去细胞同种异体移植面神经材料的制备

Scaffold of acellular tissue engineered artificial facial nerve allograft

目的构建异体组织工程化面神经,提供具有仿生结构的支架。方法取8段长5 cm、直径2 mm兔的面神经,其中2段不行处理作为正常对照,其余6段分成3组,用3%三硝基甲苯和4%脱氧胆酸钠分别萃取1、2和3次,在萃取神经和未萃取神经的中段取材,行苏木精-伊红染色、Masson染色和S-100免疫组化染色,在光镜和电镜下观察神经萃取前后形态学的变化。结果用三硝基甲苯和脱氧胆酸钠萃取后,面神经内的细胞消失,而纤维性支架结构与未经萃取的神经相仿,电镜下可见萃取后的神经由空的神经基膜管和管之间的胶原纤维构成。随着萃取次数的增加,神经内残留的S-100蛋白减少,但反复萃取后神经的支架结构受破坏。结论用三硝基甲苯和脱氧胆酸钠萃取2次可去除兔面神经的细胞而保留完整的神经基膜管和纤维支架结构,是制备具有仿生结构的组织工程化面神经支架较为理想的方法。

Objective To develop a procedure by which Schwann cells and myelin in the facial nerve could be removed while the basal lamina tubes remained intact, and to obtain an acellular scaffold for fabricating tissue-engineered facial nerve allograft in rabbits. Methods 8 facial nerves 5.0 cm long and 2.0 mm in diameter were excised from rabbits and cleaned from external debris. The nerves were treated with a solution of Triton X-100 and a solution of sodium deoxycholate at room temperature. After a final wash in water, the nerves were stored in phosphate-buffered saline （PBS, pH 7.2） at 4 degrees C. HE. luxol fast blue and fibrin staining were performed to visualize cells, myelin and basal membranes respectively and immunohistochemical staining was performed to visualize the presence of laminin, a Schwann cell lamina component, both in fresh and acellular nerve segments. To reveal overall structure better, methylene blue-fuchsin staining was performed in semithin section. The .ultrastructure of acellular and fresh nerves were observed and photographed in a transmission electron microscope. Resuits The acellular facial nerve was white long cylinder with good elasticity and ductility. HE, myelin and fibrin staining revealed that cells, axons and myelin sheath were removed and basal membrane was preserved after extraction procedure. Staining for the presence of laminin showed that the Schwann cell basal lamina component was present in the nerves after chemical treatment. Methylene blue-fuchsin staining and transmission electron microscopy showed that the myelin sheaths were absent from the extracted nerve segments and empty basal lamina tubes remained in the endoneurium. Conclusion We developed an extracted procedure with the detergents of Triton X-100 and deoxycholate, by which, axons and myelin sheaths could be removed from a rabbit facial nerve while the basal lamina tubes remained intact and an acellular nerve allograft was obtained. The lanfinin, a Schwann cell basal lamina component, can be preserved in the acellular nerve. Chemical extraction is an ideal method to prepare nerve scaffold for fabricating tissue-engineered nerve in humans.