组织工程化喉软骨的构建

Fabrication of laryngeal cartilage by means of tissue engineering technique

目的 研究组织工程化喉支架软骨的构建方法。方法 酶消化法获取幼兔肋和关节软骨细胞并进行体外培养,传代时延长胰蛋白酶作用时间3-5 min,收集第3代培养的软骨细胞用于实验;采用溶液浇铸、模压成形和颗粒滤沥方法制备喉形态聚羟基烷酸酯类[poly(3-hydroxybutyrate-co-3-hydroxyhexanoate,PHBHH)]生物材料塑形物,接种软骨细胞形成细胞-PHBHH复合物,体外共同培养1周后,分别在成兔体内用大网膜充填复合物中空部分与整体包裹、腹内成形(9只动物)和背部筋膜瓣与肌肉组织共同充填与包裹复合物、皮下成形(9只)的方法将负载软骨种子细胞的喉形态材料塑形物植入腹腔和背部,设空白对照组(每批3只)。术后一定时间取材,大体形态观察、组织学和免疫组织化学检测评估工程化喉软骨的成形与再生情况。结果 获取的软骨细胞活性为(93±2)％,传统方法为(94±2)％(P>0.05)。制备的喉形态生物材料塑形物呈中空半面喇叭状,液体(乙醇)静态容积测定孔隙率>90％。两种体内植入法均构建出喉形态组织工程化组织,大体形态维持良好,组织学和免疫组织化学均证实为软骨组织,且形态学和组织学表现两者基本一致。结论 改良培养细胞传代过程中胰酶作用程度未影响软骨细胞的成软骨性能;

Objective To explore the method of fabricating tissue engineered laryngeal cartilage. Methods The rib and articular cartilage of infant New Zealan white rabbits were harvested in sterile condition. The chondrocytes were separated by collagenase digestion and cultured in vitro for 3 passage. Serial steps of solution casting, extrusion molding and participate leaching were used to make larynx-shaped biomaterial models with poly(3-hydroxybutyrate-co- 3-hydroxyhexanoate,PHBHH) . The chondrocytes were seeded onto PHBHH scaffolds to form cell-PHBHH composites, which were subsequently in vitro for one week. After that, the measure of filling inner space of cell-PHBHH composites together with wrapping total composites using either greater omentum(n =9) or fascia flap and muscle (also n=9) in experimental groups was taken to implant the larynx-shaped biomaterial models seeded with chondrocytes into the belly and the back of adult New Zealan white rabbits. Control groups ( every group n = 3 ) were the same measure as experimental groups but without chondrocyte on PHBHH scaffolds. Finally, morphological observation, HE staining & special staining and immunohistochemical test were conducted to assess cartilage regeneration and its shape at different period following implantation. Results The rate of viable cell in the final cell suspension was (93±2) % after well-controlled prolongation of digestion trysin. Similar to that by traditional procedures (94±2)%(P>0. 05). The larynx-shaped PHBHH models with edges and corners of laryngeal cartilage made by us appeared to be hollow half-trumpet shape and its porosity was more than 90% . It showed that chondrocytes equally attached to the surface of porous PHBHH and filled within porousness with scanning electron microscopic examination. Tissue engineered larynx-shaped specimens could alternatively be harvested with the above mentioned two different implantation measures. The specimens presented to be similar to that before implantation in gross shape. It was demonstrated to be cartilaginous tissue through histological and immunohistochemical examination. Furthermore, There was nearly no difference between two kinds of tissue engineered laryngeal cartilage with two measures of implantation in morphology and histology. Conclusions The regeneration of tissue engineered cartilage in vivo is not influenced by the chondrocytes harvested by improvement of well-controlled prolonged digestion with trysin during in vitro cell culture. It seems that PHBHH may be used as scaffold in cartilage tissue engineering and wrapping together with filling method with either greater omentum or fascia flap and muscle is appropriate for fabricating tissue engineered laryngeal cartilage.