group A, particularly at 7, 14, and 21 day. Sema3A was significantly higher in osteoblasts of new bone trabecula in group A than group C, especially at 14 and 21 days (<0.05). Western blot results showed that the Sema3A had the same expression trend during fracture healing in groups A and C. However, the expression of Sema3A protein was significantly higher in group C than group A (<0.05) and in group B than group D (<0.05) at 7, 14, 21, and 28 days.
Abnormal expression of Sema3A may play a role in fracture healing after nerve injury by promoting the chondrocytes proliferation and reducing the distribution of sensory nerve fibers and osteoblast differentiation.
通过观察创伤性脑损伤（TBI）后胫骨骨折愈合过程中信号素3A（Sema3A）的表达，探讨Sema3A在神经损伤后骨折愈合中的作用机制。
选取192只8 - 10周龄、体重220 - 250 g的雌性Wistar大鼠，随机分为胫骨骨折组（A组，n = 48）、TBI组（B组，n = 48）、TBI合并胫骨骨折组（C组，n = 48）和对照组（D组，n = 48）。A组于右侧建立胫骨骨折模型；B组采用改良的Feeney法制作TBI模型；C组制作TBI合并胫骨骨折模型；D组不做处理。术后3、5、7、14、21和28天分别采集组织样本；采用苏木精 - 伊红（HE）染色、免疫组织化学染色及蛋白质免疫印迹法对骨痂组织中Sema3A进行定位及定量检测。
HE染色显示，B组和D组各时间点均无明显变化。A组和C组术后3天和5天，骨折部位无明显骨痂生长，有炎性细胞和纤维组织填充。术后7天和14天，A组和C组骨膜纤维组织长入骨折部位；A组和C组骨膜外层软骨细胞增殖，逐渐在骨折部位形成类骨质骨痂。C组软骨细胞比A组体积更大、排列更疏松、类骨质更多。与D组相比，B组骨膜紊乱，骨膜下轻度骨质增生，骨小梁无明显变化。术后21天和28天，A组和C组软骨痂逐渐被新生骨小梁替代。与A组相比，C组骨小梁排列疏松、结构紊乱、密度低，骨痂面积大，软骨细胞和类骨质少；B组与D组相似。免疫组织化学染色显示，C组软骨细胞中Sema3A表达高于A组，尤其在术后7天、14天和21天。A组新生骨小梁成骨细胞中Sema3A明显高于C组，特别是在术后14天和21天（P < 0.05）。蛋白质免疫印迹结果显示，A组和C组骨折愈合过程中Sema3A表达趋势一致。然而，术后7天、14天、21天和28天，C组Sema3A蛋白表达明显高于A组（P < 0.05），B组高于D组（P < 0.05）。
Sema3A的异常表达可能通过促进软骨细胞增殖、减少感觉神经纤维分布和成骨细胞分化，在神经损伤后骨折愈合中发挥作用。
