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头颈外科中的软骨重建:用于人类鼻中隔软骨组织工程的可吸收聚合物支架比较

Cartilage reconstruction in head and neck surgery: comparison of resorbable polymer scaffolds for tissue engineering of human septal cartilage.

作者信息

Rotter N, Aigner J, Naumann A, Planck H, Hammer C, Burmester G, Sittinger M

机构信息

Department of Otorhinolaryngology, Head and Neck Surgery, Klinikum Grosshadern, Ludwig-Maximilians University of Munich, Germany.

出版信息

J Biomed Mater Res. 1998 Dec 5;42(3):347-56. doi: 10.1002/(sici)1097-4636(19981205)42:3<347::aid-jbm2>3.0.co;2-j.

Abstract

New cell culture techniques raise the possibility of creating cartilage in vitro with the help of tissue engineering. In this study, we compared two resorbable nonwoven cell scaffolds, a polyglycolic acid/poly-L-lactic acid (PGA/PLLA) (90/10) copolymer (Ethisorb) and pure PLLA (V 7-2), with different degradation characteristics in their aptitude for cartilage reconstruction. Chondrocytes were isolated enzymatically from human septal cartilage. The single cells were resuspended in agarose and transferred into the polymer scaffolds to create mechanical stability and retain the chondrocyte-specific phenotype. The cell-polymer constructs were then kept in perfusion culture for 1 week prior to subcutaneous transplantation into thymusaplastic nude mice. After 6, 12, and 24 weeks, the specimens were explanted and analyzed histochemically on the presence of collagen (azan staining), proteoglycans (Alcian blue staining), and calcification areas (von Kossa staining). Furthermore, different collagen types (collagen type I, which is found in most tissues, but not in hyaline cartilage matrix; and collagen type II, which is cartilage specific) were differentiated immunohistochemically by the indirect immunoperoxidase technique. Vascular ingrowth was investigated by a factor VIII antibody, which is a endothelial marker. Quantification of several matrix components was performed using the software Photoshop. Significant differences were found between both nonwoven structures concerning matrix synthesis and matrix quality as well as vascular ingrowth. Ethisorb, with a degradation time of approximately 3 weeks in vitro, showed no significant differences from normal human septal cartilage in the amount of collagen types I and II 24 weeks after transplantation. Thin fibrous tissue layers containing blood vessels encapsulated the transplants. V 7-2 constructs, which did not show strong signs of degradation even 24 weeks after transplantation, contained remarkably smaller amounts of cartilage-specific matrix components. At the same time, there was vascular ingrowth even in central parts of the transplants. In conclusion, polymer scaffolds with a short degradation time are suitable materials for the development of cartilage matrix products, while longer stability seems to inhibit matrix synthesis. Thus, in vitro engineering of human cartilage can result in a cartilage-like tissue when appropriate nonwovens are used. Therefore, this method could be the ideal cartilage replacement method without the risk of infection and with the possibility of reconstructing large defects with different configurations.

摘要

新的细胞培养技术增加了借助组织工程在体外制造软骨的可能性。在本研究中,我们比较了两种可吸收的非织造细胞支架,一种聚乙醇酸/聚-L-乳酸(PGA/PLLA)(90/10)共聚物(Ethisorb)和纯PLLA(V 7-2),它们在软骨重建能力方面具有不同的降解特性。软骨细胞通过酶解法从人鼻中隔软骨中分离出来。将单细胞重悬于琼脂糖中,然后转移到聚合物支架中,以形成机械稳定性并保留软骨细胞特异性表型。然后将细胞-聚合物构建体在灌注培养中保存1周,再皮下移植到无胸腺裸鼠体内。6周、12周和24周后,取出标本,通过组织化学分析胶原蛋白(偶氮染色)、蛋白聚糖(阿尔辛蓝染色)和钙化区域(冯·科萨染色)的存在情况。此外,通过间接免疫过氧化物酶技术对不同类型的胶原蛋白(I型胶原蛋白存在于大多数组织中,但不存在于透明软骨基质中;II型胶原蛋白是软骨特异性的)进行免疫组织化学鉴别。通过因子VIII抗体(一种内皮标志物)研究血管长入情况。使用Photoshop软件对几种基质成分进行定量分析。在基质合成、基质质量以及血管长入方面,两种非织造结构之间存在显著差异。Ethisorb在体外的降解时间约为3周,移植24周后,其I型和II型胶原蛋白的含量与正常人鼻中隔软骨相比无显著差异。含有血管的薄纤维组织层包裹着移植体。V 7-2构建体即使在移植24周后也没有明显的降解迹象,其软骨特异性基质成分的含量明显较少。与此同时,即使在移植体的中央部分也有血管长入。总之,降解时间短的聚合物支架是开发软骨基质产品的合适材料,而较长的稳定性似乎会抑制基质合成。因此,当使用合适的非织造材料时,人软骨的体外工程可以产生类似软骨的组织。因此,这种方法可能是理想的软骨替代方法,没有感染风险,并且有可能重建不同形态的大缺损。

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