CHONDROCYTE MIGRATION AND REPOPULATION OF CARTILAGE

指导所提出的初步研究的工作假设是，软骨细胞能够迁移，并且这种潜力可以通过运动生长因子和/或通过补充或过度表达参与运动信息传递的细胞信号传导机制的关键组分来诱导或增强。 软骨细胞迁移的能力以前没有系统的研究，即使它可能是生长和修复过程的关键。 一些论文表明，软骨细胞在二维表面上迁移的能力至少是有限的，但这些研究没有正确地量化反应。 此外，分化的软骨细胞是否迁移尚不清楚。 细胞迁移生物学领域的新信息正在爆炸，我们相信，这是及时将这些知识应用到软骨细胞运动的研究。我们的方法将是启动研究软骨细胞迁移的二维表面，使用视频显微镜定量评估动态细胞运动参数，包括：膜的延伸率，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。 我们将探讨供体牛的年龄，从新生儿到成熟，对软骨细胞移动能力的影响。 人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。 在具体目标1中，我们将探索培养条件和基质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，我们将致力于定义最大限度地促进迁移的因素，包括使用选定的生长因子（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）进行治疗，以及运动级联中关键细胞内中间体的过表达（候选者包括早期“开关”、Cas/Crk复合物、有丝分裂原激活激酶和选定的胍三磷酸酶）。 将编码候选蛋白的cDNA通过腺病毒转染转导到软骨细胞中。 在具体目标3中，然后用荧光染料标记经工程化以最大程度迁移的软骨细胞，并将其植入软骨盘的表面上;通过使用双光子显微镜来评估和量化它们在软骨基质内渗透和迁移的能力。 注意力将被给予蛋白酶的运动软骨细胞的表达，最初的注意力将集中在MT 1-MMP（金属蛋白酶家族的成员）。 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，如果成功的话，将提供全新的方法来解决工程软骨修复的问题。

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.