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Title: [Research of acellular xenogeneic nerve combined with adipose-derived stem cells and platelet rich plasma in repair of rabbit facial nerve injury].
Author: Sun Y, Zhang R, Mao X, Zhang M.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2018 Jun 15; 32(6):736-744. PubMed ID: 29905054.
Abstract:
OBJECTIVE: To investigate the early effects of acellular xenogeneic nerve combined with adipose-derived stem cells (ADSCs) and platelet rich plasma (PRP) in repairing facial nerve injury in rabbits. METHODS: The bilateral sciatic nerves of 15 3-month-old male Sprague-Dawley rats were harvested and decellularized as xenografts. The allogeneic ADSCs were extracted from the neck and back fat pad of healthy adult New Zealand rabbits with a method of digestion by collagenase type Ⅰ and the autologous PRP was prepared by two step centrifugation. The 3rd generation ADSCs with good growth were labelled with CM-Dil living cell stain, and the labelling and fluorescence attenuation of the cells were observed by fluorescence microscope. Another 32 New Zealand rabbits were randomly divided into 4 groups and established the left facial nerve defect in length of 1 cm ( n=8). The nerve defects of groups A, B, C, and D were repaired with CM-Dil-ADSCs composite xenogeneic nerve+autologous PRP, CM-Dil-ADSCs composite xenogeneic nerve, xenogeneic nerve, and autologous nerve, respectively. At 1 and 8 weeks after operation, the angle between the upper lip and the median line of the face (angle θ) was measured. At 4 and 8 weeks after operation, the nerve conduction velocity was recorded by electrophysiological examination. At 8 weeks after operation, the CM-Dil-ADSCs at the distal and proximal ends of regenerative nerve graft segment in groups A and B were observed by fluorescence microscopy; after toluidine blue staining, the number of myelinated nerve fibers in regenerated nerve was calculated; the structure of regenerated nerve fibers was observed by transmission electron microscope. RESULTS: ADSCs labelled by CM-Dil showed that the labelling rate of cells was more than 90% under fluorescence microscope, and the labelled cells proliferated well, and the fluorescence attenuated slightly after passage. All the animals survived after operation, the incision healed well and no infection occurred. At 1 week after operation, all the animals in each group had different degrees of dysfunction. The angle θ of the left side in groups A, B, C, and D were (53.4±2.5), (54.0±2.6), (53.7±2.4), and (53.0±2.1)°, respectively; showing significant differences when compared with the healthy sides ( P<0.05). At 8 weeks after operation, the angle θ of the left side in groups A, B, C, and D were (61.9±4.7), (56.8±4.2), (54.6±3.8), and (63.8±5.8)°, respectively; showing significant differences when compared with the healthy sides and with the values at 1 week ( P<0.05). Gross observation showed that the integrity and continuity of regenerated nerve in 4 groups were good, and no neuroma and obvious enlargement was found. At 4 and 8 weeks after operation, the electrophysiological examination results showed that the nerve conduction velocity was significantly faster in groups A and D than in groups B and C ( P<0.05), and in group B than in group C ( P<0.05); no significant difference was found between groups A and D ( P>0.05). At 8 weeks after operation, the fluorescence microscopy observation showed a large number of CM-Dil-ADSCs passing through the distal and proximal transplants in group A, and relatively few cells passing in group B. Toluidine blue staining showed that the density of myelinated nerve fibers in groups A and D were significantly higher than those in groups B and C ( P<0.05), and in group B than in group C ( P<0.05); no significant difference was found between groups A and D ( P>0.05). Transmission electron microscope observation showed that the myelinated nerve sheath in group D was large in diameter and thickness in wall. The morphology of myelin sheath in group A was irregular and smaller than that in group D, and there was no significant difference between groups B and C. CONCLUSION: ADSCs can survive as a seed cell in vivo, and can be differentiated into Schwann-like cells under PRP induction. It can achieve better results when combined with acellular xenogeneic nerve to repair peripheral nerve injury in rabbits. 目的: 探讨复合脂肪来源干细胞（adipose-derived stem cells，ADSCs）的脱细胞异种神经联合富血小板血浆（platelet rich plasma，PRP）修复兔面神经损伤的早期效果。. 方法: 取 15 只 3 月龄雌性 SD 大鼠双侧坐骨神经进行脱细胞处理，作为异种神经移植体。取成年新西兰大耳白兔颈背部脂肪垫，采用Ⅰ型胶原酶单独消化法分离培养 ADSCs；兔耳静脉采血后经两步离心法提取 PRP；观察不同体积分数 PRP 对 ADSCs 增殖的影响，以及诱导其为类雪旺细胞可行性。取第 3 代 ADSCs，CM-Dil 活细胞染色剂标记后，荧光显微镜观察细胞标记及传代后荧光衰减情况。另取 32 只新西兰大耳白兔建立左侧面神经 1 cm 长缺损模型，随机分成 4 组（ n=8），A、B、C、D 组分别采用 CM-Dil-ADSCs 复合脱细胞异种神经+自体PRP、CM-Dil-ADSCs 复合脱细胞异种神经、脱细胞异种神经、自体神经移植修复神经缺损。术后 1、8 周静态下测量各组动物左侧上唇与面正中线成角（θ 角）；4、8 周神经电生理检测记录神经传导速度；8 周荧光显微镜观察 A、B 组再生神经远近端 CM-Dil-ADSCs 数量，各组再生神经甲苯胺蓝染色计数有髓神经纤维、透射电镜观察再生神经纤维结构。. 结果: 5%~20% PRP 均能促进 ADSCs 增殖，经 20% PRP诱导后细胞 S-100 免疫荧光染色呈阳性。ADSCs 经 CM-Dil 标记后荧光显微镜下观察示细胞标记率达 90% 以上，被标记细胞传代后细胞增殖良好，传代后荧光稍衰减。术后各组动物均存活至实验完成。术后 1 周各组动物面部均发生不同程度功能障碍，A、B、C、D 组左侧 θ 角分别为（53.4±2.5）、（54.0±2.6）、（53.7±2.4）、（53.0±2.1）°，均显著低于健侧（ P<0.05）；术后 8 周分别为（61.9±4.7）、（56.8±4.2）、（54.6±3.8）、（63.8±5.8）°，与健侧及术后 1 周比较差异均有统计学意义（ P<0.05）。大体观察各组动物再生神经完整性及连续性均良好。术后 4、8 周神经电生理检测示，A、D 组神经传导速度均显著快于 B、C 组，B 组快于 C 组（ P<0.05），A、D 组间比较差异无统计学意义（ P>0.05）。术后 8 周荧光显微镜观察示，A 组移植神经远、近端横断面均可见大量 CM-Dil-ADSCs 通过，B 组通过细胞相对较少。甲苯胺蓝染色示，A、D 组有髓神经纤维密度均显著高于 B、C 组，B 组高于 C 组（ P<0.05）；A、D 组间差异无统计学意义（ P>0.05）。透射电镜观察示，D 组有髓神经纤维鞘直径大、壁厚，形态规则；A 组髓鞘形态与 D 组相似；B、C 组髓鞘形态不规则，直径小、壁薄。. 结论: ADSCs 可以作为种子细胞在体内存活，并可在 PRP 诱导下分化为类雪旺细胞，与脱细胞异种神经复合后修复兔周围神经损伤可获得较好效果。.

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