Articular cartilage degradation and repair: mechanical components of matrix degradation and mechanisms of mesenchymal stem cell migration in tissue engineering [English translation]

Articular disorders are a leading cause of suffering and disability in the population over 50. Osteoarthitis (OA) is an articular pathology associated with persistent pain leading eventually to weakened and immobilized joints. OA is characterized by severe degradation of articular cartilage, covering the extremities of long bones, and by a relatively low level of inflammation compared to rheumatoid arthritis. Despite the important socio-economical consequences of AO, its pathogenesis is still misunderstood. Combined with the fact that symptoms usually appear when cartilage degradation is.generalized and irrerversible, early diagnostics are difficult and clinical therapies are very invasive and only palliative. The need for early diagnostic technologies and for adapted treatment governs the motivation behind the work of this thesis.

Aticular cartilage is a mainly composed of water and two proteins, collagen and prteoglycan. The former is a fibrillar molecule that limits the swelling pressure induced by the later. Cartilage provides a smooth near-frictionless surface between bone extremities and also distributes forces to the underlying bone, which are better suited to absorb forces then cartilage. Although mechanical stimulation is necessary for cartilage development and homeostasis, abnormal loading has also been shown to initiate cartilage degradation. Previous studies regarding load-mediated tissue degradation have mostly focused on water content, ion-induced swelling, proteoglycan metabolism and/or histological analysis of cartilage and bone, while few have provided data on possible direct collagen denaturation in situ due to loads. It has been shown and modeled that under rapid unconfined compressions, the collagen network is responsible for an important part of the tissue resistance. The objective of the study in this work was to determine if collagen could be denatured by mechanical compressions, and to study underlying mechanisms. Our main hypotheses was that mechanical compressions could compromise the integrity of the collagen network, render it less functional and possibly activate a series of collagen and proteoglycan related biochemical turnover events that are specific to mechanically loaded cartilage and not necessarily present in static cultures. To do this, we have applied a new collagen breakdown assay to precisely loaded cartilage/bone explants in order to investigate the ability of mechanical load to modulate collagen network integrity, turnover and functional mechanical properties as well as proteoglycan metabolism in this multidimensional study. We found that cyclic unconfined compression imposed on mature articular cartilage was capable of inducing a variety of changes, including immediate (in minutes) reduced mechanical properties, immediate damage (denaturation) to type II collagen, and a prolonged (for days) increase in loss of proteoglycan and collagen extracellular matrix fragments from the loaded disks to the culture medium. These results suggest that mechanical load plays an intrinsic role in the homeostatic control of collagen metabolism that is important in normal physiology as well as in cartilage degeneration.

Cartilage has a limited ability for repair; lesions often lead to progressive and generalized degradation. The second part of the thesis will focus on important events underlying cartilage regeneration in the context of tissue engineering, an emerging area of research that develops therapies using biomaterial scaffolds and/or cell transplantation in an effort to promote tissue regeneration. Targeted events of this work will be the migration of bone marrow mesenchymal stem cells (MSC). These cells, already considered essential in the natural repair of full thickness cartilage lesions, possess proliferative and differentiation patterns that make them ideal candidates for their use in tissue engineering approaches, where their potential is usually harnessed by either recruitment or transplantation. Cell migration is an important process involved in all these strategies, and its comprehension is deemed important for the rational elaboration of such techniques. It is in that context that the following studies are presented.

The objectives of the second paper published in Cell Motility and the Cytoskeleton in November 2006 was to develop a new technique that would enable a clear and detailed observation of cells migrating in a 3D environment. Most studies analyzing cell migration mechanisms are in two dimensions, and they seldom use MSC. The hypothesis was that the transmembrane migration assay, a quantitative assay for the migration response of a cell population, could be adapted to observe single cells migrating in the z axis. This new technique enabled us to simultaneously observe for the first time the main cytoskeletal components and the nucleus in a single cells migrating in three dimensions. Novel observations point to several differences compared to 2D migration, and suggests this method can be further applied to observe relationships with cytoskeleton associated proteins in terms of location and associations within the cell motile machinery. This method provides a tool to gain new insights into the cellular mechanisms of cell motility.

Finally, the objective of the third and last study presented in this thesis was to determine the effects of proteins of the extracellular matrix, namely fibronectin, vitronectin and collagen I, on the motogenic behavior of MSC. MSC from both rabbit and human origin were tested based in part on the widespread use of rabbit models for musculoskeletal related tissue engineering and repair models and their unknown correspondence to human in terms of MSC migration. Furthermore, tests were done in an optimised transmembrane assay, a rare feature in these kinds of studies. Our primary finding was that all three ECM proteins mentioned above induced a significant directed migration of MSC for both rabbit and human. Directed migration was elicited by two mechanisms, namely haptotaxis and chemotaxis. These results should permit a better understanding of the distribution of these cells in the body during wound repair and also provide biological information relevant to the rational design of cell scaffolds and of bioinstructive biomaterials in the quest to improve the performance of cartilage regenerative medicine strategies.