Experimental study on autologous injectable platelets rich fibrin combined with bone mesenchymal stem cells in treating sciatic nerve injury in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GAO Haiming , WANG Bo , CAO Jiaquan , LI Xiujun , HUANG Chenyi ,  WU Jiaqi ,  LIU Zongchao

Department of Orthopedics, the Traditional Chinese Medicine Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P.R.China;

Corresponding author：

WU Jiaqi, Email: wujiaqi15@163.com; LIU Zongchao, Email: 565409672@qq.com

Keywords：

Neural tissue engineering; injectable platelet rich fibrin; bone marrow mesenchymal stem cells; nerve injury; rat

DOI：

10.7507/1002-1892.201909061

Video：

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Objective To investigate the effectiveness of autologous injectable platelet rich fibrin (i-PRF) combined with bone marrow mesenchymal stem cells (BMSCs) for sciatic nerve injury in rats.Methods BMSCs were isolated and cultured from tibial bone marrow of Sprague Dawley (SD) neonatal rats aged 10-15 days and passaged to the 4th generation. i-PRF was prepared from posterior orbital venous blood of adult SD rats by improved low-speed centrifugation. Twenty-four adult SD rats were selected and randomly divided into 4 groups with 6 rats in each group after the sciatic nerve Ⅲ degree injury model was established by modified crush injury method. Groups A, B, C, and D were injected with BMSCs suspension+autologous i-PRF, autologous i-PRF, BMSCs suspension, and normal saline, respectively. The Basso-Beattie-Bresnahan (BBB) score was used to evaluate the recovery of neurological function of the affected limb of rats every week from 1 to 8 weeks after operation. At 2 months after operation, the rats were sacrificed and the histological changes of sciatic nerve were observed by HE staining. The microstructural changes of nerve fibers, myelin sheath, and nucleus were observed by transmission electron microscope. The expressions of N-cadherin, Nestin, and glial fibrillary acidic protein (GFAP) were detected by Western blot.Results No immune rejection or death occurred in the rats after operation. There was no significant difference in BBB scores between groups at 1 week after operation (P>0.05); at 2-8 weeks after operation, BBB scores in group A were significantly higher than those in groups B, C, and D, and in groups B, C than in group D (P<0.05), there was no significant difference between groups B and C (P>0.05). HE staining showed that the nerve fibers in group A arranged in order, without defect or demyelination; the nerve fibers in group B were not clear and slightly swollen; some of the nerve fibers in group C were disordered and demyelinated; the nerve fibers in group D were not continuous, obviously demyelinated, and some of the nerve adventitia damaged. Transmission electron microscope showed that the structure of nerve fibers in group A was clear, myelin sheath was complete, and nucleus was dense; group B was slightly less than group A; group C had fuzzy structure, demyelination, and hollowing out; group D had disorder structure, demyelination, and hollowing out, and the middle part of nerve adventitia continuity. Western blot detection results showed that there was no significant difference in the relative expression of Nestin between groups (P>0.05). The relative expression of N-cadherin was significantly lower in groups B, C, and D than in group A, in groups C and D than in group B, and in group D than in group C (P<0.05). The relative expression of GFAP was significantly lower in groups B, C, and D than in group A, in group D than in groups B and C (P<0.05); there was no significant difference between groups B and C (P>0.05).Conclusion Autologous i-PRF combined with BMSCs can effectively treat sciatic nerve tissue injury in rats.

Citation： GAO Haiming, WANG Bo, CAO Jiaquan, LI Xiujun, HUANG Chenyi, WU Jiaqi, LIU Zongchao. Experimental study on autologous injectable platelets rich fibrin combined with bone mesenchymal stem cells in treating sciatic nerve injury in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(5): 637-642. doi: 10.7507/1002-1892.201909061 Copy

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2.	石更强, 胡益. 周围神经种子细胞的研究进展. 生物医学工程学杂志, 2015, 32(2): 470-474.
3.	Fink KD, Deng P, Torrest A, et al. Developing stem cell therapies for juvenile and adult-onset Huntington's disease. Regen Med, 2015, 10(5): 623-646.
4.	Lei L, Yu Y, Han J, et al. Quantification of growth factors in advanced platelet-rich fibrin and concentrated growth factors and their clinical efficiency as adjunctive to the GTR procedure in periodontal intrabony defects. J Periodontol, 2019. [Epub ahead of print].
5.	Wang X, Yang Y, Zhang Y, et al. Fluid platelet-rich fibrin stimulates greater dermal skin fibroblast cell migration, proliferation, and collagen synthesis when compared to platelet-rich plasma. J Cosmet Dermatol, 2019, 18(6): 2004-2010.
6.	李松, 郭延伟, 王占义, 等. 兔下颌骨来源骨髓间充质干细胞的体外培养及鉴定. 上海口腔医学, 2013, 22(1): 19-24.
7.	Wang X, Zhang Y, Choukroun J, et al. Behavior of gingival fibroblasts on titanium implant surfaces in combination with either injectable-PRF or PRP. Int J Mol Sci, 2017, 18(2): pii: E331.
8.	Mourão CF, Valiense H, Melo ER, et al. Obtention of injectable platelets rich-fibrin (i-PRF) and its polymerization with bone graft: technical note. Rev Col Bras Cir, 2015, 42(6): 421-423.
9.	Gigo-Benato D, Russo TL, Tanaka EH, et al. Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve. Lasers Surg Med, 2010, 42(9): 673-682.
10.	Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci, 2003, 23(4): 1416-1423.
11.	Buckenmeyer MJ, Meder TJ, Prest TA, et al. Decellularization techniques and their applications for the repair and regeneration of the nervous system. Methods, 2019. [Epub ahead of print].
12.	Sadeghian H, Wolfe GI. Therapy update in nerve, neuromuscular junction and myopathic disorders. Curr Opin Neurol, 2010, 23(5): 496-501.
13.	Cooney DS, Wimmers EG, Ibrahim Z, et al. Mesenchymal stem cells enhance nerve regeneration in a rat sciatic nerve repair and hindlimb transplant model. Sci Rep, 2016, 6: 31306.
14.	Zhang Y, Hu W, Ma K, et al. Reprogramming of keratinocytes as donor or target cells holds great promise for cell therapy and regenerative medicine. Stem Cell Rev Rep, 2019, 15(5): 680-689.
15.	Zhu SF, Zhong ZN, Fu XF, et al. Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between human umbilical cord and placenta-derived mesenchymal stem cells. Neurosci Lett, 2013, 541: 77-82.
16.	Rosada C, Justesen J, Melsvik D, et al. The human umbilical cord blood: a potential source for osteoblast progenitor cells. Calcif Tissue Int, 2003, 72(2): 135-142.
17.	尹刚, 刘蔡钺, 林耀发, 等. 脂肪干细胞来源外泌体对周围神经损伤后再生作用的实验研究. 中国修复重建外科杂志, 2018, 32(12): 1592-1596.
18.	陈涛, 高绍莹, 郝艺, 等. 人羊膜间充质干细胞外泌体通过微小 RNA-135a 促进成纤维细胞迁移实验研究. 中国修复重建外科杂志, 2020, 34(2): 234-239.
19.	赵斌, 马剑雄, 马信龙. 组织工程周围神经血管化研究进展. 中国修复重建外科杂志, 2019, 33(8): 1029-1032.
20.	刘泉体, 兰婷, 吴佳奇. 富血小板血浆制备技术及其抗菌效应的研究进展. 重庆医学, 2019, 48(9): 1572-1575.
21.	Visser R, Arrabal PM, Santos-Ruiz L, et al. Basic fibroblast growth factor enhances the osteogenic differentiation induced by bone morphogenetic protein-6 in vitro and in vivo. Cytokine, 2012, 58(1): 27-33.
22.	Morigele M, Shao Z, Zhang Z, et al. TGF-β1 induces a nucleus pulposus-like phenotype in Notch 1 knockdown rabbit bone marrow mesenchymal stem cells. Cell Biol Int, 2013, 37(8): 820-825.
23.	Wang W, Yu Y, Jiang Y, et al. Silk fibroin scaffolds loaded with angiogenic genes in adenovirus vectors for tissue regeneration. J Tissue Eng Regen Med, 2019, 13(5): 715-728.
24.	Sunitha Raja V, Munirathnam Naidu E. Platelet-rich fibrin: evolution of a second-generation platelet concentrate. Indian J Dent Res, 2008, 19(1): 42-46.
25.	Pintus E, Baldassarri M, Perazzo L, et al. Stem cells in osteochondral tissue engineering. Adv Exp Med Biol, 2018, 1058: 359-372.
26.	Yao Y, Zheng Z, Song Q. Mesenchymal stem cells: A double-edged sword in radiation-induced lung injury. Thorac Cancer, 2018, 9(2): 208-217.
27.	郭子宽. 间充质干细胞及其临床应用中的几个问题. 中国组织工程研究, 2012, 16(1): 1-10.

1. Jiang B, Zhang P, Jiang B. Advances in small gap sleeve bridging peripheral nerve injury. Artif Cells Blood Substit Immobil Biotechnol, 2010, 38(1): 1-4.
2. 石更强, 胡益. 周围神经种子细胞的研究进展. 生物医学工程学杂志, 2015, 32(2): 470-474.
3. Fink KD, Deng P, Torrest A, et al. Developing stem cell therapies for juvenile and adult-onset Huntington's disease. Regen Med, 2015, 10(5): 623-646.
4. Lei L, Yu Y, Han J, et al. Quantification of growth factors in advanced platelet-rich fibrin and concentrated growth factors and their clinical efficiency as adjunctive to the GTR procedure in periodontal intrabony defects. J Periodontol, 2019. [Epub ahead of print].
5. Wang X, Yang Y, Zhang Y, et al. Fluid platelet-rich fibrin stimulates greater dermal skin fibroblast cell migration, proliferation, and collagen synthesis when compared to platelet-rich plasma. J Cosmet Dermatol, 2019, 18(6): 2004-2010.
6. 李松, 郭延伟, 王占义, 等. 兔下颌骨来源骨髓间充质干细胞的体外培养及鉴定. 上海口腔医学, 2013, 22(1): 19-24.
7. Wang X, Zhang Y, Choukroun J, et al. Behavior of gingival fibroblasts on titanium implant surfaces in combination with either injectable-PRF or PRP. Int J Mol Sci, 2017, 18(2): pii: E331.
8. Mourão CF, Valiense H, Melo ER, et al. Obtention of injectable platelets rich-fibrin (i-PRF) and its polymerization with bone graft: technical note. Rev Col Bras Cir, 2015, 42(6): 421-423.
9. Gigo-Benato D, Russo TL, Tanaka EH, et al. Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve. Lasers Surg Med, 2010, 42(9): 673-682.
10. Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci, 2003, 23(4): 1416-1423.
11. Buckenmeyer MJ, Meder TJ, Prest TA, et al. Decellularization techniques and their applications for the repair and regeneration of the nervous system. Methods, 2019. [Epub ahead of print].
12. Sadeghian H, Wolfe GI. Therapy update in nerve, neuromuscular junction and myopathic disorders. Curr Opin Neurol, 2010, 23(5): 496-501.
13. Cooney DS, Wimmers EG, Ibrahim Z, et al. Mesenchymal stem cells enhance nerve regeneration in a rat sciatic nerve repair and hindlimb transplant model. Sci Rep, 2016, 6: 31306.
14. Zhang Y, Hu W, Ma K, et al. Reprogramming of keratinocytes as donor or target cells holds great promise for cell therapy and regenerative medicine. Stem Cell Rev Rep, 2019, 15(5): 680-689.
15. Zhu SF, Zhong ZN, Fu XF, et al. Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between human umbilical cord and placenta-derived mesenchymal stem cells. Neurosci Lett, 2013, 541: 77-82.
16. Rosada C, Justesen J, Melsvik D, et al. The human umbilical cord blood: a potential source for osteoblast progenitor cells. Calcif Tissue Int, 2003, 72(2): 135-142.
17. 尹刚, 刘蔡钺, 林耀发, 等. 脂肪干细胞来源外泌体对周围神经损伤后再生作用的实验研究. 中国修复重建外科杂志, 2018, 32(12): 1592-1596.
18. 陈涛, 高绍莹, 郝艺, 等. 人羊膜间充质干细胞外泌体通过微小 RNA-135a 促进成纤维细胞迁移实验研究. 中国修复重建外科杂志, 2020, 34(2): 234-239.
19. 赵斌, 马剑雄, 马信龙. 组织工程周围神经血管化研究进展. 中国修复重建外科杂志, 2019, 33(8): 1029-1032.
20. 刘泉体, 兰婷, 吴佳奇. 富血小板血浆制备技术及其抗菌效应的研究进展. 重庆医学, 2019, 48(9): 1572-1575.
21. Visser R, Arrabal PM, Santos-Ruiz L, et al. Basic fibroblast growth factor enhances the osteogenic differentiation induced by bone morphogenetic protein-6 in vitro and in vivo. Cytokine, 2012, 58(1): 27-33.
22. Morigele M, Shao Z, Zhang Z, et al. TGF-β1 induces a nucleus pulposus-like phenotype in Notch 1 knockdown rabbit bone marrow mesenchymal stem cells. Cell Biol Int, 2013, 37(8): 820-825.
23. Wang W, Yu Y, Jiang Y, et al. Silk fibroin scaffolds loaded with angiogenic genes in adenovirus vectors for tissue regeneration. J Tissue Eng Regen Med, 2019, 13(5): 715-728.
24. Sunitha Raja V, Munirathnam Naidu E. Platelet-rich fibrin: evolution of a second-generation platelet concentrate. Indian J Dent Res, 2008, 19(1): 42-46.
25. Pintus E, Baldassarri M, Perazzo L, et al. Stem cells in osteochondral tissue engineering. Adv Exp Med Biol, 2018, 1058: 359-372.
26. Yao Y, Zheng Z, Song Q. Mesenchymal stem cells: A double-edged sword in radiation-induced lung injury. Thorac Cancer, 2018, 9(2): 208-217.
27. 郭子宽. 间充质干细胞及其临床应用中的几个问题. 中国组织工程研究, 2012, 16(1): 1-10.

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Experimental study on autologous injectable platelets rich fibrin combined with bone mesenchymal stem cells in treating sciatic nerve injury in rats

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