【作者】 于凤宾；

【导师】 陈德玉；

【作者基本信息】 第二军医大学 ， 外科学， 2013， 博士

【摘要】 创伤性或医源性的硬脊膜缺损在脊柱及神经外科有一定的发生率，处理不当可引发持续性脑脊液漏、低颅内压综合征、急性气道阻塞、脑脊液囊肿、粘连性蛛网膜炎、椎管内感染、甚至危及生命的化脓性脑膜炎等一系列并发症。然而对于硬脊膜缺损的治疗，由于硬脊膜的不可再生性、现有的硬脊膜替代材料均无模仿天然硬脊膜空间结构的设计理念以及缺乏理想的修复方法，导致目前在临床上不同程度的存在治疗疗程长、痛苦大、组织愈合的形式以疤痕组织为主以及痊愈的标准也仅仅是脑脊液漏停止等诸多不足。硬脊膜缺损的―高质量‖（既能实现术后硬脊膜再生、预防硬膜外粘连及瘢痕，又能实现术中有效封堵）依然是临床亟待解决的难题。要想实现硬脊膜的再生，必须先明确硬脊膜的空间结构及表观形貌特征，并以此构建与之空间构象近似的替代材料；而要想实现术中有效封堵，必须研制出一种理想的修复方法；同时，要想预防硬膜外粘连及瘢痕形成，替代材料必须兼具此功能。由于硬脊膜主要由胶原基质和成纤维细胞组成，其胶原束具有特殊的取向性结构，因此本项目提出采用具有不同功能的双层复合PLGA纳米纤维膜片和组织工程技术来构建组织工程化人工硬脊膜，结合粘合力强且具有排水性能的贻贝粘蛋白生物胶，来实现硬脊膜再生及术中有效封堵。拟构建的硬脊膜替代材料设计为双层结构：内层（修复层）专司硬脊膜的再生，利于静电纺丝法制备与天然硬脊膜结构近似的有―取向性‖（有序）的聚乳酸/乙醇酸共聚物（PLGA）纳米纤维膜，作为组织工程支架，促使种子细胞（成纤维细胞）接种后所合成的胶原束也呈取向性分布，使新生组织接近天然硬脊膜；外层（加强层）应用无―取向性‖（无序）PLGA-壳聚糖纳米纤维膜片，专司增强内层力学性能、预防硬膜外粘连及瘢痕形成；内外层之间采用非缝合胶粘技术（贻贝粘蛋白），既可将外层与硬脊膜牢固粘合成一体，又能避免粘合胶与脑脊液直接接触以及人为封闭膜体风险，以期实现术中快速、确切的封闭硬脊膜囊。研究目的：构建与羊硬脊膜空间结构近似的复合组织工程化PLGA纳米纤维膜，评价体内回植修复羊硬脊膜缺损的效果。研究方法：①获取山羊硬脊膜的表观形貌及三维结构：成年山羊硬脊膜取材，对标本进行肉眼观察、组织学观察、扫描电镜以及透射电镜观察。②3种人工硬脊膜的制备及评价：利用静电纺丝法制备有序及无序PLGA纳米纤维膜；壳聚糖溶液单面涂层无序PLGA纳米纤维膜片，制备PLGA-壳聚糖膜片；手工撕成单丝的无纺PGA纤维，以同一方向均匀缠绕于特制U型钢丝，制备有序PGA纤维膜片。对有序及无序PLGA纳米纤维膜片进行物理性能及生物相容性检测。③3种组织工程化人工硬脊膜体外构建：将体外分离、扩增的山羊皮肤成纤维细胞，作为种子细胞，分别接种于有序PLGA、无序PLGA纳米纤维膜以及有序PGA纤维膜上，观察种子细胞在支架材料上的黏附、生长及基质分泌等情况。④组织工程化人工硬脊膜修复羊硬脊膜缺损的系列实验:1）将9只成年山羊随机分为3组：有序PLGA组、无序PLGA组及有序PGA组，分别采用上述3种膜片作为内层覆盖山羊腰段硬脊膜缺损（0.6cm×0.5cm），然后以无序PLGA-壳取糖膜片作为外层，涂抹贻贝粘蛋白粘贴于硬脊膜缺损周缘；术后1月取材，对术后并发症、封堵效果、与周围组织粘连、炎性反应、材料降解以及硬脊膜的再生质量等进行评价。2）对3种膜片中修复效果最佳者进行再次分组：组织工程化材料组、单纯材料组，体内回植于6只山羊，术后3月取材，按前述方法对其进行评价。3）将实验二中效果最佳膜片再次进行山羊体内回植，评价其远期疗效（6个月）。结果：①山羊硬脊膜主要由胶原纤维及散在分布的成纤维细胞组成的半透明薄膜，胶原纤维直径介于400nm～1000nm之间，呈―取向性‖排列，微观下可见硬脊膜内外表面呈连綿不断的山峰。②采用静电纺丝法成功制备出与天然硬脊膜三维空间结构（胶原直径及排列）近似的有序的PLGA纳米纤维膜片以及无序PLGA纳米纤维膜片，二种膜片均具有良好的生物相容性及力学性能；成功制备出PLGA-壳聚糖膜片。③成功构建组织工程化有序PLGA、无序PLGA及有序PGA人工硬脊膜膜片。④术后1月取材，证实组织工程化有序PLGA膜优于组织工程化无序PLGA膜，组织工程化无序PLGA膜优于组织工程化PGA膜。⑤术后3月取材，证实组织工程化有序PLGA膜优于单纯有序PLGA膜。⑥术后6月取材，证实组织工程化有序PLGA膜体内回植再生膜的表观形貌及三维结构与天然硬脊膜高度相似。结论：复合组织工程化PLGA纳米纤维膜实现了真正意义的硬脊膜再生以及达到预防硬膜外瘢痕及粘连的目的，非缝合技术应用（贻贝粘蛋白）可显著减少术后脑脊液漏发生率。更多还原

【Abstract】 In neuro-and spinal surgey, iatrogenic or traumatic dural defects will all causeleakage of cerebrospinal fluid（CSF）and further result in serious complications. Thesecomplications include cutaneous CSF fistula, intracranial hypotension syndrome,airway obstruction, delayed wound healing, cerebrospinal fluid cyst, adhesivearachnoiditis, spinal infection and even life-threatening purulent meningitis. However,due to the non-renewablity of dura, the lack of dural substitutions that mimic naturaldural microstructure and an ideal repair method, the current treatments of duraldefects all have some shortcomings more or less, including the long course oftreatment, scar tissue formation in the defect region and CFS leakage stopping as thestandard of healing. Therefore, we propose to apply a novel two-lay compositenanofibrous membrane and fibroblasts as seed cells to reconstitute dura tissue viatissue engineering technique. The reconstructed tissue will be used to repair pre-madedura defects in sheep with the aid of mussel musin bioglue during the surgery. Thedesign of current study was based on the principle of―practical, clinical, andappliable‖. Due to the fact that natrual dura was composed of highly oriented collagenfibers as matrix lined with fibroblasts, the artificial membrane was fabricated with twolayers: the inner layer is oriented PLGA nanofiberous membrane of prepared byelectrospinning. It is used as scaffolds for the growth of fibroblasts in order toregenerate neo-dura tissue. With its similar microstructure to that of normal dura,neo-collagen fibers secreted by the seeded fibroblasts are expected to be arrangedaccordingly. The outer layer is electrospinned PLGA nanofibrous membrane coatedwith chitosan. Its main function is to provide mechanical strength of the whole designand further prevent adhesion of the neo-dura to adjacent tissue. The seamlessimmobilization of the outer layer in the defect area to the surrounding normal dura isrealized by mussel musin bioglue in the marginal strip. The two layers are alsostabilized with the same seamless technique. Hence, the risk of cerebrospinal fluidleakage can be minimized while the direct contact of cerebrospinal fluid with thebioglue is avoided. Based on the above design, efficient and quick sealing of duradefect during the operation could be realized while biological dura tissue renegeration could be achieved afterwards.Objective：To invertigate the effects of reconstituting tissue enginered artificialdura using composite PLGA nanofiber mamborane to repair dural defects in goats.Methods:①Light microscopy, scanning electron microscopy and transmissionelectron microscopy were used to gain the dural microstructure of goat.②Theoriented PLGA nanofibrous membrane and no-oriented PLGA nanofibrous membranewere prepared by electrospinning through adjusting the electrospinning parameters.And the oriented PGA fiberous membrane was also reconstituted by twinningnonwoven PGA fibers evenly in single direction at a tailor-made U-shaped wire. ThePLGA-chitosan nanofibrous membrane was prepared by the chitosan solution singlesided coating. The physical performances and biocompatibility of them were thendetected.③The fibroblasts of goats which were separated and amplificated in vitro,as seed cells, were planted in the oriented PLGA nanofibrous membrane, no-orientedPLGA nanofibrous membrane and the oriented PGA fiberous membrane respectively.The adhesion, growth and matrix secretion of fibroblasts in scaffolds were observed.④Aseries of experiments that tissue engineered artificial dural membranes repaireddural defects in goats:1) Nine adult goats were divided into3groups randomly：oriented PLGA group，non-oriented PLGA group and oriented PGA group. Each typeof the membranes mentioned above was attached to the dural defects of lumbar(0.6cm×0.5cm）in goats respectively. And then the seamless immobilization of theouter layer (PLGA-chitosan nanofibrous membrane) in the defect area to thesurrounding normal dura was realized by mussel musin bioglue in the marginal strip.All the animals were killed in1month after operation. Perioperative complications,incidence of CSF leaks, adherences to the surrounding tissue, inflammatory reactionand dural regeneration were evaluated to choose the best one.2) After selecting thebest membrane, the dural defects were repaired by adopting the tissue engineeredmembrane or single material membrane (no seed cells) in6adult goats randomly.They were evaluated after3months of surgery according to the above mentionedmethods to determine which one was better.3) The best membrane selected by thesecond experiment was given a six month observation in vivo to evaluate itslong-term effects.Results:①The dura of goat shows a translucent film which is mainly composedof highly oriented collagen fibers as matrix lined with fibroblasts. The diameter ofcollagen fibers ranges from400nm to1000nm. The microscopic surface of dura looks like the endless peaks.②The oriented PLGA membrane which has the similarmicrostructure to that of natrual dura of goat and the non-oriented PLGA membranewere successfully prepared by electrospinning. Both membranes showed goodbiocompatibility and mechanical properties.③The oriented PLGA membrane,non-oriented PLGA membrane and oriented PGA membrane were seeded with thefibroblasts of goats successfully.④The results of1month after operation confirmedthat the tissue engineered oriented PLGA membrane showed the best performanceamong the3type of membranes. And the performance of the tissue engineerednon-oriented PLGA membrane was better than that of the tissue engineered orientedPGA membrane.⑤The results of3month after operation indicated that theperformance of the tissue engineered oriented PLGA membrane was better than thatof the single oriented PLGA membrane(no seed cells).⑥A six month observation invivo showed the morphology and microtructure of regenerated membrane were highlysimilar to that of the natural dura of goat by adopting the tissue engineered orientedPLGA membrane.Conclusion: The composite tissue engineered PLGA nanofibrous membraneachieves biological repair of dural defect and realizes the purpose of preventiingepidural scar and adhesions. Meanwhile, non-suture technology application (musseladhesive protein) can reduce the incidence of CSF leakage effectively.更多还原