| Background and objectivesDural defect is a common problem in the clinics due to trauma, inflammation, tumorresection and congenital malformations. An inadequate closure of the dura after theneurosurgery may lead to complications including cerebrospinal fluid leakage, infections,meningitis and epilepsy. For these reasons, there is a need to develop novel clinical strategiesto accelerate dural closure. During the past years, various substitutes including autografts,allografts, xenografts as well as synthetic materials have been employed for dural repair.Among the autologous tissues that are commonly used for dural repair are temporalfascia, fascia lata femoris and periosteal flaps. Autologous tissues still represent the preferreddural substitutes due to their excellent biocompatibility, non-toxicity and non-immunogenicity.Furthermore, autologous tissues can be rapidly obtained and transplanted without sterilization.However, the availability of tissue with optimal size and shape for dural graft is one of themajor challenges. The use of allogous tissue such as lyophilized cadaveric dural graft hasbeen associated with Creutzfeldt-Jakob disease (CJD). Xenogenic materials such as porcineintestinal submucosa and bovine pericardium have also been used as dural substitutes.However, xenogenic materials are non-resorbable, and pose a risk of complications associatedwith infection and immunorejection.Other materials have been used as dural substitutesincluding synthetic polymers, such as silicone, ploy-lactic acid, poly-ε-caprolactone, ePTFE,polyurethane, Vicryl or nanofibrous and naturally occurring polymers, such as collagen,gelatin or fibrinogen. In recent years, hydrogels was demonstrated to be feasible to repairdural defects in animal models. Of these, alginate shows promise due to its rapid gelformation, biocompatibility and mild reactions. Synthetic materials can be degraded intonon-toxic small molecules in vivo and were expected to cause only a small risk of infection with some minor long-term adverse defects. Despite the theoretical advantages of thesynthetic materials, most of these have been rejected because of local tissue reactions,excessive scar formation, meningitic symptoms, or hemorrhage risk. Besides, collagen-baseddural substitutes have some shortcomes such as insufficient mechanical strength, limitedprotection to the central nerve system and easily cause cerebrospinal fluid leakage. Thus,engineered biocompatible materials need to be developed for dural replacement patches.The ideal material for dural repair should be safe, inexpensive and easy to handle andbeing able to enhance tissue regeneration without inducing immune response. To this end, wefabricated a multilayer biomimetic scaffold by combining the advantages of collagen anddegradable polymers. The scaffold contains three layers. The inner layer is composed of PLAto reduce tissue adhesion. The middle layer is composed of PCL and PLA, which providesmechanical strength and watertight seal. The outer layer contains collagen and PLA whichenhances tissue regeneration and provides mechanical strength, respectively. The objective ofthis study was to test this novel substitute for dural repair. We anticipate the scaffold mayprovide structural and functional properties similar to the natural dura to promote tissuerepair.Methods1. Electrostatic spinning production of different ratio of collagen, photo and PLAelectrospinning film materials, according to the mechanical properties, flexibility and stabilityof electrospinning to determine the layers of electrospinning film composition, optimizationof electrospinning parameters and produce complete stratified electrospinning membranerepair material.2. Scanning electron microscope observation of microstructure of stents, measurementbracket fiber diameter and the aperture. Measure the mechanical properties, water absorption,porosity, water proofing property, and in vitro degradation rate and compared with collagenmatrix.3. Culture HSF cells on the inside and outside layers respectively, observe theelectrospinning film on the surface of the stent cell growth situation, acridine orange staining cells determined by MTT test material compatibility and adhesion experiment andquantitative PCR and Western blot methods such as contrast, both inside and outside layergrowth of fibroblasts on main functions such as adhesion and extracellular matrix secretion,understand the inner and outer layer material impact on cell activity.4. SD rats subcutaneously implanted electrospinning film to verify the biocompatibility,HE staining and scanning electron microscopy (SEM) observation of electrospinningmembrane surface and interior of cell growth. Build New Zealand rabbit dural defect inanimal models, electrospinning film implanted as dura substitute material experiment, verifythe material of the animals into effect, HE staining to observe internal cell growth ofelectrospinning film, embedded parts of the brain HE dyeing observation data of actual effectand influence.Results1. Electrospinning parameters optimization and electrospinning film mechanicalproperties test to determine the composition of the complex film of electrospinning. PLA forlining materials; PLA/PCL (1/1) electrospinning filmin the experimental group has the largesttensile strength, and elongation at break and the softness is better, for middle materials;6%wtcollagen/PLA(4/1) electrospinning solution group in guarantee under the premise ofmaximizing the collagen content were able to get stable electrospinning fibers, used for outerlayer material. Through continuous stratified electrospinning technology making complexfilm of electrospinning, via electrospinning time to control the thickness of the material, theinner electrospinning time2h,4h,6h to the outer, middle electrostatic spinning aftercompletion of the electrospinning film thickness is about150to300microns.2. Electrospinning film and collagen matrix on the mechanical properties, porosity, waterabsorption, water proofing property comparison test, electrospinning film variousperformance has improved significantly.3. HSF cells can grow in the electrospinning film on the inner and outer layer materialand the normal value, acridine orange staining and determined by MTT detection showincremental growth of cells on the outer material is better than that of inner layer and culture plate control; Adhesive experiments show that the outer material is conducive to cell adhesion;Quantitative PCR and Western blot results show that in the outer layer material on the growthof cells on the secretion of extracellular matrix function is better than that of inner layer andthe culture plate.4. Rats subcutaneously implanted tissue cells can both inside and outside the materialafter electrospinning film layer with attach and grow, HE staining showed in the outer layermaterial the lining material has more compact internal fibroblast cells and epidermoid cells.Build New Zealand rabbit dural defect in animal models, implantation of complex film ofelectrospinning material replacement animals recovered well postoperatively, no incisioninfection and intracranial infection occurred signs. Electrospinning film material from thesurface and brain tissue has no obvious adhesion, electrospinning outer membrane materialwith the surrounding tissue adhesion, peel resistance, there is a certain graft site and thesurrounding brain tissue surface color is normal, no obvious injury, edema, inflammatoryreaction. Electrospinning film HE dyeing visible material outer lining more fiber andepidermoid cells.cause it; Embedded parts of the brain HE staining showed electrospinningfilm with their own fascia in the control group there were no significant differences, all didnot appear obvious signs of inflammation and hyperplasia of granulation tissue.ConclusionsThis topic using the electrostatic spinning technology made with good ultrastructure ofnew complex layer of dura substitute materials, its structure mimics the natural dura mater,ingredients for the inner PLA,50%PLA50%PCL middle,50%collagen20%PLA outer,three layer thickness ratio is about syntactic sugar for1:2:3, overall thickness is about150to300microns. Electrospinning membrane compared with clinical commonly used collagenmatrix material on the mechanical properties, water absorption, porosity, water proofingproperty is improved obviously. Electrospinning film was verified by means of cell culture invitro cell compatibility, confirmed the material of outer contain active ingredients tostrengthen adhesion of fibroblasts and collagen secretion, and other functions, through ratsubcutaneous implant and dura mater defect in New Zealand rabbits implanted experiment verify the histocompatibility of electrospinning film, confirmed the material adhesion did notproduce obvious lining and the surrounding tissue and collagen outer have promote cellgrowth. This new type of complex film of electrospinning as dura substitute material has theprospect of application. |
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