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Title: [Experimental study on autologous injectable platelets rich fibrin combined with bone mesenchymal stem cells in treating sciatic nerve injury in rats].
Author: Gao H, Wang B, Cao J, Li X, Huang C, Wu J, Liu Z.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2020 May 15; 34(5):637-642. PubMed ID: 32410433.
Abstract:
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. 目的: 探讨自体注射型富血小板纤维蛋白（injectable platelet rich fibrin，i-PRF）联合 BMSCs 治疗大鼠坐骨神经损伤的疗效。. 方法: 取 10～15 日龄 SD 乳鼠胫骨骨髓分离培养 BMSCs 至第 4 代备用。取 24 只成年 SD 大鼠眶后静脉血采用改良低速离心法制备 i-PRF 后，采用改良挤压损伤法制作坐骨神经Ⅲ度损伤模型后随机分为 4 组，每组 6 只。A、B、C、D 组分别于损伤部位鞘膜内注射 BMSCs 悬液+自体 i-PRF、自体 i-PRF、BMSCs 悬液、生理盐水。术后 1～8 周每周采用 BBB 评分法评价大鼠患肢神经功能恢复情况；术后 2 个月处死各组大鼠取材，行 HE 染色观察坐骨神经组织结构变化，透射电镜观察神经纤维、髓鞘、细胞核等细微结构改变，Western blot 检测 N-cadherin、巢蛋白（Nestin）、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）表达。. 结果: 术后大鼠均未发生免疫排斥反应及死亡。术后 1 周各组大鼠 BBB 评分比较差异无统计学意义（ P>0.05）；术后 2～8 周，A 组 BBB 评分显著高于 B、C、D 组，B、C 组显著高于 D 组（ P<0.05），B、C 组间差异无统计学意义（ P>0.05）。HE 染色示，A 组神经纤维排列整齐，无缺损及脱髓鞘改变；B 组神经纤维结构欠清晰，轻度肿胀；C 组神经纤维部分结构紊乱，局部脱髓鞘改变；D 组神经纤维连续性欠佳，明显脱髓鞘改变，神经外膜部分缺损。透射电镜观察示，A 组神经纤维结构清晰，髓鞘完整，细胞核致密；B 组较 A 组稍次之；C 组神经纤维结构模糊，有脱髓鞘改变；D 组神经纤维结构紊乱，有脱髓鞘改变，神经外膜连续性中断。Western blot 检测示，各组 Nestin 蛋白相对表达量比较差异均无统计学意义（ P>0.05）。B、C、D 组 N-cadherin 蛋白相对表达量显著低于 A 组，C、D 组低于 B 组，D 组低于 C 组，差异均有统计学意义（ P<0.05）。B、C、D 组 GFAP 蛋白相对表达量显著低于 A 组，D 组显著低于 B、C 组，差异有统计学意义（ P<0.05）；B、C 组间差异无统计学意义（ P>0.05）。. 结论: 自体 i-PRF 与 BMSCs 联合使用可有效治疗大鼠坐骨神经损伤。.

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