Promotion of transplanted collagen scaffolds combined with brain-derived neurotrophic factor for axonal regeneration and motor function recovery in rats after transected spinal cord injury_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Xi ¹ , FAN Yongheng ² , XIAO Zhifeng ² , LI Xing ² , YANG Bin ² , ZHAO Yannan ² , HOU Xianglin ² , HAN Sufang ² ,  DAI Jianwu ^1,2

1. Institute of Combined Injury, College of Military Preventive Medicine, Army Medical University (Third Military Medical University), Chongqing, 400038, P.R.China;
2. National State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100190, P.R.China;

Corresponding author：

DAI Jianwu, Email: jwdai@genetics.ac.cn

Keywords：

Collagen scaffold; collagen binding domain; brain-derived neurotrophic factor; spinal cord injury; motor function; rat

DOI：

10.7507/1002-1892.201803094

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ObjectiveTo evaluate the effect of the combination of collagen scaffold and brain-derived neurotrophic factor (BDNF) on the repair of transected spinal cord injury in rats.MethodsThirty-two Sprague-Dawley rats were randomly divided into 4 groups: group A (sham operation group), T₉, T₁₀ segments of the spinal cord was only exposed; group B, 4-mm T₉, T₁₀ segments of the spinal cord were resected; group C, 4-mm T₉, T₁₀ segments of the spinal cord were resected and linear ordered collagen scaffolds (LOCS) with corresponding length was transplanted into lesion site; group D, 4-mm T₉, T₁₀ segments of the spinal cord were resected and LOCS with collagen binding domain (CBD)-BDNF was transplanted into lesion site. During 3 months after operation, Basso-Beattie-Bresnahan (BBB) locomotor score assessment was performed for each rat once a week. At 3 months after operation, electrophysiological test of motor evoked potential (MEP) was performed for rats in each group. Subsequently, retrograde tracing was performed for each rat by injection of fluorogold (FG) at the L₂ spinal cord below the injury level. One week later, brains and spinal cord tissues of rats were collected. Morphological observation was performed to spinal cord tissues after dehydration. The thoracic spinal cords including lesion area were collected and sliced horizontally. Thoracic spinal cords 1 cm above lesion area and lumbar spinal cords 1 cm below lesion area were collected and sliced coronally. Coronal spinal cord tissue sections were observed by the laser confocal scanning microscope and calculated the integral absorbance (IA) value of FG-positive cells. Horizontal tissue sections of thoracic spinal cord underwent immunofluorescence staining to observe the building of transected spinal cord injury model, axonal regeneration in damaged area, and synapse formation of regenerated axons.ResultsDuring 3 months after operation, the BBB scores of groups B, C, and D were significantly lower than those of group A (P<0.05). The BBB scores of group D at 2-12 weeks after operation were significantly higher than those of groups B and C (P<0.05). Electrophysiological tests revealed that there was no MEP in group B; the latencies of MEP in groups C and D were significantly longer than that in group A (P<0.05), and in group C than in group D (P<0.05). Morphological observation of spinal cord tissues showed that the injured area of the spinal cord in group B extended to both two ends, and the lesion site was severely damaged. The morphologies of spinal cord tissues in groups C and D recovered well, and the morphology in group D was closer to normal tissue. Results of retrograde tracing showed that the gray matters of lumbar spinal cords below the lesion area in each group were filled with FG-positive cells; in thoracic spinal cords above lesion sites, theIA value of FG-positive cells in coronal section of spinal cord in group A was significantly larger than those in groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), but no significant difference was found between groups C and D (P>0.05). Immunofluorescence staining results of spinal cord tissue sections selected from dorsal to ventral spinal cord showed transected injured areas of spinal cords which were significantly different from normal tissues. The numbers of NF-positive axons in lesion center of group A were significantly larger than those of groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05).ConclusionThe combined therapeutic approach containing LOCS and CBD-BDNF can promote axonal regeneration and recovery of hind limb motor function after transected spinal cord injury in rats.

Citation： CHEN Xi, FAN Yongheng, XIAO Zhifeng, LI Xing, YANG Bin, ZHAO Yannan, HOU Xianglin, HAN Sufang, DAI Jianwu. Promotion of transplanted collagen scaffolds combined with brain-derived neurotrophic factor for axonal regeneration and motor function recovery in rats after transected spinal cord injury. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(6): 650-659. doi: 10.7507/1002-1892.201803094 Copy

1.	Hagg T, Oudega M. Degenerative and spontaneous regenerative processes after spinal cord injury. J Neurotrauma, 2006, 23(3-4): 264-280.
2.	McDonald JW, Sadowsky C. Spinal-cord injury. Lancet, 2002, 359(9304): 417-425.
3.	Thuret S, Moon LD, Gage FH. Therapeutic interventions after spinal cord injury. Nat Rev Neurosci, 2006, 7(8): 628-643.
4.	Shrestha B, Coykendall K, Li Y, et al. Repair of injured spinal cord using biomaterial scaffolds and stem cells. Stem Cell Res Ther, 2014, 5(4): 91.
5.	Haggerty AE, Oudega M. Biomaterials for spinal cord repair. Neurosci Bull, 2013, 29(4): 445-459.
6.	Li X, Han J, Zhao Y, et al. Functionalized collagen scaffold implantation and cAMP administration collectively facilitate spinal cord regeneration. Acta Biomater, 2016, 30: 233-245.
7.	Fan C, Li X, Xiao Z, et al. A modified collagen scaffold facilitates endogenous neurogenesis for acute spinal cord injury repair. Acta Biomater, 2017, 51: 304-316.
8.	Xing L, Jin H, Zhao Y, et al. A functionalized collagen scaffold neutralizing the myelin inhibitory molecules promoted neurites outgrowth in vitro and facilitated spinal cord regeneration in vivo. ACS Appl Mater Interfaces, 2015, 7(25): 13960-13971.
9.	Li X, Zhao Y, Cheng S, et al. Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair. Biomaterials, 2017, 137: 73-86.
10.	Lu P, Wang Y, Graham L, et al. Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell, 2012, 150(6): 1264-1273.
11.	Bowling H, Bhattacharya A, Klann E, et al. Deconstructing brain-derived neurotrophic factor actions in adult brain circuits to bridge an existing informational gap in neuro-cell biology. Neural Regen Res, 2016, 11(3): 363-367.
12.	Han S, Wang B, Jin W, et al. The linear-ordered collagen scaffold-BDNF complex significantly promotes functional recovery after completely transected spinal cord injury in canine. Biomaterials, 2015, 41: 89-96.
13.	Lai BQ, Che MT, Du BL, et al. Transplantation of tissue engineering neural network and formation of neuronal relay into the transected rat spinal cord. Biomaterials, 2016, 109: 40-54.
14.	Han Q, Sun W, Lin H, et al. Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats. Tissue Eng Part A, 2009, 15(10): 2927-2935.
15.	Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol, 1996, 139(2): 244-256.
16.	Li X, Liu S, Zhao Y, et al. Training neural stem cells on functional collagen scaffolds for severe spinal cord injury repair. Advanced Functional Materials, 2016, 26(32): 5835-5847.
17.	Xiao Z, Chen B, Dai JW. Building the regenerative microenvironment with functional biomaterials for spinal cord injury repair. J Spine, 2016. doi:10.4172/2165-7939.S7-005.
18.	Siebert JR, Eade AM, Osterhout DJ. Biomaterial approaches to enhancing neurorestoration after spinal cord injury: strategies for overcoming inherent biological obstacles. Biomed Res Int, 2015, 2015: 752572. doi: 10.1155/2015/752572.
19.	Agbay A, Edgar JM, Robinson M, et al. Biomaterial strategies for delivering stem cells as a treatment for spinal cord injury. Cells Tissues Organs, 2016, 202(1-2): 42-51.
20.	Li X, Tan J, Xiao Z, et al. Transplantation of hUC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury. Sci Rep, 2017, 7: 43559.
21.	Kadoya K, Tsukada S, Lu P, et al. Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury. Neuron, 2009, 64(2): 165-172.
22.	Nakajima H, Uchida K, Kobayashi S, et al. Rescue of rat anterior horn neurons after spinal cord injury by retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene. J Neurotrauma, 2007, 24(4): 703-712.
23.	Han Q, Jin W, Xiao Z, et al. The promotion of neural regeneration in an extreme rat spinal cord injury model using a collagen scaffold containing a collagen binding neuroprotective protein and an EGFR neutralizing antibody. Biomaterials, 2010, 31(35): 9212-9220.
24.	陈亚亮, 李莉, 高秀来. BDA 法神经束路追踪技术. 首都医科大学学报, 2002, 23(3): 258-260.
25.	Kulik P, Zacharko-Siembida A, Arciszewski MB. The localization of primary efferent sympathetic neurons innervating the porcine thymus-a retrograde tracing study. Acta Vet Brno, 2017, 86(2): 117-122.
26.	Zhang W, Xu D, Cui J, et al. Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways. Microsc Res Tech, 2017, 80(2): 260-266.

1. Hagg T, Oudega M. Degenerative and spontaneous regenerative processes after spinal cord injury. J Neurotrauma, 2006, 23(3-4): 264-280.
2. McDonald JW, Sadowsky C. Spinal-cord injury. Lancet, 2002, 359(9304): 417-425.
3. Thuret S, Moon LD, Gage FH. Therapeutic interventions after spinal cord injury. Nat Rev Neurosci, 2006, 7(8): 628-643.
4. Shrestha B, Coykendall K, Li Y, et al. Repair of injured spinal cord using biomaterial scaffolds and stem cells. Stem Cell Res Ther, 2014, 5(4): 91.
5. Haggerty AE, Oudega M. Biomaterials for spinal cord repair. Neurosci Bull, 2013, 29(4): 445-459.
6. Li X, Han J, Zhao Y, et al. Functionalized collagen scaffold implantation and cAMP administration collectively facilitate spinal cord regeneration. Acta Biomater, 2016, 30: 233-245.
7. Fan C, Li X, Xiao Z, et al. A modified collagen scaffold facilitates endogenous neurogenesis for acute spinal cord injury repair. Acta Biomater, 2017, 51: 304-316.
8. Xing L, Jin H, Zhao Y, et al. A functionalized collagen scaffold neutralizing the myelin inhibitory molecules promoted neurites outgrowth in vitro and facilitated spinal cord regeneration in vivo. ACS Appl Mater Interfaces, 2015, 7(25): 13960-13971.
9. Li X, Zhao Y, Cheng S, et al. Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair. Biomaterials, 2017, 137: 73-86.
10. Lu P, Wang Y, Graham L, et al. Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell, 2012, 150(6): 1264-1273.
11. Bowling H, Bhattacharya A, Klann E, et al. Deconstructing brain-derived neurotrophic factor actions in adult brain circuits to bridge an existing informational gap in neuro-cell biology. Neural Regen Res, 2016, 11(3): 363-367.
12. Han S, Wang B, Jin W, et al. The linear-ordered collagen scaffold-BDNF complex significantly promotes functional recovery after completely transected spinal cord injury in canine. Biomaterials, 2015, 41: 89-96.
13. Lai BQ, Che MT, Du BL, et al. Transplantation of tissue engineering neural network and formation of neuronal relay into the transected rat spinal cord. Biomaterials, 2016, 109: 40-54.
14. Han Q, Sun W, Lin H, et al. Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats. Tissue Eng Part A, 2009, 15(10): 2927-2935.
15. Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol, 1996, 139(2): 244-256.
16. Li X, Liu S, Zhao Y, et al. Training neural stem cells on functional collagen scaffolds for severe spinal cord injury repair. Advanced Functional Materials, 2016, 26(32): 5835-5847.
17. Xiao Z, Chen B, Dai JW. Building the regenerative microenvironment with functional biomaterials for spinal cord injury repair. J Spine, 2016. doi:10.4172/2165-7939.S7-005.
18. Siebert JR, Eade AM, Osterhout DJ. Biomaterial approaches to enhancing neurorestoration after spinal cord injury: strategies for overcoming inherent biological obstacles. Biomed Res Int, 2015, 2015: 752572. doi: 10.1155/2015/752572.
19. Agbay A, Edgar JM, Robinson M, et al. Biomaterial strategies for delivering stem cells as a treatment for spinal cord injury. Cells Tissues Organs, 2016, 202(1-2): 42-51.
20. Li X, Tan J, Xiao Z, et al. Transplantation of hUC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury. Sci Rep, 2017, 7: 43559.
21. Kadoya K, Tsukada S, Lu P, et al. Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury. Neuron, 2009, 64(2): 165-172.
22. Nakajima H, Uchida K, Kobayashi S, et al. Rescue of rat anterior horn neurons after spinal cord injury by retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene. J Neurotrauma, 2007, 24(4): 703-712.
23. Han Q, Jin W, Xiao Z, et al. The promotion of neural regeneration in an extreme rat spinal cord injury model using a collagen scaffold containing a collagen binding neuroprotective protein and an EGFR neutralizing antibody. Biomaterials, 2010, 31(35): 9212-9220.
24. 陈亚亮, 李莉, 高秀来. BDA 法神经束路追踪技术. 首都医科大学学报, 2002, 23(3): 258-260.
25. Kulik P, Zacharko-Siembida A, Arciszewski MB. The localization of primary efferent sympathetic neurons innervating the porcine thymus-a retrograde tracing study. Acta Vet Brno, 2017, 86(2): 117-122.
26. Zhang W, Xu D, Cui J, et al. Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways. Microsc Res Tech, 2017, 80(2): 260-266.

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Promotion of transplanted collagen scaffolds combined with brain-derived neurotrophic factor for axonal regeneration and motor function recovery in rats after transected spinal cord injury

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