Martinez Inigo, Elvenes Jan, Olsen Randi, Bertheussen Kjell, Johansen Oddmund
Department of Orthopaedic Surgery, Institute of Clinical Medicine, University of Tromsø, 9037 Tromsø, Norway.
Cell Transplant. 2008;17(8):987-96. doi: 10.3727/096368908786576499.
The main purpose of this work has been to establish a new culturing technique to improve the chondrogenic commitment of isolated adult human chondrocytes, with the aim of being used during cell-based therapies or tissue engineering strategies. By using a rather novel technique to generate scaffold-free three-dimensional (3D) structures from in vitro expanded chondrocytes, we have explored the effects of different culture environments on cartilage formation. Three-dimensional chondrospheroids were developed by applying the hanging-drop technique. Cartilage tissue formation was attempted after combining critical factors such as serum-containing or serum-free media and atmospheric (20%) or low (2.5%) oxygen tensions. The quality of the formed microtissues was analyzed by histology, immunohistochemistry, electron microscopy, and real-time PCR, and directly compared with native adult cartilage. Our results revealed highly organized, 3D tissue-like structures developed by the hanging-drop method. All culture conditions allowed formation of 3D spheroids; however, cartilage generated under low oxygen tension had a bigger size, enhanced matrix deposition, and higher quality of cartilage formation. Real-time PCR demonstrated enhanced expression of cartilage-specific genes such us collagen type II and aggrecan in 3D cultures when compared to monolayers. Cartilage-specific matrix proteins and genes expressed in hanging-drop-developed spheroids were comparable to the expression obtained by applying the pellet culture system. In summary, our results indicate that a combination of 3D cultures of chondrocytes in hanging drops and a low oxygen environment represent an easy and convenient way to generate cartilage-like microstructures. We also show that a new specially tailored serum-free medium is suitable for in vitro cartilage tissue formation. This new methodology opens up the possibility of using autogenously produced solid 3D structures with redifferentiated chondrocytes as an attractive alternative to the currently used autologous chondrocyte transplantation for cartilage repair.
这项工作的主要目的是建立一种新的培养技术，以改善分离的成人人类软骨细胞的软骨形成能力，旨在用于基于细胞的治疗或组织工程策略。通过使用一种相当新颖的技术，从体外扩增的软骨细胞生成无支架三维（3D）结构，我们探索了不同培养环境对软骨形成的影响。通过应用悬滴技术培养出三维软骨球。在结合含血清或无血清培养基以及大气（20%）或低（2.5%）氧张力等关键因素后，尝试形成软骨组织。通过组织学、免疫组织化学、电子显微镜和实时PCR分析所形成微组织的质量，并直接与天然成人软骨进行比较。我们的结果显示，通过悬滴法形成了高度有序的三维组织样结构。所有培养条件都能形成三维球体；然而，在低氧张力下生成的软骨尺寸更大、基质沉积增强且软骨形成质量更高。实时PCR表明，与单层培养相比，三维培养中软骨特异性基因如II型胶原蛋白和聚集蛋白聚糖的表达增强。悬滴培养形成的球体中表达的软骨特异性基质蛋白和基因与应用微团培养系统获得的表达相当。总之，我们的结果表明，软骨细胞悬滴三维培养与低氧环境相结合是生成软骨样微结构的一种简单便捷的方法。我们还表明，一种新的特制无血清培养基适用于体外软骨组织形成。这种新方法为使用自体产生的、带有再分化软骨细胞的固体三维结构开辟了可能性，作为目前用于软骨修复的自体软骨细胞移植的一种有吸引力的替代方法。
