聚羟基烷酸酯聚合物负载软骨细胞修复同种异体喉软骨缺损

摘要

背景：软骨组织工程基础研究相当深入，但在耳鼻咽喉科实际应用研究颇少，探索组织工程技术简便实用的喉软骨修复方法是值得研究的课题。目的：比较多孔海绵状聚羟基丁酸酯与聚羟基己酸酯共聚物生物材料负载软骨细胞体外培养形成的初期组织工程软骨组织与体内植入一定时期形成的较成熟组织工程软骨组织修复同种异体甲状软骨缺损的效果。方法：收集体外培养第3代乳兔(3 d龄)软骨细胞，以多孔海绵状聚羟基丁酸酯与聚羟基己酸酯共聚物生物材料为细胞外基质，采用组织工程技术制备细胞-材料复合物，共同体外培养形成初级组织工程软骨组织后直接应用于成兔甲状软骨缺损的修复(实验组A，n=5)或将初级组织工程软骨组织体内植入一定时期形成较成熟组织工程软骨再应用于甲状软骨缺损的修复(实验组B，n=5)。设单纯聚羟基丁酸酯与聚羟基己酸酯共聚物材料修复组(对照A组，n=4)和单纯软骨细胞修复组(对照B组，n=4)作为对照。分别于术后4周(实验B组)和8周(实验A组、对照A组、对照B组)取材，对甲状软骨缺损修复效果进行大体和组织学评价。结果与结论：两者大体支架形态基本一致，修复区与原有软骨均相续平坦，无凹陷及缺损。但实验A组存在界面无细胞区，修复区基质分泌不丰富；实验B组界面区有细胞生长，基质分泌良好。两者炎细胞浸润均不明显。对照组修复区凹陷，呈暗红色软组织充填，组织学及特殊染色检查未发现软骨样结构及其分泌的基质成分。结果表明在有免疫力的动物体内，初级组织工程软骨组织直接应用与体内植入后再应用均能有效修复同种异体甲状软骨缺损，无明显免疫反应；相同时期内，应用较成熟组织工程软骨组织修复效果优于应用初级组织工程软骨组织。然而，直接应用初级组织工程软骨组织可节省时间、成本、工作量及操作环节，避免二次皮下手术的痛苦，是比较实用的方法之一。%BACKGROUND:A great development has been achieved in essential research on tissue engineered cartilage. However, its real application in otolaryngology has been rarely reported. It is faced with the topic to explore the simple and convenient method of repairing laryngeal cartilage by tissue engineering technique. OBJECTIVE:To compare the effect of porous spongy poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) seeded with chondrocytes or using senior tissue engineered cartilage in repairing al ogenic thyroid cartilage defects.METHODS:Chondrocytes at passage 3 were harvested from infant rabbits within 3 days. Porous spongy poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) seeded with chondrocytes composites were made by tissue engineering technique. The chondrocyte-poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) composites were co-cultured in vitro to form junior tissue engineered cartilage. And then respectively used for repairing the thyroid cartilage defects and directly transplanted with junior tissue engineered cartilage (experimental group A, n=5), or firstly the junior tissue engineered cartilage to be implanted subcutaneously for a period of time to further maturity for relative senior tissue engineered cartilage and secondly to be transplanted (experimental group B, n=5) into adult New Zealand white rabbits. Simple poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) sponge scaffold (control group A, n=4) and chondrocyte suspensions(control group B, n=4) were used as reparative materials in defect areas as control groups. Final y, the reparative effect was respectively studied grossly and histological y at 4 weeks (experimental group B) and 8 weeks (experimental group A, control group A and control group B) after transplantation. RESULTS AND CONCLUSION:The cartilage defects were wel repaired in the experimental groups. It was smooth between the reparative area and original cartilage without dents and defects. Both were similar grossly. But few chondrocytes at interfacial region between the reparative area and original cartilage and poor matrices were observed in the experimental group A. A Few chondrocytes and more matrices were observed in the experimental group B. Inflammatory cellinfiltration was not obvious in two experimental groups. Control groups showed soft tissue of dark-red color accompanied with local concave in gross specimens. Histological examination and special staining showed there were no cartilage-like structure and secretion of matrix components. The results showed that it is possible to repair thyroid cartilage defect using junior tissue engineered cartilage directly or junior tissue engineered cartilage after in vitro implantation in al ograft rabbits with immunity, and the immunoreaction is not obvious;in the same period, the repairing effect of mature tissue engineered cartilage is better than that of junior tissue engineered cartilage. However, application of junior tissue engineered cartilage directly can save time, costs, workload and operational link, and avoid the pain from secondary skin surgery, which is one of the more practical approaches.