Züchtung menschlichen Knorpelgewebes mit Hilfe einer Perfusionskammer
Züchtung menschlichen Knorpelgewebes mit Hilfe einer Perfusionskammer
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Züchtung menschlichen Knorpelgewebes mit Hilfe einer Perfusionskammer
DOI：
10.1055/s-2007-997199
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发表时间：
1994
影响因子：
1
通讯作者：
G. Burmester
中科院分区：
文献类型：
--
作者：
J. Bujía;M. Sittinger;C. Hammer;G. Burmester
在耳鼻喉科领域，软骨移植通常用于重建骨骼缺损。软骨细胞生长和分化的知识现在可以用来设计用于移植的软骨组织。第一个条件是软骨细胞除了能够产生新的软骨基质外，还保持其分化的表型。本研究的目的是建立一个三维培养系统，在体外形成的重要软骨移植。通过用胶原酶和透明质酸酶消化软骨基质来分离软骨细胞。在包埋在"低熔点"琼脂糖中后，将软骨细胞置于灌注培养室中以向培养物提供恒定的营养供应。蠕动泵以30分钟的开/关间隔运行。使用添加2%FCS和50微克/毫升抗坏血酸的Ham's F12作为培养基。使用对I型和II型胶原特异性的单克隆抗体来抑制细胞和基质合成。甲苯胺蓝和偶氮染料染色显示蛋白多糖和胶原的合成。在所述培养条件下，软骨细胞保持分化的表型（II型胶原蛋白的表达），并合成胶原蛋白和蛋白聚糖。基质产物的积累是在细胞周实现的。2 - 8周后，获得的组织表现出优异的组织学外观，显示出软骨组织的典型特征。结果表明，灌注室允许在体外快速制造一块用于移植的纯软骨组织。
In the field of otolaryngology cartilage grafting is commonly performed to reconstruct skeletal defects. Knowledge of chondrocyte growth and differentiation can now be used to engineer cartilage tissue for grafting. The first condition is that chondrocytes maintain their differentiated phenotype besides being able to produce a new cartilage matrix. The target of this study was to develop a three-dimensional culture system for in-vitro formation of vital cartilage transplants. Chondrocytes were isolated by digesting the cartilage matrix with collagenase and hyaluronidase. After embedding in "low-melting" agarose, the chondrocytes were placed into a perfusion culture chamber to provide a constant supply of nutrients to the cultures. The peristaltic pump was operated with on/off intervals of 30 min. Ham's F12 supplemented with 2% FCS and 50 micrograms/ml ascorbic acid was employed as culture medium. Monoclonal antibodies specific to collagens type I and type II were used to characterise cells and matrix synthesis. Synthesis of proteoglycans and collagens was achieved using toluidine blue and azan staining. Under the described culture conditions, the chondrocytes maintained a differentiated phenotype (expression of collagen type II) with synthesis of collagens and proteoglycans. An accumulation of matrix products was achieved pericellularly. After 2-8 weeks the obtained tissue exhibited an excellent histological appearance showing the typical features of cartilage tissue. The results show that the perfusion chamber allows a quick in-vitro fabrication of a piece of pure cartilage tissue for transplantation.