Objective: 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).
Methods: 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, n=48), TBI group (group B, n=48), TBI with tibia fracture group (group C, n=48), and control group (group D, n=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.
Results: 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 (P<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 (P<0.05) and in group B than group D (P<0.05) at 7, 14, 21, and 28 days.
Conclusions: 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.
目的: 通过观察脑信号蛋白3A（Semaphorin 3A，Sema3A）在创伤性颅脑损伤（traumatic brain injury，TBI）后胫骨骨折愈合中的表达变化，探讨Sema3A在神经损伤后骨折愈合中的作用机制。.
方法: 取8～10周龄Wistar雌性大鼠192只，体质量220～250 g，随机分为胫骨骨折组（A组）、TBI组（B组）、TBI伴胫骨骨折组（C组）、对照组（D组），每组48只。A组制备右侧胫骨骨折模型，B组采用改进后的Feeney法自由落体颅脑损伤装置制备TBI模型，C组制备TBI合并胫骨骨折模型，D组不作处理。分别于术后3、5、7、14、21、28 d处死8只动物取材，采用HE染色、免疫组织化学染色及Western blot法对骨痂组织中Sema3A进行定位、定量检测。.
结果: HE染色示D组各时间点未见明显变化。术后3、5 d， A、C组骨折断端无明显骨痂生长，可见炎性细胞及纤维组织填充；B组与D组无差异。7、14 d，A、C组纤维组织自骨膜下向断端间隙生长，骨外膜软骨细胞增生，逐渐向断端生成软骨痂，C组与A组相比骨外膜软骨细胞体积大、排列疏松，类骨质多；B组与D组相比，骨膜稍紊乱，骨膜下骨质轻度增生，骨小梁未见明显差异。21、28 d，A、C组软骨痂逐渐被新生骨小梁代替，C组与A组相比，骨小梁排列疏松、结构紊乱、密度低，骨痂面积较大，残存少量软骨细胞及类骨质；B组与D组比较无明显差异。免疫组织化学检测示，Sema3A阳性染色在C组软骨细胞表达高于A组，尤其在7、14、21 d；Sema3A阳性染色在A组新生骨小梁成骨细胞表达较C组高，尤其在14、21 d；差异均有统计学意义（P＜0.05）。Western blot检测示，Sema3A在A、C组骨折愈合过程中表达趋势相同，术后7、14、21、28 d C组Sema3A蛋白相对表达量高于A组（P＜0.05）；B组Sema3A蛋白相对表达量在术后7、14、21、28 d显著高于D组（P＜0.05）。.
结论: Sema3A在神经损伤后骨折愈合中表达异常，可能通过促进软骨细胞增殖、降低感觉神经纤维分布及成骨细胞分化，在神经损伤后骨折愈合中发挥作用。.
Keywords: Chondrocytes; Fracture healing; Semaphorin 3A; Sensory nerve; Traumatic brain injury.