| 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. |
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