| Objective:Dural defects caused by intracranial tumor erosion,craniocerebral surgery and severe trauma are common in clinics.The dural repair is often needed to prevent cerebrospinal fluid leakage,intracranial infection and tissue adhesion to protect the brain.Over the past 100 years,scholars have been finding dural repair methods and exploring materials for dural substitutes.Autologous,allogeneic,xenograft and synthetic materials are largely applied for dural repairs and have their benefits and drawbacks.For example,autologous tissue has the advantages of no immune rejection,avoiding the possibility of transplantation infection-related pathogens,and less economic burden,but the choices related to the size and shape of materials are limited.Additional surgery is required that increase the trauma and pain of patients.Allogeneic materials are not widely used because of their limited sources in clinics.Xenobiotic materials are widely used in clinics,but their biocompatibility is needed to be improved.Recently,synthetic polymer materials have emerged as cheaper with no potential risk of pathogen infections,but there are still deficiencies in biocompatibility.Extracellular matrix materials have become a research hotspot in tissue engineering as they are used more often in clinics because of their good biocompatibility and retaining the whole three-dimensional structure.In this study,the porcine dura mater was used as the original material to prepare different configurations of decellularized extracellular matrix by combining a variety of acellular methods and freeze-drying processes,so as to explore new dural substitutes.1.Discovering an acellular scheme suitable for porcine dura mater and evaluating the acellular effect,histological morphology,content of main components,and mechanical properties;2.Evaluate the biocompatibility of two new materials with different configurations;3.Evaluate the repair effect and safety of the new materials by repairing the dural defect in rabbits.Methods: 1.Explore the combined application of the physical method(hypotonic fluid swelling cell membrane),chemical methods(Triton X-100,SDS),biological methods(DNase and RNase)and other decellularization methods to obtain the proper scheme for porcine dura mater.Porcine dural extracellular matrix was dried with or without lyophilized to prepare new decellularized dura mater(DM)and lyophilized-decellularized dura mater(L-DM).The residual contents of DNA,α-gal antigen epitope,collagen,glycosaminoglycan and residual situation of cells,their basic tissue structure was observed through histology.The scanning electron microscope and atomic force microscope were used to evaluate the mechanical properties and surface ultrastructural properties of the two new dural substitutes.2.The biocompatibility of the two kinds of dural substitutes was analyzed both in vivo and in vitro.SDS residue was detected,and the extract of DM and L-DM were prepared for cytotoxicity test.L929 cells were co-cultured with the two kinds of new dural substitutes to evaluate the colonization and migration of cells on the surface of DM and L-DM.The sensitization and rejection of the new dural substitutes were evaluated by a subcutaneous sensitization test in rabbits and a subcutaneous implantation test in Sprague Dawley rats.3.The rabbit models of dural defect were prepared,and the dural defect was repaired with autologous skull periosteum(PM),DM and L-DM.Rabbits of each group were sacrificed by deeply anaesthetizing at 2 and 12 months after the operation.The recovery of the dura mater and the complications were evaluated by head magnetic resonance examination in vivo.Blood samples were taken for the hematological tests to evaluate blood components and liver and kidney function changes.A histological examination was performed to evaluate the effect of dural substitutes on dural repair and vital organs(such as liver,kidney,and spleen).Results: 1.No visible nuclear components were found in the two groups of DM and L-DM by HE staining and DAPI staining.After decellularized treatment,d ECM retained the basic collagen fiber structure which was identified by Masson staining.2.The DNA content of fresh porcine dura was 240.61 ± 19.04ng/mg.The content of DNA was 29.88± 3.27ng/mg in DM group and 30.11 ± 5.73ng/mg in L-DM group.DNA agarose gel electrophoresis showed no obvious DNA bands in DM or L-DM.The content of α-gal epitope siginificantly reduced from 13.363 ± 1.697 ng/mg in fresh porcine dura mater to3.388 ± 0.421 ng/mg after decellularization.The elimination rate of α-gal was 74.60%.The content of collagen in porcine dura before decellularization was 571.94 ± 92.11 μg /mg,and it increased upto 818.04 ± 153.16 μg / mg after decellularization.The content of glycosaminoglycan in porcine dura was 3.108 ± 0.104 μg/ mg and was 2.399 ± 0.296 μg /mg after decellularization.There was a significant difference between them(P < 0.05),but most glycosaminoglycans were still retained.In the mechanical experiment,the fracture stress of DM group and L-DM group were 3.368 ± 0.413 MPa and 1.307 ± 0.247 MPa,and the fracture strain were 18.24 ± 3.82% and 12.24 ± 3.59% respectively(P <0.05).Scanning electron microscopy(SEM)showed that the three-dimensional structures of DM and L-DM were highly similar to that of human dura mater.The surface roughness Sq of DM and L-DM were 42.50 ± 11.55 nm and 57.80 ± 17.86 nm respectively.2.The residual SDS of the DM and L-DM were 0.00053 ± 0.00029 and0.00048 ± 0.00007%,which were lower than the standard of concentration safety dose of50 mg / L(concentration of 0.005%).CCK-8 cytotoxicity test verified that DM and L-DM had no obvious cytotoxicity and slightly promoted cell proliferation.SEM showed that fibroblasts colonized and migrated well on the surfaces of the two kinds of new dura substitutes.In the rat model implanted subcutaneously in the back,DM and L-DM did not cause strong immune rejection.It was observed that the L-DM group began ECM reconstruction after 14 days of operation,which was earlier than the DM group,and the degradation rates of DM and L-DM were matched with the reconstruction rates of ECM.3.The rabbit dural defect model compared the repair effects of autologous skull periosteum(PM),DM and L-DM.According to the head MRI,there were no abscess,hydrocephalus,and liquefaction complications after the dural repair operation,confirming DM and L-DM’s good effect.No inflammation,subcutaneous abscess formation,cerebrospinal fluid leakage and other abnormal manifestations were found in the incision of the three groups,and there were no behavioral changes such as convulsion,limb paralysis and abnormal gait in rabbits.Histological examination showed that DM and L-DM were not completely degraded at two months after the operation,and the reconstruction of new ECM tissue along the fiber bundle of DM and L-DM was directional;at 12 months after the operation,the reconstruction of dural ECM was nearly completed,but a small amount of residues were still seen in DM and L-DM to maintain the stability of the repaired area.DM and L-DM had no significant effect on liver,kidney,spleen and blood system.Conclusion: 1.In this study,the two kinds of new dural substitutes with different configurations prepared by the decellularization scheme were developed to meet the decellularization standard of biomaterials.The three-dimensional structures of DM and L-DM were similar to that of the human dura mater.2.The new dural substitutes had good biocompatibility.The repair of a dural defect in rabbits obtained conclusive effect,and the long-term implantation was safe and reliable,with no significant impact on important organs and blood systems. |
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