用组织工程方法桥接周围神经缺损的实验研究

目的 寻找修复周围神经缺损的新的有效替代材料。方法 将体外培养扩增的许旺细胞接种在生物可吸收材料聚羟基乙酸纤维支架上培养２周,形成一种新的神经桥接物,并对其进行了观察和动物移植试验。结果 发现接种的许旺细胞可在聚羟基乙酸纤维上良好吸附、迁移形成一种新型的、非管状的、具有三维纵向细胞排列和导层粘蛋白通道的组织工程化神经桥接物,用其修复近交的Ｗｉｓｔａｒ大鼠１５ｍｍ坐骨神经缺损时,取得了接近自体神经移     

许旺细胞在人工神经支架材料上三维培养的体外活性研究

目的　探讨许旺细胞在组织工程支架材料上体外三维培养的生长活性。　方法　对聚羟基乙酸和聚乳酸的共聚物细丝支架进行生物学修饰 ,把原代培养的许旺细胞悬液接种在支架材料上 ,用MTT法、单细胞凝胶电泳法及显微分光光度和显微图像联合法测定许旺细胞的生长活性。　结果　聚羟基乙酸和聚乳酸的共聚物细丝支架上的许旺细胞各项活性指标与正常培养的许旺细胞比较 ,差异无显著性 (P >0 0 5 ) ,实验组许旺细胞DNA无损伤。　结论　聚羟基乙酸和聚乳酸的共聚物细丝适于构建具有许旺细胞活性的组织工程化人工神经。     

聚乳酸微丝在外周神经内的降解性研究

目的 研究聚乳酸微丝在神经干内的降解特点以及在降解过程中对外周神经的影响，探讨其是否适合作为组织工程化外周神经的支架。方法 在20只SD大鼠两侧的坐骨神经内分别植入聚乳酸微丝(实验侧)和Vicryl微丝(对照侧)，于术后1、4、8和12周观察微丝的形态和神经组织结构的变化以及植入部位的组织和细胞反应。结果 聚乳酸微丝植入神经干后在表面和内部同时发生崩裂，至12周时已大部分降解；在降解过程中始终伴随着巨噬细胞活跃的吞噬活动；两种微丝植入神经干后均引起类似的轻度异物炎性反应；在聚乳酸微丝的降解过程中，神经束和神经纤维的组织结构没有明显变化。结论 聚乳酸微丝在外周神经内的降解时间超过12周；微丝在降解时引起的轻度非特异性炎性反应对神经组织没有不良影响。     

利用组织工程技术预制兔耳廓软骨的实验研究

组织工程化软骨的形成与许多因素有关，但其中最主要的因素是细胞培养支架的选择。以聚乳酸包埋的聚羟基乙酸无纺网是应用较为广泛的支架之一，但亲水性差，对细胞吸附力弱，细胞基质产生少是其不足之处。我们选择了卵磷脂、多聚赖氨酸及聚乳酸分别和共同包埋聚羟基乙酸，...     

人工组织神经移植物辅加神经生长因子修复大鼠坐骨神经缺损

目的 探索壳聚糖与聚乙醇酸联合制成的人工组织神经移植物辅加神经生长因子（NGF）修复大鼠外周神经缺损的可能性。方法 壳聚糖套管和聚乙醇酸纤维制备成人工组织神经移植物并辅加NGF，修复大鼠的坐骨神经10mm缺失。术后24周，进行足迹试验，电生理学测试，形态学观察。用自体神经，硅胶管以及壳聚糖套管与聚乙醇酸纤维等分别作为对照组，结果 术后24周内，实验动物均未出现明显炎症及排斥反应。实验组的各检测指标均优于除自体神经组外的各对照。结论 壳聚糖和聚乙醇酸与外周神经组织有良好的生物相容性；辅加NGF的人工组织神经移植物对修复缺损的神经具有良好的桥梁作用和促神经生长作用。     

生物可降解材料聚乳酸/聚羟基乙酸复合壳聚糖在人工冠状动脉血管支架制备中的应用

第一代金属裸支架和第二代涂层支架介入治疗冠状动脉粥样硬化性心脏病(冠心病)已得到广泛应用。由于长期存在金属支架异物刺激及其携带的药物扰乱血管壁各层细胞生长,引起支架内再狭窄和血管栓塞,远期仍有较多的主要心血管不良事件发生和需要再血管化治疗。因此,由聚酯、聚碳酸酐及聚磷酸酯等高分子材料制备的完全可生物降解吸收支架及药物洗脱支架应运而生,其中聚乳酸(poly-lactic acid,PLA)、聚羟基乙酸(poly-glycolicacid,PGA)、壳聚糖、聚己内酯(poly-caprolactone,PCL)及一些共聚物如聚乳酸/聚羟基乙酸共聚物(poly-lactic-co-glycolic acid,PLGA)材料制备的心血管植入支架的安全性、组织及血液相容性已得到证实,然而这些支架具有各自的缺点,如PLA降解较慢质硬易断裂柔韧性不足,PGA降解较快质软支撑力不足,支架降解太快或者太慢,均难以达到有效支撑,支架植入后容易出现血管损伤、弹性回缩,导致血管再狭窄及血栓形成,远期效果不佳。通过优化组合不同摩尔比的PLA和PGA及壳聚糖涂层,可以获得具有更好的生物相容性、适度的降解速率(约3～6个月完全降解)、足够的机械强度、较低的炎症反应和伸展度良好的复合材料,从而为制备完全生物可降解冠状动脉支架奠定实验基础。     

修饰后的聚羟基乙酸和聚乳酸共聚物支架与人胚雪旺细胞联合培养的实验研究

目的：探索人体组织工程神经构建的方法。方法：应用鼠尾胶和层粘蛋白包埋纤维状的聚羟基乙酸和聚乳酸共聚物支架与人胚雪旺细胞培养2周，观察细胞在支架上的吸附迁移和生长情况，结果：人雪旺细胞能在共聚物纤维上迁移包裹，雪旺细胞均匀分布于以鼠尾胶和层粘蛋白共同包埋后的纤维之间及吸附在纤维表面，且基质分泌旺盛，结论：人胚雪细胞在修饰后的polyglytin纤维上得到大量扩增，形成的三维立体结构具有人工神经的基本特性。     

利用组织工程技术再生软骨组织的实验研究

目的 在有免疫力的动物体兔体内探索组织工程化软骨生成的影响因素。 方法 经不同物质修猸的聚羟基乙酸支架与软骨细胞体外培养,观察基质产生情况,并将细胞支架复合物体内回植,观察软骨的生成,并进行组织学及超微结构评价。结果 以孵磷脂、多聚赖氨酸及聚乳酸共同修饰的聚羟基乙酸与软骨细胞体外培养,结果基质产生旺盛,体内回植后软骨生成良好。 结论 细胞支架复合物体外培养期间有基质产生,是组织工程化软骨生成的前提     

甲壳素涂层并预置引导纤维神经导管的实验研究

目的探讨甲壳素涂层并预置引导纤维神经导管修复周围神经缺损的效果。方法成年雌性SD大鼠24只，无菌条件下切断双侧坐骨神经，制成14mm的大鼠坐骨神经缺损模型。根据不同修复材料随机分为4组，每组6只。A组：甲壳素涂层并预置引导纤维的聚乳酸聚羟基乙酸共聚物（polyglycolic-lacticacid，PGLA）神经导管；B组：甲壳素涂层的PGLA神经导管；C组：单纯PGLA神经导管；D组：自体神经移植作为对照。术后4周和12周行大体观察、肌电图检查、S-100免疫组织化学染色和组织学观察，图像分析评价修复效果。结果术后4周A、B及C组观察到神经导管中有新生轴索通过，再生神经发育不成熟；D组近段有髓神经纤维均匀疏散分布，远段未见明显再生神经束形成。术后12周各组再生神经已通过神经导管长入远端，肌电图、S100免疫组织化学染色和图像分析结果表明A组再生神经轴突数量及再生神经质量优于B、C组，差异有统计学意义（P〈0．05）。D组移植神经轴突直径、髓鞘厚度、纤维密度与A、B及C组比较，差异有统计学意义（P〈0．05），但D组近段神经髓鞘部分空泡样变性及脱髓鞘改变，远段再生神经束形成少。结论甲壳素涂层并预置引导纤维的神经导管能有效修复周围神经缺损。     

冻干骨和PLGA材料表面软骨细胞增殖的形态学观察比较

目的了解两种材料表面软骨细胞的增殖特性预测形成组织工程化关节软骨的可能性。方法获取幼兔关节软骨细胞,种植于两种材料表面,培养观察细胞生长情况。结果冻干骨和聚羟基乙酸聚乳酸共聚物(PLGA)材料表面细胞均有粘附增殖及基质分泌,但后者明显优于前者。结论冻干骨表面粘合PLGA材料种植软骨细胞有可能形成组织工程化关节软骨。     

骨髓干细胞诱导分化构建组织工程神经

[目的]探讨组织工程化神经修复周围神经缺损的作用。[方法]以DMEM为培养基体外诱导人骨髓基质干细胞分化为雪旺细胞，与细胞外基质及可降解聚乳酸导管构建组织工程化神经；建立坐骨神经缺损10mm的Wistar大鼠动物模型，A组：经诱导骨髓基质干细胞分化雪旺细胞与天然细胞外基质（extra cellular matrix，ECM）凝胶及可降解聚乳酸导管构建组织工程化神经桥接神经缺损；B组：单纯将ECM凝胶注入可降解聚乳酸导管桥接神经缺损；C组：自体神经移植组。术后12周进行神经电生理检测、新生神经组织学观察和轴突计数等检测坐骨神经功能恢复情况。[结果]1．诱导后骨髓基质干细胞呈梭形、胞核大、周围有光晕、突起细长呈纵形排列，GAFP及S-100免疫组织化学染色阳性。动物模型各组经移植术后12周，再生神经已通过缺损区长至神经远端。A组、C组组织学及电生理检测指标均优于B组（PAB=0．021，PBC=0．001），A组与C组无显著性差异（PAC=0．065）；A、B组聚乳酸导管降解吸收明显。[结论]骨髓基质干细胞在体外可诱导分化为雪旺氏细胞，利用其与细胞外基质及可降解聚乳酸导管构建组织工程化神经可以修复周围神经缺损。     

己丁糖处理神经吻合口促进神经再生的实验研究

目的:神经离断伤经显微手术修复后,神经吻合口粘连常可阻碍神经再生。本研究利用己丁糖防止粘连的作用并与其他方法进行对比,探索促进神经再生的办法。方法:将48只兔随机分为4组:己丁糖组、强的松龙组、静脉包裹组及单纯缝合组。将兔的胫神经切断后吻合,吻合口分别用己丁糖、强的松龙处理、静脉包裹及不作处理。于术后2、4、10、16周行肉眼观察及光镜检查,第16周行电镜、电生理检查及作轴突图像分析。结果:己丁糖在瘢痕床上的使用可有效防止瘢痕对修复段神经的压迫、粘连和固定,强的松龙也有防止神经粘连的作用,二者优于静脉包裹和单纯缝合组。经统计学处理,差异有显著性意义(P<0.01)。结论:术中使用己丁糖、强的松龙处理神经吻合口,能有效地防止周围神经粘连,促进周围神经再生,最大限度地恢复周围神经的功能。     

In vitro and in vivo evaluation of a biodegradable chitosan-PLA composite peripheral nerve guide conduit material

Chitosan, a nature biodegradable material, has good biocompatibility but poor physical properties to serve as a nerve conduit. In this study, polylactic acid (PLA) was added to chitosan to form a composite material with improved intensity and elasticity, to be used as nerve conduits. The chitosan-PLA nerve conduits were fabricated with a mold casting/infrared dehydration technique. The constituent ratio of PLA and chitosan of 1:5 (v:v) was chosen to give the composite material both good mechanical properties and good biocompatibility. An in vitro cytotoxicity test showed that the chitosan-PLA material was not cytotoxic. The conduits were proved biodegradable and had many micropores to allow permeability. We evaluated chitosan-PLA nerve conduits as a guidance channel to repair 10 mm gaps in rat sciatic nerves. Nerve autograft and silicon conduits were used as the control. After 12 weeks, the regenerating nerves in three groups succeeded in passing through the nerve gap and reinnervating the muscle. Assessments, including ECG, histomorphometric evaluation, and weighing of triceps calf muscle, showed that the functional recovery of sciatic nerve was better in chitosan-PLA conduit group than in the silicon conduit group (P < 0.05), but the differences between the chitosan-PLA conduit group and the nerve autograft group were not significant (P > 0.05). Therefore, the chitosan-PLA guide proved to be a promising nerve conduit.     

壳聚糖与Ⅰ型胶原复合制作新型人工神经支架材料及其性能研究

目的 探讨以壳聚糖复合Ⅰ型胶原蛋白结合冷冻干燥技术制备新型人工神经支架材料.方法 将壳聚糖与Ⅰ型胶原蛋白分别溶于0.05 mol/L的醋酸溶液中,利用冷冻干燥技术制备新型人工神经支架材料,并以紫外线照射的方法使其交联.经扫描电镜观察内部结构的排列规律及走行,比较其与周围神经的异同,测量其孔径大小、计算孔隙率等指标.观察在0.01 mol/L磷酸盐缓冲液中30 d体外降解率.并对材料进行力学及细胞毒性实验.结果 构建的材料为均匀圆柱状,内部为孔径均匀且平行排列的微观结构,其微孔直径为60-130μm,孔隙率为83.30%,体外降解率为16.95%,具有较好的力学性能与周阡司组织无毒性反应.结论 使用壳聚糖复合Ⅰ型胶原蛋白结合冷冻干燥技术,制备出具有良好三维空间构型的新型人工神经支架材料,其牛物相容性良好,具有应用潜力.     

Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve

The purpose of this study was to investigate the effects of combined application of human amniotic membrane wrapping and hyaluronic acid injection on peripheral nerve scarring in an adult rat model. After circumferential epineurectomies of right sciatic nerves were performed in each of 72 rats, nerves were randomly divided into three groups: (1) nothing was done additional to epineurectomy; (2) the epineurectomized segment was wrapped with human amniotic membrane; and (3) the epineurectomized segment was wrapped with human amniotic membrane and hyaluronic acid was injected inside. The extent of adhesions and perineurial scar thickness was macroscopically and histologically evaluated at 4 and 12 weeks. The least adhesion and perineurial scar tissue were observed in nerves treated with human amniotic membrane wrapping and hyaluronic acid injection. This combination appears to be safe and effective in preventing perineurial scar formation after peripheral nerve surgery in rats.     

神经生长因子促再生周围神经血管生成的实验研究

[目的]证实神经生长因子（NGF）具有促进大鼠再生坐骨神经中的血管生成作用：[方法]用单通道的砷胶管桥接75只Wistar大鼠1cm的坐骨神经缺损，在桥接管内给药，并将它们随机分为三组：生理盐水组、医用几丁糖组、NGF＋医用几丁糖组，每组25只，在5个不同时相点（0，2，7，14，30d），用免疫组化的方法检测大鼠再生坐骨神经新生血管内皮细胞中CD34的表达。[结果]随着时间的增加（30d以内），CD34的表达呈逐渐增长的趋势，其中在0.2d时，坐骨神经中没有CD34表达：7、14、30d时，NGF＋医用几丁糖组的CD34阳性染色显著多于医用几丁糖和生理盐水对照组（P〈0．01），医用几丁糖组和生理盐水组间并无显著差异（P〉0．05）：结论]NGF能促进再生周围神经的血管生成；NGF能促进毛细血管的生成。     

Sutureless nerve repair with laser-activated chitosan adhesive: a pilot in vivo study

OBJECTIVE: The anastomosis of peripheral nerves is a demanding procedure that has potential complications due to foreign body reactions elicited by sutures. In this study, the sutureless in vivo anastomosis of rat tibial nerves was successfully performed, using for the first time a chitosan-based laser-activated adhesive. The nerve thermal damage caused by the laser irradiation was quantitatively assessed. MATERIALS AND METHODS: A novel adhesive composed of chitosan, indocyanine green, acetic acid, and water, was fabricated in thin sheets. Its adhesive strength was tested in vitro by bonding strips (surface area approximately 20 mm2, thickness approximately 20 microm) onto rat sciatic nerves and sheep intestine by laser activation with low fluence ( approximately 50 J/cm2), using a fiber-coupled diode laser (n = 13). The tensile strength of the adhesive/tissue bonds was measured after tissue repair. The chitosan adhesive was then used to perform sutureless anastomosis of tibial nerves in vivo (n = 6). Adhesive strips were also bonded in vivo onto intact rat sciatic nerves (n = 6) in order to quantitatively assess, by counting myelinated axons, the thermal damage induced by the laser. RESULTS: The adhesive bonded well to tissue with a tensile strength of 12.5 +/- 2.6 KPa (mean +/- SD; n = 13). The in vivo anastomosed nerves were in continuity 3 d after surgery. Axon counting showed the number and morphology of myelinated axons were normal proximally ( approximately 96%) compared with intact nerves (100%). Axon demyelination was observed at the operation site ( approximately 49%) and distally ( approximately 27%), and was attributed to laser-induced thermal damage. CONCLUSIONS: Nerve anastomosis, performed by the laser-adhesive procedure, was successful 3 d postoperatively. Proximal myelinated axons were not significantly damaged by the low laser fluence.     

Repairing a 35-mm-long median nerve defect with a chitosan/PGA artificial nerve graft in the human: a case study

We have developed a chitosan/polyglycolic acid (PGA) artificial nerve graft which was previously used for bridge implantation of dog sciatic nerves across 30-mm long defects. Here we describe a clinical trial of this graft for repairing a 35-mm-long median nerve defect at elbow of a human patient. During the 3-year follow-up period, functional recovery of the injured median nerve was assessed by pinch gauge test, hydraulic hand dynamometry, static two-point discrimination and touch test with monofilaments, in couple with electrophysiological examinations. The motor and sensory function of the median nerve demonstrated an ongoing recovery postimplantation, reaching M4 and S3+ levels during the follow-up period. The results indicate that the chitosan/PGA artificial nerve graft could be used for surgical repair of larger defects in major peripheral nerves at a higher level in the human.     

Effects of hyaluronic acid on peripheral nerve scarring and regeneration in rats

The purpose of this experimental study was to investigate the effects of topical applications of hyaluronic acid on peripheral nerve scarring and regeneration in an adult rat model. After the right sciatic nerves of 48 rats were transected and immediately repaired, nerves were randomly divided into two groups. Nerves to which were applied hyaluronic acid comprised the experimental group, and nerves to which were applied saline comprised the control group. Perineural scarring was evaluated at 4 and 12 weeks macroscopically and histologically. Nerves treated with hyaluronic acid demonstrated significant reduction in perineural scar thickness (P < 0.05, Student's t-test). Histomorphologic nerve analysis, electrophysiologic studies, muscle mass evaluation, and serial functional walking-track analysis were performed for evaluation of peripheral nerve regeneration at 12 weeks. The results showed better conduction velocities, increased axon-fiber diameter, and faster functional recovery in hyaluronic acid-treated nerves (P < 0.05, Student's t-test). In conclusion, hyaluronic acid appears to be effective in preventing perineural scar formation, resulting in enhancement of peripheral nerve regeneration.