Modified Chitin/Bacterial Cellulose Bilayer Dura Substitute

The dura mater,a bilayer membrane covers the brain tissue and the spine,is the ultimate barrier to prevent cerebrospinal fluid leakage and keep intracranial pressure stable.Traumatic,neurosurgical and postoperative complications can cause dura tears or injuries.The aim of this study is to develop a novel bilayer dura substitute by simulating the human dura mater structure.It consists of a dense layer that prevents cerebrospinal fluid leakage and a porous layer that promotes tissue regeneration.The derivative of natural macromolecule chitin was used as the matrix material,bacterial cellulose（BC）was used as a reinforcing material,and glutaraldehyde（GA）and citric acid（CA）were used as porous surface cross-linking agents to prepare modified chitin/BC bilayer dura substitute.The main research contents and results were as follows:1. High molecular weight chitosan（CS）was prepared fromβ-chitin by concentrated alkali deacetylation and ultrasonic degradation,the obtained CS with deacetylation degree（DD）of85.95%±0.52%and viscosity average molecular weight（Mv）of 1.00×10⁶,0.94×10⁶,0.85×10⁶,0.80×10⁶ and 0.74×10⁶,respectively.O-carboxymethyl chitin（O-CMCH）was made fromα-chitin by alkalization-chitin chloroacetic acid substitution method,the obtained O-CMCH with DD of 41%±1%and substitution degree（DS）of 59%±2%.2. The dense layer was made of CS and BC mixed solution by facile solvent casting methods,the effects of BC content and Mv of CS on the properties of the CS/BC films were investigated.The results showed that CS and BC have good compatibility,the surfaces of CS/BC films were densely packed,all films were flexible,tough and suitable for surgical cutting.The introduction of BC overcomed the defect of pure CS film that was easy to bend in water and poor mechanical properties.CS/BC films can meet the mechanical performance requirements of tensile strength≥5 MPa and elongation at break≥10%,they had a certain ability to prevent cell adhesion and good thermal stability,suitable for high temperature humid heat sterilization,no leakage was observed under the hydrostatic pressure（approximately 6times the internal pressure in adults）for 30 minutes.The prepared CS/BC film,which using CS with Mv of about 0.85×10⁶ and adding 14 wt.%of BC,had best performance and was suitable as a dense layer of a bilayer dura substitute,the swelling ratio was 115%±2%,the dry tensile strength was 111±5 MPa,the dry elongation at break was 23%±1%,the wet tensile strength was 24±1 MPa,the wet elongation at break was 32%±1%,the breaking strength retention（BSR）was 72.7%±1.9%after 4 weeks of in vitro enzymolysis,and the mass residual rate was76.5%±1.5%after 8 weeks of in vitro enzymolysis.3. GA crosslinked O-CMCH porous membrane was prepared by freeze-drying method,the optimal condition of GA crosslinked O-CMCH was discussed.BC was introduced to construct O-CMCH/BC porous membrane,the effect of BC content on the morphology and properties of GA crosslinked O-CMCH/BC porous membrane were discussed.The results showed that the crosslinked O-CMCH porous membrane,which was prepared by 0.30 wt.%GA,had the best properties and the sol gel transition process was easy to control.After BC addition,the swelling rate of porous membrane increased slightly,and the mechanical properties were improved.The cross-linked O-CMCH/BC porous membrane with 0.11 wt.%BC had the best properties,which was suitable for providing support for cell adhesion,migration and growth,the average pore size was 127±51μm,the porosity was 97.5%.±0.4%,the swelling rate was 3,398%±83%,the degree of skeleton cross-linking was 96.87%±2.04%,the tensile strength was 1.42±0.04MPa,the elongation at break was 9.7%±0.5%,the mass residual rate was 46.8%±4.4%after5 weeks of in vitro enzymolysis,the enzymolysis performance in vitro meet the medical requirements.4. CA crosslinked O-CMCH porous membrane was prepared by freeze-drying method without using potentially toxic catalysts and dehydrating agents.The optimal conditions of CA crosslinking O-CMCH were discussed.The results showed that there was not only physical cross-linking between CA and O-CMCH,but also chemical cross-linked at high temperatures to form ester bonds.CA were added at m _O-CMCH:m _CA=5:2 and the crosslinking occured at145℃,the obtained O-CMCH porous membrane had the best performance.BC was added to construct O-CMCH/BC porous membrane,and the effect of BC content and crosslinking time on the morphology and properties of O-CMCH/BC membrane were discussed.The results showed that BC enhanced the porosity,swelling rate,the degree of skeleton cross-linking and the mechanical properties of the porous membrane.Prolonging the cross-linking time had no significant promotion on the degree of skeleton cross-linking.The O-CMCH/BC porous membrane containing 0.11%BC and cross-linking for 20 minutes had the best performance.It was suitable as a porous layer for a bilayer patch.Its pore size was 136±34μm,the porosity was 95.45%±0.76%,the swelling rate was 2,042%±62%,the degree of skeleton cross-linking was 87.7%±1.3%,the tensile strength was 0.50±0.05 MPa,the elongation at break was 7.9%±0.6%,and the mass residual rate was 5.4%±3.4%after 8 days of in vitro enzymolysis.5. According to the preparation conditions of the optimal dense surface and porous surface,GA and CA were used as the cross-linking agents of porous surface respectively.The porous surface sol was poured into the dense surface and then lyophilized.Nine kinds of double-layer dural patches with different thickness were prepared and their properties were tested.The results showed that the bilayer dura substitute had a flat and tidy surface,flexible and light-transmissive.The porous layer and the dense layer were tightly connecting with each other without excessively large cavities.The pore size of porous layer was 90～200μm.The porous distribution was uniform and the connectivity was high.The bilayer dura substitutes can be bent and cut at will,which was easy for surgical operation.The bilayer dura substitutes with dense surface thickness of≥0.04 mm and a total thickness of≤400μm had better performances.All GA crosslinked bilayer dura substitutes can meet the requirements of tensile strength≥5 MPa and elongation at break≥10%,was better than the CA crosslinked bilayer dura substitutes.6. The bilayer dura substitute preparation method used in this experiment was simple and effective.The prepared bilayer dura substitutes had good cell compatibility,no cytotoxicity,and meet the requirements of Class III medical devices in vitro cytotoxicity.The test group showed significant differences in cell proliferation compared with the control group,indicated that the bilayer dura substitute can not only provide support for cell proliferation and migration,but also promote cell proliferation and growth;In the mouse peritoneal environment,the bilayer dura substitutes can keep themself from deforming and shrinking,without causing tissue lesions,necrosis and chronic inflammation,the tissue inflammation was mild.The dense layer of bilayer dura substitute had certain anti-tissue adhesion properties.The porous layer of bilayer dura substitute can promote the growth and reproduction of autologous cells,moreover,it provided some tissue adhesion effect after water absorption and gelation,which can further improve the tightness of the seal.The bilayer dura substitutes had good histocompatibility.