【作者】 孙安科；

【导师】 陈文弦；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2001， 博士

【摘要】 喉、气管病损所致的软骨缺损的修复目前仍无理想的方法。常规的治疗措施主要靠自体、异体软骨及人工替代品来实现软骨支架的修复与重建，虽然有一定的治疗效果，但存在供体再损伤，材料来源有限，与受体形状不相配，免疫排斥及异物反应和难以与机体融合等问题。因此，探索一种新的修复重建方法仍是耳鼻咽喉-头颈外科医师面临的重要任务之一。近年来兴起的组织工程技术为解决这些问题提供了新途径。组织工程是融合生命科学、材料学、工程学和化学的一门边缘学科。其基本思想是用所需组织或器官的细胞作种子细胞，经体外培养扩增后将高浓度有活力的细胞接种到一种生物相容性好、可生物降解、具有预制形态和空间结构的合成聚合物或天然基质材料中。再将此细胞-材料复合物植入体内缺损部位，在生物支架材料逐步降解吸收过程中，种植的细胞增生繁殖、不断分化，形成新的具有特殊功能和形态的相应组织和器官，达到修复缺损和重建功能的目的。 组织工程化软骨形成的重要条件之一是必须获得大量的软骨种子细胞。自体软骨取材量小不足以产生足够的细胞，体外培养扩增，反复传代易致细胞“去分化”（dedifferentiation）而失去软骨细胞性质。取材量大不仅造成供区严重损伤，而且有时是不可能的。同种异体软骨来源广，易获取，细胞经过消化分离及体外培养有可能消弱其免疫原性。因此，有望成为构建组织工程化软骨的充裕细胞来源。本研究以同种异体软骨细胞作为种子细胞，探讨在有免疫力的动物体内构建预定形态组织工程化软骨的可能性，研究同种异体软骨细胞一支架材料复合物及新形成的同种异体工程化软骨I 第四军医大学98级博士论文 一 在喉软骨缺损修复和功能重建中应用的可行性及其效果。为软骨组织工程 技术在耳鼻咽喉科的可能应用提供资料，奠定基础。 材料与 方法 一、同种异体预定形态组织工程化软骨的构建 取1周龄乳兔肋软骨，剪切后用＊型胶原酶消化分离获得软骨细胞， 置含200胎牛血清的HaM－12培养基在37℃、50COZ和饱和湿度条件下 培养，收集传第 2I代的软骨细胞，调整浓度达 SX 10＼ml，接种子塑为方 形片状和“C”形管状经多聚赖氨酸处理的 PGA三维支架材料上，将软骨 细胞IGA复合物体外培养 7q 0天，然后种植于成年兔皮下。分别于 4周、 8周和 12周取材，行大体和组织学观察（HE染色、Masson三色染色和阿 丽辛蓝过碘酸雪夫（AB／PAS）染色），对工程化预定形态软骨的构建情况进 行评价。在了解组织工程化软骨形成的同时，观察PGA的降解情况。 二、同种异体组织工程化软骨修复喉软骨缺损 将种植于成年兔皮下的方型片状同种异体软骨细胞PGA支架材料复 合物，4周取材用于修复实验＊ 只雄性新西兰大白兔O七月龄，体重2．5咤 士0．skg卜随机分为实验组O 只）和对照组O只X3％戊巴比妥按30m叭m 静脉麻醉。无茵条件下暴露甲状软骨，在一侧甲状软骨板上造成 0石cm X 0．scm的缺损区，内外软骨膜一并去除，保留喉内粘膜。然后植入同等大 下 小组织工程化软骨（实验组和单纯PGA材料（对照组卜分别于术后4周、8 周和12周取材，观察修复区大体和组织学情况，评估修复效果。 三、软骨细胞－PGA复合物修复喉软骨缺损 雄性新西兰大白兔 18只O七月龄，体重2．skg土0．skg），随机分成实验 组（只X对照A组（只抨对照B组征只卜与实验二同法在一侧甲状软 2 第四军医大学98缀博土论文 一 骨板上造成0石cm X 0．scm的缺损区，植入同等大小体外培养71 天的软 骨细胞IGA复合物（实验组贞单纯PGA支架材料（对照A组），对照B组 缺损区注入 200 ul软骨细胞悬液脓度为 6 XI ovml、术后观察动物一般情 I 况，并分别于4周、吕周和12周取材，对缺损区修复愈合情况进行大体和 组织学评估，并观察PGA的降解性。v 结 果 一、体外培养细胞观察 原代软骨细胞贴壁时间较长，约48～72h，传代后细胞贴壁所需时间 逐渐缩短。软骨细胞接种后，细胞呈球形，以串珠状或层状吸附于PGA 纤维表面。软骨细胞IGA复合物体外培养1周，可见细胞分泌的基质呈更多还原

【Abstract】 Nowadays, no perfect methods are performed to repair cartilage defects caused by diseases or traumas in larynx and trachea. Although autologous, allogeneic cartilage and artificial products are usually used as reparative materials to reconstruct laryngeal and tracheal function, these methods still remain unsolved problems which are the damage to donor, lack of enough cartilage, no suitable shape for recipient, immune rejection from recipient, foreign body reaction and difficult blending with recipient. Therefore, it is still an important task for the otorhinolaryngeal workers to explore a new way of cartilage reconstruction. In recent years, tissue engineering technique provides a effective approach for solving these problems. Tissue engineering is a multidisciplinary scientific field merging with biology, material science, engineering and chemistry. Its main idea is to harvest minimal quantity of tissue cells, and then expand them in vitro. Next, the cells are seeded onto a3. Laryngeal cartilage defects repair with chondrocyte-PGA complexes.18 adult male New Zealand White Rabbits (age:4~6 months, weight : 2.5kg?.5kg) were at random divided into experimental groups(n=6). 0.6cmX 0.5cm cartilage defect sites were created just as experiment 2.Then,defects were repaired using chondrocyte-PGA complexes in experimental groups;in control A groups,defects were repaired using single PGA biomaterials.200 ul chondrocyte suspension with cellular density of 6X 107/ml was injected into defect sites in control B groups.Finally.the reparative effect was studied on gross and histology in 4,8 and 12 weeks after surgery.Results1. Chondrocytes culture in vitroIt took about 48-72 hours to adhere to the dishs for primary chondrocytes.During subculture.the time gradually became shorter.After seeding chondrocytes onto PGA scaffolds,cells maintained their spherical shape and adhered to the PGA fibers in the form of a string of beads or multiplayer.A number of matrices like web amoung fibers were observed around one week after culture in vitro.2. Gross and histology ofallogeneic tissue engineered cartilage4 weeks after transplantation,the specimens were excised. On gross examination, sheets and "C" shaped specimens appeared milk white cartilage tissue of approximately the same shape as the original configuration. Histologicalanalysis.the immature chondrocytes had been regenerated in allograft animals. Some mild inflammatory cells surrounding the new cartilage and a few PGA fibers were observed(HE stain ). Light-green staining of collagen and purple-blue staining of sulfated glycosamioglycan(GAG) within the cartilage matrix were detected. 8 weeks after transplantation, mature cartilage similar to native cartilage structure was found.The formation of lacunae in which the single or multiple chondrocytes were enclosed was observed .More collagen and sulfated GAG were found.Less evidence of inflammatory cells response and residual PGA fibers were watched. 12 weeks after transplantation,more mature cartilage was obtained.There was no evidence of neovascular formation. No chondrocytes and their matrice were also seen in control groups.3. Observation ofreparative effect -with tissue enginnered cartilage4 weeks after operation,gross specimens showed that laryngeal thyroid cartilage defects were well repaired using allogeneic tissue engineered cartilage as reparative material. Histological examination found a few chondrocytes and matrices between the reparative sites with tissue engineered cartilage and normal cartilage.But chondrocytes was smaller in repartive sites than that in native cartilage sites.8 and 12 weeks after operation,more chondrocytes and matrice were observed in reparative and interfacial areas.Few inflammatory cells were found.Only connective tissue existed in reparative sites of control groups.4. Observation ofreparative effect with chondrocyte-PGA complexes4 weeks after operation, gross inspection showed that cartilage defect areas were initially repaired .Histolog更多还原