玉米蛋白基硬膜封合胶在硬膜缺损修复中应用的实验研究

Application of zein-based dural sealant in the repair of dural defects

中文关键词:  硬膜缺损  脑脊液漏  玉米蛋白  硬膜封合胶

英文关键词:Dural defect  Cerebrospinal fluid leakage  Zein  Dural sealant

中文摘要:

  【摘要】 目的：观察新型的玉米蛋白基硬膜封合胶生物相容性并验证其相关生物安全性及水环境下的黏附效果，同时观察其在兔硬脑膜及大鼠硬脊膜缺损模型中的修复效果。方法：制备出一种新型玉米蛋白基硬膜封合胶并冻干成补片状及部分研磨后使用。利用PC12细胞的细胞计数试剂盒-8（cell counting Kit-8，CCK-8）试验、活死染色试验、兔红细胞溶血试验验证玉米蛋白基硬膜封合胶在细胞水平的生物安全性，通过大鼠皮下降解试验探究其可吸收性能。通过改良的美国材料与试验协会（American Society for Testing and Materials，ASTM）F2392猪硬脊膜体外模型和兔硬脑膜体内模型验证玉米蛋白基硬膜封合胶的爆破压力并与纤维蛋白胶进行比较。在兔硬脑膜缺损修复模型中，依据处理方式将造模成功的日本大耳白兔随机分为空白组、纤维蛋白胶组、玉米蛋白基硬膜封合胶组。术后HE染色观察硬膜缺损处新生纤维的生长及修复情况；MRI观察脑脊液漏的密封情况；酶联免疫吸附测定法（enzyme-linked immunosorbent assay，ELISA）评估硬膜封合效果对脑脊液中环氧合酶-2（cyclooxygenase-2，COX-2）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）等炎症因子表达情况的影响。在大鼠硬脊膜缺损模型中，采用同样的分组模式，并观察不同组对脑脊液漏的密封效果。结果：玉米蛋白基硬膜封合胶制备成功，且其CCK-8实验结果证明其不同浓度、不同时间点的细胞活力均在80%以上，溶血率均低于5%，活死染色可见活细胞率高于90%。体内降解试验可见玉米蛋白基硬膜封合胶在7～10d内完全降解且HE染色提示无明显炎症反应，其爆破压约为92.2±12.3cmH2O，大于在相同条件下纤维蛋白胶的爆破压36.0±3.2cmH2O。在兔硬脑膜缺损模型中，玉米蛋白基硬膜封合胶HE染色可见较空白组及纤维蛋白胶组更快及更致密的新生纤维组织长入，MRI提示玉米蛋白基硬膜封合胶较空白组及纤维蛋白胶组有着更可靠的脑脊液漏密封效果，且其脑脊液中COX-2、TNF-α等炎症因子表达水平均更低。在大鼠硬脊膜缺损模型中，大体观察可见玉米蛋白基硬膜封合胶较空白组及纤维蛋白胶组有着更可靠脑脊液漏密封效果。结论：玉米蛋白基硬膜封合胶生物相容性良好且可以利用其出色的水环境黏附性能更好地密封脑脊液漏，并为成纤维细胞提供更加良好的生理环境，从而更好地完成硬膜缺损封合。

英文摘要:

  【Abstract】 Objectives: To observe the biocompatibility of a novel zein-based dural sealant and verify its related biosafety and adhesive effect in aqueous environments, and observe its repair efficacy in rabbit dura mater and rat spinal dura mater defects. Methods: A novel zein-based dural sealant was fabricated, then lyophilized into patch form or partially ground for application. The biocompatibility of zein-based dural sealant was verified at the cellular level via cell counting Kit-8(CCK-8) assay, live/dead staining assay on PC12 cells, and hemolysis assay on rabbit red blood cells. Its absorbable properties were investigated through subcutaneous degradation experiments. The burst pressure of the zein-based dural sealant was validated using a modified porcine spinal dura mater in vitro model and a rabbit cranial dura mater in vivo model following ASTM F2392 standard, with a parallel comparison to fibrin glue. For the rabbit dural defect repair model, successfully modeled Japanese white rabbits were randomly divided into three groups based on treatment: blank group, fibrin glue group, and zein-based sealant group. Postoperatively, hematoxylin-eosin(HE) staining was used to observe the growth and repair of new fibers at the dural defect site; MRI examination was performed to assess CSF leakage sealing; And enzyme-linked immunosorbent assay(ELISA) was employed to evaluate the impact of sealing efficacy on the expression of inflammatory factors(including COX-2 and TNF-α) in CSF. The same grouping strategy was adopted in the rat spinal dura mater defect model to observe CSF leakage sealing effects across groups. Results: The zein-based dural sealant was successfully prepared. CCK-8 assay results showed that cell viability remained above 80% across all concentrations and time points; Hemolysis rate was below 5%; And Calcein/PI staining indicated a viable cell rate exceeding 90%. In vivo degradation experiments demonstrated complete resorption of the sealant within 7-10 days, with no significant inflammatory response observed via HE staining. The burst pressure of the zein-based sealant was approximately 92.2±12.3cmH2O, which was significantly higher than that of fibrin glue(36.0±3.2cmH2O) under the same conditions. In the rabbit dural defect model, HE staining revealed faster and denser ingrowth of new fibrous tissue in the zein-based sealant group compared with the blank and fibrin glue groups. MRI results confirmed more reliable CSF leakage sealing in the zein-based sealant group, accompanied by lower expression levels of inflammatory factors(COX-2 and TNF-α) in CSF. In the rat spinal dura mater defect model, gross observation also verified superior CSF leakage sealing performance of the zein-based sealant relative to the other two groups. Conclusions: The zein-based dural sealant exhibits excellent biocompatibility. Its outstanding adhesive performance in aqueous environments enables more effective sealing of CSF leaks, and it provides a favorable physiological microenvironment for fibroblasts, thereby achieving superior repair of dural defects.