CHANGES OF SEMAPHORIN 3A EXPRESSION IN HEALING OF TIBIA FRACTURE AFTER TRAUMATIC BRAIN INJURY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIZhengzheng ^1,2 , ZHAOJunwei ^1,2 , YIZhigang ^1,2 , LUOWei ^1,2 , LIKang ^1,2 ,  WANGYuliang ¹ , WANGJing ² , ANLiping ² , MAJinglin ²

1. Department of Orthopedics, Lanzhou University Second Hospital, Lanzhou Gansu, 730030, P. R. China;
2. Orthopedics Key Laboratory of Gansu Province, Lanzhou University Second Hospital, Lanzhou Gansu, 730030, P. R. China;

Corresponding author：

WANGYuliang, Email: wyl60918@sina.com

Keywords：

Traumatic brain injury; Fracture healing; Semaphorin 3A; Chondrocytes; Sensory nerve

DOI：

10.7507/1002-1892.20160251

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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. Conclusion 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.

Citation： LI Zhengzheng, ZHAO Junwei, YI Zhigang, LUO Wei, LI Kang, WANG Yuliang, WANG Jing, AN Liping, MA Jinglin. CHANGES OF SEMAPHORIN 3A EXPRESSION IN HEALING OF TIBIA FRACTURE AFTER TRAUMATIC BRAIN INJURY. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1225-1232. doi: 10.7507/1002-1892.20160251 Copy

1.	Hayashi M, Nakashima T, Taniguchi M, et al. Osteoprotection by semaphorin 3A. Nature, 2012, 485(7396):69-74.
2.	Fukuda T, Takeda S, Xu R, et al. Sema3A regulates bone-mass accrual through sensory innervations. Nature, 2013, 497(7450):490-493.
3.	Hashimoto M, Ino H, Koda M, et al. Regulation of semaphorin 3A expression in neurons of the rat spinal cord and cerebral cortex after transection injury. Acta Neuropathol, 2004, 107(3):250-256.
4.	Zhang L, Zhang L, Mao Z, et al. Semaphoring 3A:an association between traumatic brain injury and enhanced osteogenesis. Med Hypotheses, 2013, 81(4):713-714.
5.	夏群, 苗军, 张继东, 等. 神经系统对骨折愈合的调节. 中华创伤杂志, 2001, 17(9):572-574.
6.	Gomez C, Burt-Pichat B, Mallein-Gerin F, et al. Expression of Semaphorin-3A and its receptors in endochondral ossification:potential role in skeletal development and innervation. Dev Dyn, 2005, 234(2):393-403.
7.	Okubo M, Kimura T, Fujita Y, et al. Semaphorin 3A is expressed in human osteoarthritic cartilage and antagonizes vascular endothelial growth factor 165-promoted chondrocyte migration:an implication for chondrocyte cloning. Arthritis Rheum, 2011, 63(10):3000-3009.
8.	Feeney DM, Boyeson MG, Linn RT, et al. Responses to cortical injury:I. Methodology and local effects of contusions in the rat. Brain Res, 1981, 211(1):67-77.
9.	陈旭红, 白露, 顾航宇, 等. 创伤性脑损伤对骨折愈合影响动物模型的建立与评估. 北京大学学报(医学版), 2012, 44(6):831-837.
10.	Chen J, Li Y, Wang L, et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci, 2001, 189(1-2):49-57.
11.	Nakamura H, Aoki K, Masuda W, et al. Disruption of NF-kappaB1 prevents bone loss caused by mechanical unloading. J Bone Miner Res, 2013, 28(6):1457-1467.
12.	Kolodkin AL, Matthes DJ, Goodman CS. The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell, 1993, 75(7):1389-1399.
13.	Vachkov IH, Huang X, Yamada Y, et al. Inhibition of axonal outgrowth in the tumor environment:involvement of class 3 semaphorins. Cancer Sci, 2007, 98(8):1192-1197.
14.	Ito D, Nojima S, Kumanogoh A. The role of semaphorin family in immune systems. Nihon Rinsho Meneki Gakkai Kaishi, 2014, 37(1):1-10.
15.	Tufro A. Semaphorin3a signaling, podocyte shape, and glomerular disease. Pediatr Nephrol, 2014, 29(4):751-755.
16.	Liang Y, Wang W, Huang J, et al. Potential role of Semaphorin 3A and its receptors in regulating aberrant sympathetic innervation in peritoneal and deep infiltrating endometriosis. PLoS One, 2015, 10(12):e146027.
17.	Zhang Y, Kerns JM, Anderson DG, et al. Sensory neurons and fibers from multiple spinal cord levels innervate the rabbit lumbar disc. Am J Phys Med Rehabil, 2006, 85(11):865-871.
18.	Tolofari SK, Richardson SM, Freemont AJ, et al. Expression of semaphorin 3A and its receptors in the human intervertebral disc:potential role in regulating neural ingrowth in the degenerate intervertebral disc. Arthritis Res Ther, 2010, 12(1):R1.
19.	Tanelian DL, Barry MA, Johnston SA, et al. Semaphorin Ⅲ can repulse and inhibit adult sensory afferents in vivo. Nat Med, 1997, 3(12):1398-1401.
20.	Behar O, Golden JA, Mashimo H, et al. Semaphorin Ⅲ is needed for normal patterning and growth of nerves, bones and heart. Nature, 1996, 383(6600):525-528.
21.	屈墨羱, 胡静, 李运峰. 神经轴突导向分子Semaphorin 3A对骨质疏松模型大鼠骨折愈合的影响. 广东医学, 2016, 37(2):173-177.
22.	邱雨, 任嫒姝, 庞琴, 等. 信号素3A对成牙骨质细胞系OCCM-30增殖和矿化功能影响的实验研究. 口腔医学研究, 2016, 32(5):445-448.
23.	Parker MW, Guo HF, Li X, et al. Function of members of the neuropilin family as essential pleiotropic cell surface receptors. Biochemistry, 2012, 51(47):9437-9446.
24.	Montolio M, Messeguer J, Masip I, et al. A semaphorin 3A inhibitor blocks axonal chemorepulsion and enhances axon regeneration. Chem Biol, 2009, 16(7):691-701.
25.	Minor KH, Bournat JC, Toscano N, et al. Decorin, erythroblastic leukaemia viral oncogene homologue B4 and signal transducer and activator of transcription 3 regulation of semaphorin 3A in central nervous system scar tissue. Brain, 2011, 134(Pt 4):1140-1155.
26.	Jiang SX, Whitehead S, Aylsworth A, et al. Neuropilin 1 directly interacts with Fer kinase to mediate semaphorin 3A-induced death of cortical neurons. J Biol Chem, 2010, 285(13):9908-9918.

1. Hayashi M, Nakashima T, Taniguchi M, et al. Osteoprotection by semaphorin 3A. Nature, 2012, 485(7396):69-74.
2. Fukuda T, Takeda S, Xu R, et al. Sema3A regulates bone-mass accrual through sensory innervations. Nature, 2013, 497(7450):490-493.
3. Hashimoto M, Ino H, Koda M, et al. Regulation of semaphorin 3A expression in neurons of the rat spinal cord and cerebral cortex after transection injury. Acta Neuropathol, 2004, 107(3):250-256.
4. Zhang L, Zhang L, Mao Z, et al. Semaphoring 3A:an association between traumatic brain injury and enhanced osteogenesis. Med Hypotheses, 2013, 81(4):713-714.
5. 夏群, 苗军, 张继东, 等. 神经系统对骨折愈合的调节. 中华创伤杂志, 2001, 17(9):572-574.
6. Gomez C, Burt-Pichat B, Mallein-Gerin F, et al. Expression of Semaphorin-3A and its receptors in endochondral ossification:potential role in skeletal development and innervation. Dev Dyn, 2005, 234(2):393-403.
7. Okubo M, Kimura T, Fujita Y, et al. Semaphorin 3A is expressed in human osteoarthritic cartilage and antagonizes vascular endothelial growth factor 165-promoted chondrocyte migration:an implication for chondrocyte cloning. Arthritis Rheum, 2011, 63(10):3000-3009.
8. Feeney DM, Boyeson MG, Linn RT, et al. Responses to cortical injury:I. Methodology and local effects of contusions in the rat. Brain Res, 1981, 211(1):67-77.
9. 陈旭红, 白露, 顾航宇, 等. 创伤性脑损伤对骨折愈合影响动物模型的建立与评估. 北京大学学报(医学版), 2012, 44(6):831-837.
10. Chen J, Li Y, Wang L, et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci, 2001, 189(1-2):49-57.
11. Nakamura H, Aoki K, Masuda W, et al. Disruption of NF-kappaB1 prevents bone loss caused by mechanical unloading. J Bone Miner Res, 2013, 28(6):1457-1467.
12. Kolodkin AL, Matthes DJ, Goodman CS. The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell, 1993, 75(7):1389-1399.
13. Vachkov IH, Huang X, Yamada Y, et al. Inhibition of axonal outgrowth in the tumor environment:involvement of class 3 semaphorins. Cancer Sci, 2007, 98(8):1192-1197.
14. Ito D, Nojima S, Kumanogoh A. The role of semaphorin family in immune systems. Nihon Rinsho Meneki Gakkai Kaishi, 2014, 37(1):1-10.
15. Tufro A. Semaphorin3a signaling, podocyte shape, and glomerular disease. Pediatr Nephrol, 2014, 29(4):751-755.
16. Liang Y, Wang W, Huang J, et al. Potential role of Semaphorin 3A and its receptors in regulating aberrant sympathetic innervation in peritoneal and deep infiltrating endometriosis. PLoS One, 2015, 10(12):e146027.
17. Zhang Y, Kerns JM, Anderson DG, et al. Sensory neurons and fibers from multiple spinal cord levels innervate the rabbit lumbar disc. Am J Phys Med Rehabil, 2006, 85(11):865-871.
18. Tolofari SK, Richardson SM, Freemont AJ, et al. Expression of semaphorin 3A and its receptors in the human intervertebral disc:potential role in regulating neural ingrowth in the degenerate intervertebral disc. Arthritis Res Ther, 2010, 12(1):R1.
19. Tanelian DL, Barry MA, Johnston SA, et al. Semaphorin Ⅲ can repulse and inhibit adult sensory afferents in vivo. Nat Med, 1997, 3(12):1398-1401.
20. Behar O, Golden JA, Mashimo H, et al. Semaphorin Ⅲ is needed for normal patterning and growth of nerves, bones and heart. Nature, 1996, 383(6600):525-528.
21. 屈墨羱, 胡静, 李运峰. 神经轴突导向分子Semaphorin 3A对骨质疏松模型大鼠骨折愈合的影响. 广东医学, 2016, 37(2):173-177.
22. 邱雨, 任嫒姝, 庞琴, 等. 信号素3A对成牙骨质细胞系OCCM-30增殖和矿化功能影响的实验研究. 口腔医学研究, 2016, 32(5):445-448.
23. Parker MW, Guo HF, Li X, et al. Function of members of the neuropilin family as essential pleiotropic cell surface receptors. Biochemistry, 2012, 51(47):9437-9446.
24. Montolio M, Messeguer J, Masip I, et al. A semaphorin 3A inhibitor blocks axonal chemorepulsion and enhances axon regeneration. Chem Biol, 2009, 16(7):691-701.
25. Minor KH, Bournat JC, Toscano N, et al. Decorin, erythroblastic leukaemia viral oncogene homologue B4 and signal transducer and activator of transcription 3 regulation of semaphorin 3A in central nervous system scar tissue. Brain, 2011, 134(Pt 4):1140-1155.
26. Jiang SX, Whitehead S, Aylsworth A, et al. Neuropilin 1 directly interacts with Fer kinase to mediate semaphorin 3A-induced death of cortical neurons. J Biol Chem, 2010, 285(13):9908-9918.

Chinese Journal of Reparative and Reconstructive Surgery

CHANGES OF SEMAPHORIN 3A EXPRESSION IN HEALING OF TIBIA FRACTURE AFTER TRAUMATIC BRAIN INJURY

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