Yanaga Clinic and Tissue Culture Laboratory, Chuo-ku, Fukuoka, Japan.
Tissue Eng Part A. 2012 Oct;18(19-20):2020-9. doi: 10.1089/ten.TEA.2011.0370. Epub 2012 Jul 3.
We have developed a unique method that allows us to culture large volumes of chondrocyte expansion from a small piece of human elastic cartilage. The characteristic features of our culturing method are that fibroblast growth factor-2 (FGF2), which promotes proliferation of elastic chondrocytes, is added to a culture medium, and that cell-engineering techniques are adopted in the multilayered culture system that we have developed. We have subsequently discovered that once multilayered chondrocytes are transplanted into a human body, differentiation induction that makes use of surrounding tissue occurs in situ, and a large cartilage block is obtained through cartinogenesis and matrix formation. We have named this method two-stage transplantation. We have clinically applied this transplantation method to the congenital ear defect, microtia, and reported successful ear reconstruction. In our present study, we demonstrated that when FGF2 was added to elastic chondrocytes, the cell count increased and the level of hyaluronic acid, which is a major extracellular matrix (ECM) component, increased. We also demonstrated that these biochemical changes are reflected in the morphology, with the elastic chondrocytes themselves producing a matrix and fibers in vitro to form a natural scaffold. We then demonstrated that inside the natural scaffold thus formed, the cells overlap, connect intercellularly to each other, and reconstruct a cartilage-like three-dimensional structure in vitro. We further demonstrated by immunohistochemical analysis and electron microscopic analysis that when the multilayered chondrocytes are subsequently transplanted into a living body (abdominal subcutaneous region) in the two-stage transplantation process, neocartilage and neoperichondrium of elastic cartilage origin are regenerated 6 months after transplantation. Further, evaluation by dynamic mechanical analysis showed the regenerated neocartilage to have the same viscoelasticity as normal auricular cartilage. Using our multilayered culture system supplemented with FGF2, elastic chondrocytes produce an ECM and also exhibit an intercellular network; therefore, they are able to maintain tissue integrity post-transplantation. These findings realized a clinical application for generative cartilage surgery.
我们开发了一种独特的方法，可以从小面积的人弹性软骨中培养大量的软骨细胞扩增。我们培养方法的特点是在培养基中添加促进弹性软骨细胞增殖的成纤维细胞生长因子-2（FGF2），并采用我们开发的多层培养系统中的细胞工程技术。随后，我们发现一旦多层软骨细胞被移植到人体内，就会在原位进行利用周围组织的分化诱导，通过软骨生成和基质形成获得大的软骨块。我们将这种方法命名为两阶段移植。我们已经将这种移植方法临床应用于先天性耳缺陷、小耳畸形，并报告了成功的耳朵重建。在本研究中，我们证明了当 FGF2 被添加到弹性软骨细胞中时，细胞数量增加，细胞外基质（ECM）的主要成分之一透明质酸的水平增加。我们还证明这些生化变化反映在形态上，弹性软骨细胞本身在体外产生基质和纤维，形成天然支架。然后，我们证明在所形成的天然支架内，细胞重叠，细胞间相互连接，在体外重建出类似软骨的三维结构。我们通过免疫组织化学分析和电子显微镜分析进一步证明，在两阶段移植过程中，当多层软骨细胞随后被移植到活体（腹部皮下区域）中时，在移植后 6 个月会再生出弹性软骨来源的新生软骨和新生软骨膜。此外，动态力学分析的评估表明，再生的新生软骨具有与正常耳廓软骨相同的粘弹性。使用我们补充了 FGF2 的多层培养系统，弹性软骨细胞产生细胞外基质并表现出细胞间网络；因此，它们能够在移植后保持组织完整性。这些发现实现了生成性软骨手术的临床应用。
