| Dura mater is a kind of thick, tough bilayer membrane tissue which situates between the brain surface and the inner side of the skull. Its main function is to protect and support the brain. Under the injury factors from inside and outside of the body, dural defect usually exists. In order to prevent the adhesion between brain and surrounding tissues, treat the leakage of cerebrospinal and intracranial infection,provide accommodation and protection to nerve tissue, dura mater should be patched up as soon as possible.Bacterial cellulose( BC) which incubates and ferments from Acetobacter belongs to the nano-scale β-D- glucose. Due to many excellent physical and chemical properties,it has been proved to be the good dural substitute materials in rabbit experiments of dural defect at the perspective of pathology and molecular biology.The purpose of the fist part of this study is to use BC to patch up dural defect in rabbit.Then,comprehensively evaluate the function and effect of viable transplantation by osology. Explore the feasibility of BC being used as artificial dura mater.Optimization of bacterial cellulose(OBC) is the optimization products by electrospinning and oxidation technique in order to make up the defect of nano-porosity, heterogeneity distribution and biodegradability, including electrospinning bacterial cellulose(EBC) and oxidized bacterial cellulose(OXBC).The purpose of the second part of this study is to introduce the method of preparation of OBC and use it to patch up dural defect in rabbit. Then,comprehensively evaluate the safety and biocompatibility of viable transplantation by macroscopic observation and histopathology to compare to BC.The purpose of the third part of this study is to use OBC to patch up dural defectin rabbit.Then,comprehensively evaluate the function and effect of viable transplantation molecular biology to compare to BC. Explore the performance of OBC being used as artificial dura mater.SectionⅠ Patch Dural Defect in Rabbit to Explore the Influence of the Inflammation-related Indicators in Inflammation-related Indicators OBJECTIVE : In order to detect the changes of cerebrospinal fluid related indicators and explore the influence of local inflammatory reaction after transplantation, we create dural defect model in rabbit and apply different materials to patch up.METHODS : 20 male rabbits were randomly divided into four groups, which are A,B, C, D.( 5 in each group) Bilateral skull and dura(A,B,C) of rabbits were removed by surgery. Group A are not repaired. Group B are repaired by commercially available artificial dura. Group C are repaired by BC. Group D are control group which retain their dura. The puncture through the cisterna magna is executed at 3,7,14,30,90 days after the operation for all groups to extract the cerebrospinal fluid in order to detect inflammation-related indicators: Bacterial smear, WBC, Glucose and Protein.RESULTS : All animals are successfully extracted the cerebrospinal fluid.inflammation-related indicators: Group A occur the positive results of bacterial smear in each one rabbit(day 7/14), and others reveal negative results. Compare with group D. Group A: WBC mildly increased(day 3/90) and moderately elevated(day 7/14/90).Glucose mildly decreased(day 3/30/90) and moderately decreased(day 7/14). Protein mildly increased(day 90) and and moderately elevated(day 3/7/14/30)(P ≤ 0.05).Group B: WBC and protein mildly increased(day3/30)(P ≤0.05). Glucose mildly decreased(day 3/30)(P≤0.05). Group C: WBC and protein mildly increased(day 3)(P≤0.05). Glucose mildly decreased(day 3)(P≤0.05). The rise degree of WBC and Protein in group B are greater than group C(day 30)(P≤0.05). The reduction degree of Glucose in group B are greater than group C(day 3)(P≤0.05).CONCLUSIONS : BC repair rabbit dural defect, which can effectively prevent cerebrospinal fluid leakage and reduce the incidence of central nervous system infection. It has good biocompatibility which is an ideal biological dura substitute materials.Section Ⅱ Preparation of Optimized Bacterial Cellulose and Observation of the Histological Feature after Repairing Dural Defect with Different BC OBJECTIVE :Finishing the preparation of OBC,then use them to repair the dural defect in rabbit in order to observe general and pathological changes after transplantation.METHODS : Prepare the EBC and OXBC by electrospinning and oxidation technique. 60 rabbits were randomly divided into three groups(A,B,C)( 20 in each group). Bilateral skull and dura of rabbits were removed by surgery. Group A is repaired by EBC. Group B is repaired by OXBC. Group C is repaired by BC. All animals in each group(elected 5 randomly) were executed at 3,7,14,21 days after the operation. General and histological observation were performed.RESULTS : All animals were alive normally for a long time without the presence of rejection and toxic reaction. Different BCs can evenly cover on the surface of brain without adhesion. Microscopically, fibrous connective tissue proliferated on outer side of BC. New blood vessels appeared.CONCLUSIONS : OBC belong to non toxic biological materials. They can repair dural defect in rabbit, which also may avoid the adhesion to the brain tissue and effectively prevent the occurrence of cerebrospinal fluid leakage and epilepsy.Section Ⅲ Detect Inflammatory Cytokine in Tissue after Repairing the Dural Defect with Different BC OBJECTIVE : We apply different BC to patch up the dural defect in rabbit. Then weobserve the changes of histopathology and detect early inflammatory cytokine in tissues after implantation.METHODS : 40 male rabbits are randomly divided into 2 groups(A and B)( 20 in each group). Bilateral skull and dura of rabbits were removed by surgery. A is replaced by EBC and BC on each side. B is replaced by OXBC and BC on each side.The samples were collected at 3, 7, 14, 21 days after the operation. In order to compare the degree of inflammation in these two materials after transplantation, we apply realtime fluorescence quantitative polymerase chain reaction( RT- PCR) to detect the content of inflammatory cytokines such as Tumor Necrosis Factor- α( TNF- α), Interleukin- 6( IL- 6) and Interleukin- 1β( IL- 1β).We also apply Western Blot( WB) to detect the content of inducible nitric oxide synthase( i NOS)and cyclo- oxygenase 2( COX- 2) in patching regions.RESULTS : RT- PCR of Group A : The expression level of IL- 6( day 3), IL-1β( day 7), TNF- α( day 14) and IL- 6 and IL- 1β( day 21) in EBC are lower than BC( P ≤ 0.05). WB : The expression level of i NOS(day3/14/21) in EBC is lower than BC( P ≤ 0.05), especially on day 21(P < 0.01). The expression level of COX- 2 in EBC is lower than BC on day 7 and day 21( P < 0.05),especially on day21(P < 0.01).RT- PCR of Group B: The expression level of IL-1β( day 3), IL-6( day 7), IL-1β( day 14) and TNF- α, IL- 6 and IL- 1β( day 21) in OXBC are lower than BC( P ≤ 0.05), especially TNF- α on day 21(P < 0.01). WB : The expression level of i NOS(day7/14/21) in OXBC is lower than BC( P ≤ 0.05), especially on day 21(P < 0.01). The expression level of COX- 2 in OXBC is lower than BC on day3, day 14 and day 21( P < 0.05), especially on day 21(P < 0.01).CONCLUSIONS : The early inflammatory response of OBC are slight than normal BC and they may become more ideal dural substitute materials for curing dural defect. |
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