复合他克莫司聚乳酸-乙醇酸缓释导管修复大鼠胫神经缺损

背景：虽然单纯聚乳酸-乙醇酸导管修复大鼠神经缺损可部分恢复大鼠神经功能,但神经直径、再生纤维数量、髓鞘成熟度及功能恢复上均较自体神经移植差。 目的：观察复合他克莫司的聚乳酸-乙醇酸缓释导管修复大鼠胫神经缺损的可行性。 方法：制作SD大鼠右侧胫神经缺损模型,随机分为3组,分别植入自体胫神经、单纯聚乳酸-乙醇酸导管及复合他克莫司的聚乳酸-乙醇酸缓释导管修复。植入后3,6,12周行坐骨神经功能指数检查、电生理检查、组织学观测、腓肠肌湿质量测量。 结果与结论：植入后第6,12周复合他克莫司的聚乳酸-乙醇酸缓释导管组、自体胫神经组坐骨神经功能指数检查、电生理检查、组织学观测、腓肠肌湿质量测量结果优于单纯聚乳酸-乙醇酸导管组(P < 0.05),自体胫神经组、复合他克莫司的聚乳酸-乙醇酸缓释导管组比较差异无显著性意义。说明复合他克莫司的聚乳酸-乙醇酸缓释导管桥接修复大鼠胫神经缺损可明显促进断端神经的再生,在晚期功能恢复上取得接近自体神经移植的效果。 关键词：聚乳酸-乙醇酸;他克莫司;神经导管;大鼠;坐骨神经 doi:10.3969/j.issn.1673-8225.2012.12.003     

聚乳酸/神经生长因子缓释导管修复周围神经缺损实验研究

目的：观察聚乳酸／神经生长因子(PDLLA／NGF)缓释导管对大鼠坐骨神经缺损再生的促进作用及其性能、降解过程。方法：SD大鼠60只，随机分为4组：自体神经移植组(A组)，单纯PDLLA导管组(B组)，单纯导管加导管内一次性给药组(C组)，缓释导管组(D组)，每组15只。制作坐骨神经10mm缺损模型，分别用自体神经移植、单纯PDLLA导管、PDLLA／NGF缓释导管桥接修复。于第1、2、3月后行大体观察、三头肌湿重恢复率测量、电生理检测、组织学和图象分析对比。结果：同时间段组间比较，除三头肌恢复率低以外，D组再生神经取得了和A组相似的效果，明显比B组和C组恢复好。结论：PDLLA／NGF缓释导管桥接修复大鼠坐骨神经缺损，能够有效促进神经再生，效果接近自体神经移植。     

聚乳酸聚羟基乙酸共聚物三维神经导管修复周围神经缺损

背景:异体神经移植由于存在难以消除的宿主免疫排斥反应,限制了其使用,许多学者试图用其他组织替代来弥补以上不足,但效果均不满意。目前,尚没有研制出公认的效果满意的人工神经,自体神经移植至今仍被认为是最佳选择。目的:观察聚乳酸聚羟基乙酸共聚物(PLGA,85:15)三维神经导管修复大鼠周围神经缺损的可行性,及神经导管内微丝支架的作用和不同数量微丝对神经再生的影响。方法:40只成年SD大鼠随机数字表法分为4组,制作大鼠12mm的左侧坐骨神经缺损模型,用该导管桥接大鼠12mm的坐骨神经缺损。A组:PLGA神经导管组;B组:PLGA神经导管内纵形放入20根PLGA微丝;C组:PLGA神经导管内纵形放入40根PLGA微丝;D组:自体神经移植组。A、B、C组神经导管内均注入层粘蛋白+神经生长因子混合液。造模后动态观察大鼠肌肉萎缩、跛行情况,测量神经导管内再生神经的传导速度、小腿三头肌湿质量恢复率。对再生神经中1/3段行组织学观察及图像分析以评价神经修复的效果。结果与结论:造模后各组再生神经均已通过神经导管长入远端,B、D组再生神经较A、C组粗大;再生神经的运动神经传导速度B组和D组明显快于A组和C组(P0.05);A组、C组肌肉萎缩最明显,而B组、D组肌肉萎缩较轻且肌肉萎缩程度基本相当。病理图像分析神经纤维计数以D组最多,B组次之,而与A组、C组相比差异均有显著性意义(P0.05),B组与D组的再生神经纤维数量及成熟程度均要明显优于A组和C组。提示新型的PLGA三维神经导管能有效引导SD大鼠坐骨神经长过12mm的神经缺损,是一种较理想的神经导管;神经导管内微丝支架能有效引导神经再生,数量过多反而可能抑制神经再生。     

复合转化生长因子-β的组织工程化周围神经修复坐骨神经缺损

探讨了转化生长因子-β(Transforming growth factor-β,TGF-β)在周围神经缺损修复中的作用。将50 ng·ml-1 TGF-β加入体外培养的雪旺细胞(Sehwann cells,SC)中,MTT和流式细胞仪观测到TGF-β能够明显促进SC增殖;ELISA方法检测到TGF-β组上清神经生长因子(NGF)含量高于对照组(P<0．05);将牛去细胞基质(Bovine acellular matrix,BAM)、SC、血清和培养基按一定的比例混合,注入聚乳酸羟基乙酸共聚物(PLGA)神经导管去修复15 mm坐骨神经缺损。30只SD大鼠分为3组,实验组:PLGA导管 SC TGF-β;空白组PLGA导管 SC;和自体神经移植组。16周后通过电生理、透射电镜等检测方法显示各组坐骨神经均得到再生,修复效果实验组与自体神经移植组无显著性差异,均优于空白组。TGF-β一方面可以明显促进SC的增殖,另一方面可以增强SC分泌NGF的功能。因此周围神经修复过程中使用外源性的TGF-β对修复周围神经缺损有较好的疗效。     

人工组织神经移植物对大鼠陈旧性坐骨神经缺损(60天)的修复作用

目的 观察人工组织神经移植物对陈旧性大鼠坐骨神经缺损的修复作用.方法 切除成年SD大鼠部分左侧坐骨神经,饲养60d形成陈旧性坐骨神经缺损后,以人工组织神经移植物修复缺损,同时设自体神经修复和不修复两对照组.修复术后3个月做神经-肌复合电位、腓肠肌湿重及再生神经形态学检测.结果 人工组织神经移植物修复组实验侧的运动神经传导速度、腓肠肌湿重、移植物远侧再生神经有髓神经纤维髓鞘厚度等结果与自体神经修复组相似.不修复对照组则未记录到神经-肌复合电位,未观察到再生神经纤维结构,腓肠肌湿重明显小于人工组织神经移植组和自体神经移植组.结论 人工组织神经移植物在一定程度上能修复缺损60d的大鼠坐骨神经.     

胶原蛋白-硫酸肝素神经生物支架材料的制备

背景：有报道胶原-氨基聚糖材料基体植入周围神经组织节段性损伤处,可以促进神经轴突修复再生。硫酸肝素是细胞外基质的重要组成部分。 目的：制备胶原蛋白-硫酸肝素神经组织工程生物支架,观察其生物相容性并用于神经损伤的修复。 方法：将胶原蛋白-硫酸肝素悬浊液冷冻干燥,通过显微物镜、扫描电镜观察内部孔隙结构,测量孔的大小。选取SD大鼠12只制备5 mm坐骨神经神经缺损模型,随机分为3组。硫酸肝素复合胶原支架移植桥接组、空白对照组(旷置)、正常对照组(自体神经移植)。移植术后经神经功能学观形态学检测其修复疗效。 结果与结论：胶原蛋白复合硫酸肝素支架材料具有纵行的、平行排列的微管结构,高度仿生神经的内部空间三维结构。组织学染色、电镜观察证实再生神经纤维成功地长入组织工程材料内。部分大鼠运动功能恢复良好。结果提示,胶原蛋白复合硫酸肝素材料组织相容性良好,可以促进神经的再生并可用于神经损伤的修复。     

应用胶原-壳聚糖桥接管引导大鼠坐骨神经再生

目的:观察胶原-壳聚糖桥接管促进大鼠坐骨神经损伤后再生与修复的作用。方法:用壳聚糖和胶原蛋白按1∶3的比例采用冷冻干燥法制成胶原-壳聚糖复合导管;将20只雄性Wistar大鼠随机分为2组,胶原-壳聚糖导管组和硅胶管组,切断坐骨神经,建立10mm的缺损动物模型;分别用胶原-壳聚糖导管和硅胶管进行桥接。于术后不同时间对2组动物进行大体观察,并于术后14周进行电生理、组织学及逆行示踪检测,比较2组大鼠坐骨神经的再生和功能恢复情况。结果:术后14周2组动物坐骨神经再生和功能恢复情况的各项检测指标显示,胶原-壳聚糖桥接组明显优于硅胶管桥接组。结论:胶原-壳聚糖导管可用来桥接损伤神经,在周围神经缺损修复方面具有良好的应用前景。     

几丁糖与聚乳酸合成生物材料修复周围神经缺损

背景：单纯几丁糖材料制成的神经导管机械强度较差,易于塌陷,不利于再生神经的生长。 目的：观察几丁糖与聚乳酸复合物修复大鼠周围神经缺损的可行性。 方法：取30只SD大鼠,制作单侧坐骨神经缺损模型,随机均分为3组,分别采用自体神经、硅胶导管及几丁糖与聚乳酸复合导管修复神经缺损,修复后12周,观察桥接神经外观、表面粘连情况及有无神经瘤生成等,检测大鼠神经传导速度、动作电位波幅及潜伏期,苏木精-伊红染色观察坐骨神经桥接物中段神经再生轴突数量及再生神经横截面积,称量大鼠完整小腿三头肌湿质量。 结果与结论：修复后12周,3组再生神经均通过5 mm神经缺损间隙,硅胶管组形成神经瘤,其余两组均未出现神经瘤;自体神经组再生神经直径大于几丁糖-聚乳酸组、硅胶管组(P < 0.05),几丁糖-聚乳酸组再生神经直径大于硅胶管组(P < 0.05);几丁糖-聚乳酸组、自体神经组可见排列整齐的高密度再生轴突,再生轴突数量多于硅胶管组(P < 0.05),且神经传导速度、动作电位波幅、小腿三头肌湿质量显著大于硅胶管组(P < 0.05),潜伏期低于硅胶管组(P < 0.05)。表明几丁糖-聚乳酸复合导管可促进缺损周围神经的再生。  中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

PNGF导管复合骨髓间充质干细胞修复大鼠12 mm坐骨神经缺损

目的　观察RGD多肽接枝聚（乳酸-羟基乙酸-L-赖氨酸）/聚乳酸/β-磷酸三钙/神经生长因子（PRGD/PDLLA/β-TCP/NGF,PNGF）缓释导管复合骨髓间充质干细胞(Bone marrow derived mesenchymal stem cells, BMSCs)构建组织工程化人工神经,修复大鼠12 mm坐骨神经缺损的效果。  方法    雄性Wistar大鼠30只, 随机分为3组,每组10只,左后肢制作12 mm坐骨神经缺损模型,分别行单纯PNGF导管桥接（A）、PNGF导管复合BMSCs桥接（B）、自体神经移植（C）,所有大鼠左侧为实验侧,右侧为正常自身对照侧。术后3个月行大体观察、坐骨神经功能指数、电生理检测、小腿三头肌湿重恢复率测量、新生神经及靶肌肉组织学观察等检测坐骨神经功能恢复情况。  结果　术后3个月取材时见导管管壁变薄,表面血管化良好,管内有再生神经通过,直径较正常神经细。坐骨神经功能指数的检测结果显示PNGF导管复合BMSCs高于单纯PNGF导管组（P＜0.05）,PNGF导管复合BMSCs组神经传导速度恢复率、小腿三头肌湿重恢复率、有髓神经纤维数量和直径均优于单纯PNGF导管组（P＜0.01）,取得与自体神经移植组相似的效果。  结论　PNGF缓释导管复合BMSCs桥接修复大鼠坐骨神经缺损, 能够有效促进神经再生, 效果接近自体神经移植。     

合成可生物降解神经导管修复损伤周围神经：生物相容性良好

背景：临床对周围神经损伤进行修复治疗的时候,可以利用自体神经进行治疗或者利用不同材质的神经导管进行治疗。 目的：探索合成可生物降解材料神经导管在周围神经损伤修复中的应用效果。 方法：48只新西兰大白兔,随机等分为3组,自体神经移植组、硅胶导管组和可降解神经导管组。各组动物切除10 mm坐骨神经,构建坐骨神经缺损动物模型,并分别利用自体神经、硅胶导管以及可降解神经导管进行坐骨神经修复。 结果与结论：造模后3周,硅胶导管组兔运动神经传导效果、小腿三头肌肌肉湿质量恢复率比自体神经移植组差,但可降解神经导管组兔运动神经传导效果、小腿三头肌肌肉湿质量恢复率与自体神经移植组接近。造模后12周时,自体神经移植组中存在大量呈均匀排列的有髓神经纤维,可降解神经导管组中可见大量分布不均匀的再生有髓神经纤维,而硅胶导管组中存在少量呈不均匀排列的髓神经纤维。表明合成可生物降解材料神经导管在周围神经损伤修复中可以获得与自体神经较为接近的良好效果。  中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Preparation of electrospun PLGA-silk fibroin nanofibers-based nerve conduits and evaluation in vivo

With advances in technical methodology, the grafting of biocompatible conduits may become a viable alternative for the reconstruction of nerve gaps. In this study, electrospinning was used to fabricate nerve conduits (NCs) from poly(L-lactide-coglycolide)-silk fibroin. Conduits or autograft nerves were employed to bridge 10 mm defects in the sciatic nerves of Sprague-Dawley rats. Six weeks after the operation, morphological and functional assessment showed that nerve conduits from PLGA-silk fibroin grafts promoted the regeneration of peripheral nerves. The effects were similar to those obtained using nerve autografts. This method offers a promising alternative to the use of nerve autografts.     

The effect of high outflow permeability in asymmetric poly(dl-lactic acid-co-glycolic acid) conduits for peripheral nerve regeneration

This study attempted to accelerate the peripheral nerve regeneration, using the high outflow rate of asymmetric poly(dl-lactic acid-co-glycolic acid) (PLGA) nerve conduits. Asymmetric PLGA nerve conduits of monomer ratio 85/15 were prepared by immersion-precipitation method to serve as possible materials. In this study, mandrels were immersed into a 20% (wt/wt) of PLGA/1,4-dioxane solution and precipitated in a non-solvent bath followed by freeze-drying. Different concentrations of isopropyl alcohol (95%, 40% and 20%) were used as precipitation baths where non-asymmetric (95%) and asymmetric (40% and 20%) conduits could easily form. The asymmetric nerve conduits that consisted of macrovoids on the outer layer, and interconnected micropores in the inner sublayer, possessed characters of larger outflow rate than inflow rate. The asymmetric conduits were implanted to 10mm right sciatic nerve defects in rats. Autografts, silicone and non-asymmetric PLGA conduits were performed as the control and the contrast groups. Implanted graft specimens of all groups were harvested for histological analysis at 4 and 6 weeks following surgery. The asymmetric PLGA conduits maintained a stable supporting structure and inhibited exogenous cells invasion during entire regeneration process. Asymmetric PLGA conduits were found to have statistically greater number of regenerated axons at the midconduit and distal nerve site of implanted grafts, as compared to the silicone and non-asymmetric groups at 4 and 6 weeks. Of interest was that the results of 4 weeks in asymmetric groups were better than the non-asymmetric groups at 6 weeks in number of axons. According to the results of permeability, the asymmetric structure in the conduit wall seemed to enhance the removal of the blockage of the waste drain from the inner inflamed wound in the early stage, which may have improved the efficacy of the peripheral nerve regeneration. The asymmetric structure could be adequately employed in the future as optimal nerve conduits in peripheral nerve regeneration.     

The effect of aligned core-shell nanofibres delivering NGF on the promotion of sciatic nerve regeneration

Recent bioengineering strategies for peripheral nerve regeneration have been focusing on the development of alternative treatments for nerve repair. In this study, we incorporated nerve growth factor (NGF) into aligned core-shell nanofibres by coaxial electrospinning, and reeled the scaffold into aligned fibrous nerve guidance conduits (NGCs) for nerve regeneration study. This aligned PLGA/NGF NGC combined physical guidance cues and biomolecular signals to closely mimic the native extracellular matrix (ECM). The effect of this aligned PLGA/NGF NGC on the promotion of nerve regeneration was evaluated in a 13-mm rat sciatic nerve defect using functional and morphological analysis. After 12 weeks implantation, the results of electrophysiological and muscle weight examination demonstrated that the functional recovery of the regenerated nerve in the PLGA/NGF NGC group was significantly better than that in the PLGA group, yet had no significant difference compared with the autograft group. The toluidine blue staining study showed that more nerve fibres were regenerated in the PLGA/NGF group, while the electron microscopy study indicated that the regenerated nerve in the PLGA/NGF group was more mature than that in the PLGA group. This study demonstrated that the aligned PLGA/NGF could greatly promote peripheral nerve regeneration and have a potential application in nerve regeneration.     

Effects of unidirectional permeability in asymmetric poly(DL-lactic acid-co-glycolic acid) conduits on peripheral nerve regeneration: an in vitro and in vivo study

The high outflow permeability of the nerve conduit used to emit the drained waste generated from the traumatized host nerve stump is critical in peripheral nerve regeneration. Our earlier studies have established that asymmetric conduits fulfill the basic requirements for use as nerve guide conduits. In this study, the inflow characteristics of optimal nerve conduits were further examined using in vivo and in vitro trials. Various asymmetric poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits were controlled by modifying precipitation baths using 0, 20, and 95% isopropyl alcohol, with high-porosity (permeability), medium-porosity (high outflow and low inflow), and low-porosity (permeability), respectively. In the in vitro trial, the Schwann cells and fibroblasts were seeded on either side of the asymmetric PLGA films in a newly designed coculture system that simulated the repaired nerve conduit environment. The results of the directional permeable films indicated the statistically significant proliferation of Schwann cells and the inhibition of the division of fibroblasts in lactate dehydrogenase release and inhibition of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction, compared with the other films. In the in vivo trial, the PLGA conduits seeded with Schwann cells were implanted into 10 mm right sciatic nerve defects in rats. After 6 weeks, implanted conduits were harvested. Histological examination verified that directional permeable conduits had markedly more A-type and B-type myelin fibers in the midconduit and distal nerve. In this work, the directional transport characteristics were established as an extremely important factor to the design and development of optimal nerve guide conduits in peripheral nerve regeneration.     

The Effect of Aligned Core–Shell Nanofibres Delivering NGF on the Promotion of Sciatic Nerve Regeneration

Recent bioengineering strategies for peripheral nerve regeneration have been focusing on the development of alternative treatments for nerve repair. In this study, we incorporated nerve growth factor (NGF) into aligned core–shell nanofibres by coaxial electrospinning, and reeled the scaffold into aligned fibrous nerve guidance conduits (NGCs) for nerve regeneration study. This aligned PLGA/NGF NGC combined physical guidance cues and biomolecular signals to closely mimic the native extracellular matrix (ECM). The effect of this aligned PLGA/NGF NGC on the promotion of nerve regeneration was evaluated in a 13-mm rat sciatic nerve defect using functional and morphological analysis. After 12 weeks implantation, the results of electrophysiological and muscle weight examination demonstrated that the functional recovery of the regenerated nerve in the PLGA/NGF NGC group was significantly better than that in the PLGA group, yet had no significant difference compared with the autograft group. The toluidine blue staining study showed that more nerve fibres were regenerated in the PLGA/NGF group, while the electron microscopy study indicated that the regenerated nerve in the PLGA/NGF group was more mature than that in the PLGA group. This study demonstrated that the aligned PLGA/NGF could greatly promote peripheral nerve regeneration and have a potential application in nerve regeneration.     

PDLLA/NGF复合膜用于自体神经移植促进神经再生实验研究

目的：探讨外消旋聚乳酸／神经生长因子(PDLLA／NGF)复合膜用于自体神经移植是否具有促进神经再生作用。方法：雌性Wistar大白鼠16只，随机分为2组：A组(自体神经移植)8只；B组(自体神经移植并包绕PDLLA／NGF复合膜)8只。显露每只动物的右侧坐骨神经，A组将坐骨神经切除10mm长一段，以外膜缝合法行原位移植；B组与A组同样行神经原位移植并在远近两个神经缝接部位各包绕1块PDLLA／NGF复合膜(含NGF总量为400u)。6个月后观察比较两组神经再生情况。结果：B组的小腿三头肌湿重恢复率、运动神经传导速度、再生的有髓神经纤维数量、直径、轴突直径、髓鞘厚度均优于A组：结论：PDLLA／NGF复合膜包绕自体神经移植的神经缝接部位具有促进神经再生作用.     

A synthetic oxygen carrier in fibrin matrices promotes sciatic nerve regeneration in rats

Tissue-engineering nerve conduits have been studied for a long time in bridging large nerve defects. However, the low oxygen availability within the nerve conduits, which results in death of migratory Schwann cells (SC) or loss of the newly formed tissue’s function, is still an obstacle for axonal regeneration. Thus, it was hypothesized that an oxygen-enriched conduit would enhance axonal regeneration and functional recovery in vivo. To address this issue, perfluorotributylamine (PFTBA) enriched fibrin hydrogel was prepared and injected into collagen–chitosan conduits. The conduit containing PFTBA-enriched fibrin hydrogel was then used to bridge a 12-mm sciatic nerve defect in rats. The control rats were bridged with collagen–chitosan conduits filled with fibrin matrices without PFTBA. It was found that axonal regeneration and functional recovery in the combined PFTBA group were significantly higher than those in the control group without PFTBA. Further investigations showed that the mRNA and protein levels of S-100, brain-derived neurotrophic factor and nerve growth factor were enhanced by PFTBA at 1 and 3 weeks after surgery. However, the mRNA and protein levels of vascular endothelial growth factor were in a similar range between the combined PFTBA group and the control group without PFTBA. In addition, immunohistochemical results showed that the morphological appearances of regenerated nerve and survival of SC were enhanced by PFTBA at 4 and 12 weeks after surgery. In conclusion, PFTBA-enriched nerve conduit is capable of enhancing axonal regeneration, which provides a new avenue for achieving better functional recovery in the treatment of nerve defect.     

新型仿生人工神经导管诱导犬周围神经再生过程的组织学观察**

背景：前期动物实验已经证明,精氨酸-甘氨酸-天冬氨酸多肽接枝聚(羟基乙酸-L-赖氨酸-乳酸)/β-磷酸三钙/神经生长因子导管可以修复大鼠坐骨神经10 mm缺损,该材料具有良好的生物相容性和细胞亲和性。 目的：观察精氨酸-甘氨酸-天冬氨酸多肽接枝聚(羟基乙酸-L-赖氨酸-乳酸)/聚乳酸/β-磷酸三钙/神经生长因子导管诱导犬周围神经再生过程的组织学变化。 方法：取健康成年杂种犬12只,建立30 mm腓总神经缺损模型,用精氨酸-甘氨酸-天冬氨酸多肽接枝聚(羟基乙酸-L-赖氨酸-乳酸)/聚乳酸/β-磷酸三钙/神经生长因子人工神经导管进行桥接修复。 结果与结论：修复后3个月时在神经远端即可发现新生神经纤维,随着时间的推移,再生神经轴突直径及髓鞘厚度不断增加,9个月时再生神经中可见较多发育成熟的有髓神经纤维。胫骨前肌损伤后出现逐渐萎缩,修复后6,9个月萎缩肌肉逐渐恢复。从组织学角度证实了精氨酸-甘氨酸-天冬氨酸多肽接枝聚(羟基乙酸-L-赖氨酸-乳酸)/聚乳酸/β-磷酸三钙/神经生长因子导管可修复大动物粗大神经缺损。