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.
| Morales, T I (2007) Chondrocyte moves: clever strategies? Osteoarthritis Cartilage 15:861-71 |
| Chang, C; Lauffenburger, D A; Morales, T I (2003) Motile chondrocytes from newborn calf: migration properties and synthesis of collagen II. Osteoarthritis Cartilage 11:603-12 |
