CHONDROCYTE MIGRATION AND REPOPULATION OF CARTILAGE

指导这项初步研究的工作假设是，软骨细胞能够迁移，这种潜力可以由运动生成生长因子诱导或增强，和/或通过补充或过度表达参与运动生成信息传递的细胞信号机制的关键组件来诱导或增强。软骨细胞的迁移能力以前还没有得到系统的研究，尽管它可能对生长和修复过程至关重要。一些论文表明，软骨细胞在二维表面上的迁移能力至少是有限的，但这些研究并没有适当地量化这种反应。此外，目前还不清楚分化的软骨细胞是否会迁移。细胞迁移生物学领域充满了新的信息，我们相信，将这些知识应用于软骨细胞运动性的研究是及时的。我们的方法将是启动软骨细胞在二维表面上迁移的研究，使用视频显微镜来定量评估动态细胞运动参数，包括：膜延伸率、迁移速度和定向持久性。细胞的主要来源将是牛的关节表面，但人类软骨细胞将被选择性地研究。我们将探索供体牛从出生到成熟的年龄对软骨细胞移动能力的影响。人类软骨细胞将主要用于回答骨关节炎软骨细胞是否获得运动性的问题。在特定的目标1中，我们将探索培养条件和基质对软骨细胞在二维表面上运动的影响。在具体目标2中，我们的目标是确定最大限度地促进迁移的因素，包括用选定的生长因子(主要是肝细胞生长因子、表皮生长因子和骨形态发生蛋白-2)的治疗，以及在致动级联中的关键细胞内中间产物的过度表达(候选包括早期的“开关”、CaS/Crk复合体、丝裂原激活的激酶和选定的胍三磷酸酶)。编码候选蛋白的cDNA将通过腺病毒转染法导入软骨细胞。在具体目标3中，设计为最大限度迁移的软骨细胞随后将被标记荧光染料并植入软骨盘表面；将使用双光子显微镜评估和量化它们在软骨基质中的穿透和迁移能力。将关注运动性软骨细胞的蛋白水解酶的表达；最初的关注将集中在MT1-MMPs(金属蛋白酶家族的成员)上。这些研究应该为软骨细胞的迁移潜力提供有价值的见解，并为进一步的研究打开大门，如果研究成功，将提供全新的方法来解决工程化软骨修复的问题。

The working hypothesis guiding the proposed pilot investigation is that chondrocytes are able to migrate and that this potential can be induced or enhanced by motogenic growth factors, and/or by replenishment or overexpression of key components of the cellular signaling machinery involved in the transmition of the motogenic message. The ability of chondrocytes to migrate has not been systematically studied before, even though it may be critical for growth and repair processes. A few papers show that chondrocytes have at least a limited ability to migrate on 2-dimensional surfaces, but these studies do not properly quantify the response. Further it is not clear whether differentiated chondrocytes migrate. The field of cell migration biology is exploding with new information, and we believe that it is timely to apply this knowledge to the study of chondrocyte motility. Our approach will be to initiate studies of chondrocyte migration on 2-dimensional surfaces, using videomicroscopy to quantitatively assess dynamic cell motion parameters, including: membrane extension rates, speed of migration, and directional persistence. The primary source of cells will be the articular surfaces of bovines, but human chondrocytes will be selectively studied. We will explore the effect of age of the donor bovine, from newborn to mature, on the ability of chondrocytes to move. Human chondrocytes will be used primarily to answer the question of whether osteoarthritic chondrocytes acquire motility. In specific aim 1, we will explore the effect of culture conditions and matrix substrata on chondrocyte motility on 2-dimensional surfaces. In specific aim 2 we will aim at defining factors that maximally promote migration, including treatment with selected growth factors (primarily hepatocyte growth factor, epidermal growth factor, and bone morphogenetic protein-2), and overexpression of critical intracellular intermediates in the motogenic cascade (candidates include the early "switch", Cas/Crk complex, mitogen activated kinases and selected guanidine triphosphatases). The cDNAs encoding the candidate proteins will be transduced into chondrocytes by adenoviral transfections. In specific aim 3, chondrocytes engineered to maximally migrate will then be tagged with a fluorescent dye and implanted on the surface of cartilage disks; their ability to penetrate and migrate within the cartilage matrix will be assessed and quantified by use of 2-photon microscopy. Attention will be given to the expression of proteinases by motile chondrocytes; initial attention will be focused on MT1-MMP (a member of the metalloproteinase family). These studies should provide valuable insights into the migratory potential of chondrocytes, and open the door to further investigations that, if successful, will provide radically new approaches to tackle the problem of engineering cartilage repair.