EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS WITH ACELLULAR MUSCLE BIOSCAFFOLDS ON REPAIR OF ACUTE HEMI-TRANSECTION SPINAL CORD INJURY IN RATS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WEI Xiangke ¹ , WEN Yimin ¹ , ZHANG Tao ² , LI Han ²

1Department of Spine Surgery, Lanzhou General Hospital, Lanzhou Millitary Region of Chinese PLA, Lanzhou Gansu, 710050, P.R.China;;
2the Second Clinical College of Medicine of Lanzhou University. Corresponding author: WEN Yimin, E-mail: wenyimin007@163.com;

Corresponding author：

Keywords：

Bone marrow mesenchymal stem cells; Acellular muscle bioscaffold; Co-transplantation; Spinal cord injury; Rat

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Objective To investigate the effects of allogenic transplantation of acellular muscle bioscaffolds (AMBS) seeded with bone marrow mesenchymal stem cells (BMSCs) on the repair of acute hemi-transection spinal cord injury (SCI) in rats. Methods AMBS were prepared by reformed chemical approach and sterilized by compound cold sterilization; BMSCs were harvested by density gradient centrifugation and cultured with adherent method. The 3rd generation BMSCs labeled by Hoechst 33342 were injected into AMBS to construct the BMSCs-AMBS composite scaffolds; the biocompatibility was observed under scanning electron microscope (SEM) and fluorescence microscope in vitro at 14 days. Forty-eight adult female Sprague Dawley rats were used to build SCI model by hemi-transecting at T9-11 level, then randomly divided into 4 groups (n=12). Defects were repaired with BMSCs-AMBS composite scaffolds, BMSCs, and AMBS in groups A, B, and C, respectively; group D was blank control by injecting PBS. At 1, 2, 3, and 4 weeks after surgery, the functional recovery of the hind limbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. At 4 weeks after surgery, HE staining and immunofluorescent assay were adopted. Results Masson staining and HE staining showed that AMBS was mainly of the collagen fibers in parallel arrange, without muscle fibers. After 14 days of BMSCs and AMBS co-culture, a large number of survival BMSCs labeled by Hoechst 33342 were seen under fluorescence microscope; SEM showed that BMSCs grew and attached to the inner surfaces of AMBS. At 2-4 weeks, the BBB score in group A was significantly higher than that in groups B, C, and D (P lt; 0.05), and it was significantly lower in group D than in the other 3 groups (P lt; 0.05); at 4 weeks, the BBB score in group B was significantly higher than that in group C (t=10.352, P=0.000). HE staining revealed that the area of spinal cord cavity after SCI was markedly smaller in group A than in the other 3 groups; immunofluorescent assay showed that more neurofilament 200 positive fibers and Nestin positive cells were detected in group A than in groups B, C, and D, but glial fibrillary acidic protein (GFAP) positive cells significantly decreased. The integral absorbance (IA) values of GFAP were 733.01 ± 202.04, 926.42 ± 59.46, 1 069.37 ± 33.42, and 1 469.46 ± 160.53 in groups A, B, C, and D, respectively; the IA value of group A was significantly lower than that of groups B, C, and D (P lt; 0.05), and it was significantly higher in group D than in groups A, B, and C (P lt; 0.05). Conclusion With relatively regular internal structures and good biocompatibility, AMBS can inhibit glial scar and enhance the survival, migration, and differentiation of BMSCs, so AMBS is the ideal nature vector for cell transplantation. Co-transplantation of AMBS and BMSCs has synergistic effect in treating SCI, it can promote rat motor function recovery.

Citation： WEI Xiangke,WEN Yimin,ZHANG Tao,LI Han. EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS WITH ACELLULAR MUSCLE BIOSCAFFOLDS ON REPAIR OF ACUTE HEMI-TRANSECTION SPINAL CORD INJURY IN RATS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(11): 1362-1368. doi: Copy

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2.	Stokols S, Tuszynski MH. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials, 2006, 27(3): 443-451.
3.	Zurita M, Otero L, Aguayo C, et al. Cell therapy for spinal cord repair: optimization of biologic scaffolds for survival and neural differentiation of human bone marrow stromal cells. Cytotherapy, 2010, 12(4): 522-537.
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5.	Itosaka H, Kuroda S, Shichinohe H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering. Neuropathology, 2009, 29(3): 248-257.
6.	汪大彬, 文益民, 蓝旭, 等. 壳聚糖-藻酸盐支架复合BMSCs修复急性脊髓损伤的实验研究. 中国修复重建外科杂志, 2010, 24(2): 190-196.
7.	Arai T, Kanje M, Lundborg G, et al. Axonal outgrowth in muscle grafts made acellular by chemical extraction. Restor Neurol Neurosci, 2000, 17(4): 165-174.
8.	Mligiliche N, Kitada M, Ide C. Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve. Arch Histol Cytol, 2001, 64(1): 29-36.
9.	Carlson EC, Carlson BM. A method for preparing skeletal muscle fiber basal laminae. Anat Rec, 1991, 230(3): 325-331.
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14.	Sanes JR, Marshall LM, McMahan UJ. Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J Cell Biol, 1978, 78(1): 176-198.
15.	徐新智, 孙磊, 胡蕴玉. 肌基膜管桥接周围神经缺损的电生理研究. 中华物理医学与康复杂志, 2000, 22(2): 119-120.
16.	Keilhoff G, Pratsch F, Wolf G, et al. Bridging extra large defects of peripheral nerves: possibilities and limitations of alternative biological grafts from acellular muscle and Schwann cells. Tissue Eng, 2005, 11(7-8): 1004-1014.
17.	张秀英, 薛辉, 孙皎, 等. 肌基膜管植入对大鼠脊髓半横断损伤模型的血管化作用. 吉林大学学报: 医学版, 2011, 37(5): 784-787.
18.	张秀英. 肌基膜管移植大鼠脊髓半切损伤模型血管生成情况的研究. 长春: 吉林大学, 2007.
19.	Fansa H, Schneider W, Wolf G, et al. Host responses after acellular muscle basal lamina allografting used as a matrix for tissue engineered nerve grafts. Transplantation, 2002, 74(3): 381-387.
20.	宋宇, 刘佳梅, 薛辉, 等. 携带神经干细胞肌基膜管组织工程支架中神经干细胞的存活分化. 中国组织工程研究与临床康复, 2010, 14(47): 8751-8754.
21.	殷迪. 人羊膜上皮细胞与肌基膜管支架相容性的实验研究. 长春: 吉林大学, 2008.
22.	贾彬, 李杰, 贺西京, 等. 嗅黏膜嗅鞘细胞与肌基膜管联合移植修复脊髓损伤. 中国组织工程研究与临床康复, 2008, 12(51): 10041-10044.
23.	王鸿飞, 郑连杰, 张书琴, 等. 牛脑提取液与肌基膜管结合对脊髓损伤修复及再生的影响. 中国临床康复, 2003, 7(32): 4336-4337.
24.	李培建, 胥少汀. 神经生长因子及其结合肌基膜管移植修复脊髓横断性损伤的组织学观察. 中华神经外科杂志, 2000, 16(6): 367-370.
25.	张红旭, 胥少汀, 吴霞, 等. 肌基膜管结合神经生长因子修复脊髓缺损的实验研究. 中华骨科杂志, 1995, 15(1): 32-35, T003.

1. Yang JT, Kuo YC, Chiu KH. Peptide-modified inverted colloidal crystal scaffolds with bone marrow stromal cells in the treatment for spinal cord injury. Colloids Surf B Biointerfaces, 2011, 84(1): 198-205.
2. Stokols S, Tuszynski MH. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials, 2006, 27(3): 443-451.
3. Zurita M, Otero L, Aguayo C, et al. Cell therapy for spinal cord repair: optimization of biologic scaffolds for survival and neural differentiation of human bone marrow stromal cells. Cytotherapy, 2010, 12(4): 522-537.
4. Gros T, Sakamoto JS, Blesch A, et al. Regeneration of long-tract axons through sites of spinal cord injury using templated agarose scaffolds. Biomaterials, 2010, 31(26): 6719-6729.
5. Itosaka H, Kuroda S, Shichinohe H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering. Neuropathology, 2009, 29(3): 248-257.
6. 汪大彬, 文益民, 蓝旭, 等. 壳聚糖-藻酸盐支架复合BMSCs修复急性脊髓损伤的实验研究. 中国修复重建外科杂志, 2010, 24(2): 190-196.
7. Arai T, Kanje M, Lundborg G, et al. Axonal outgrowth in muscle grafts made acellular by chemical extraction. Restor Neurol Neurosci, 2000, 17(4): 165-174.
8. Mligiliche N, Kitada M, Ide C. Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve. Arch Histol Cytol, 2001, 64(1): 29-36.
9. Carlson EC, Carlson BM. A method for preparing skeletal muscle fiber basal laminae. Anat Rec, 1991, 230(3): 325-331.
10. 汪大彬, 文益民, 蓝旭, 等. 壳聚糖-藻酸盐多通道支架材料细胞相容性研究. 中国矫形外科杂志, 2010, 18(10): 836-839.
11. Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J Neurosurg, 1977, 47(4): 577-581.
12. Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant, 2007, 40(7): 609-619.
13. Lu P, Jones LL, Tuszynski MH. BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp Neurol, 2005, 191(2): 344-360.
14. Sanes JR, Marshall LM, McMahan UJ. Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J Cell Biol, 1978, 78(1): 176-198.
15. 徐新智, 孙磊, 胡蕴玉. 肌基膜管桥接周围神经缺损的电生理研究. 中华物理医学与康复杂志, 2000, 22(2): 119-120.
16. Keilhoff G, Pratsch F, Wolf G, et al. Bridging extra large defects of peripheral nerves: possibilities and limitations of alternative biological grafts from acellular muscle and Schwann cells. Tissue Eng, 2005, 11(7-8): 1004-1014.
17. 张秀英, 薛辉, 孙皎, 等. 肌基膜管植入对大鼠脊髓半横断损伤模型的血管化作用. 吉林大学学报: 医学版, 2011, 37(5): 784-787.
18. 张秀英. 肌基膜管移植大鼠脊髓半切损伤模型血管生成情况的研究. 长春: 吉林大学, 2007.
19. Fansa H, Schneider W, Wolf G, et al. Host responses after acellular muscle basal lamina allografting used as a matrix for tissue engineered nerve grafts. Transplantation, 2002, 74(3): 381-387.
20. 宋宇, 刘佳梅, 薛辉, 等. 携带神经干细胞肌基膜管组织工程支架中神经干细胞的存活分化. 中国组织工程研究与临床康复, 2010, 14(47): 8751-8754.
21. 殷迪. 人羊膜上皮细胞与肌基膜管支架相容性的实验研究. 长春: 吉林大学, 2008.
22. 贾彬, 李杰, 贺西京, 等. 嗅黏膜嗅鞘细胞与肌基膜管联合移植修复脊髓损伤. 中国组织工程研究与临床康复, 2008, 12(51): 10041-10044.
23. 王鸿飞, 郑连杰, 张书琴, 等. 牛脑提取液与肌基膜管结合对脊髓损伤修复及再生的影响. 中国临床康复, 2003, 7(32): 4336-4337.
24. 李培建, 胥少汀. 神经生长因子及其结合肌基膜管移植修复脊髓横断性损伤的组织学观察. 中华神经外科杂志, 2000, 16(6): 367-370.
25. 张红旭, 胥少汀, 吴霞, 等. 肌基膜管结合神经生长因子修复脊髓缺损的实验研究. 中华骨科杂志, 1995, 15(1): 32-35, T003.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS WITH ACELLULAR MUSCLE BIOSCAFFOLDS ON REPAIR OF ACUTE HEMI-TRANSECTION SPINAL CORD INJURY IN RATS

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