Glycosaminoglycan Dependent Isolation of Mesenchymal Chondroprogenitor Populations from Human Bone Marrow

Abstract

Osteoarthritis (OA) is a chronic disease of joints characterised by progressive destruction of articular cartilage resulting in painful, limited joint movement. Cartilage has a limited ability to self-repair due to low chondrocyte motility and proliferative rates, and is further complicated by the absence of blood vessels for recruitment of circulating cells. Current clinical therapies do not result in full regeneration of healthy cartilage tissue. The long-term success of cartilage repair will therefore depend on regenerative methodologies resulting in the restoration of articular cartilage that closely duplicates the native tissue. For cell-based therapies, the optimal cell source must be readily accessible with easily isolated, abundant cells capable of collagen type II and sulfated proteoglycan production in appropriate proportions. Although a cell source with these therapeutic properties remains elusive, mesenchymal stem cells (MSCs) show promise of reproducing the structural or biomechanical properties of healthy articular cartilage. Current knowledge of and selection techniques for chondroprogenitors within the MSC population are relatively limited. This study focuses on methods for their isolation and activation. As cartilage is a tissue composed primarily of extracellular matrix (ECM) surrounding chondrocytes, it was hypothesised that there is a sub-population of progenitor cells in bone marrow that are primed towards the chondrogenic pathway with pre-requisite receptors for extracellular matrix (ECM) molecules. Consequently, chondroprogenitors could be isolated from bone marrow via their specific adhesion to cartilaginous ECM proteins. In this study hyaluronan (HA) and chondroitin-6-sulfate (CS) were used to select cells directly from bone marrow by coating tissue culture plastic or by adding in solution to unprocessed marrow. Various methods were undertaken to isolate this putative population of chondroprogenitors such as isolating the early adherent (EA) and late adherent (LA) cells and the sub-populations present as slow adherent cells in the EA and LA marrow fractions. Extracellular matrix-mediated isolation of cells, specifically the exposure of MSCs to a specific ECM molecule adhered to tissue culture plastic and subsequent re-plating onto non-coated flasks resulted in a 9-fold higher chondrogenic ability compared to the traditionally isolated plastic adhered cells. These ECM isolated cells retained their tri-lineage potential but the increase in differentiation potential was a chondrogenic phenomenon only. Further analysis suggested that this was not a specific selection of chondroprogenitors but an activation of a chondro-specific pathway within the ECM isolated MSCs. This study has not only elucidated a process enabling the isolation of a highly chondrogenic population of cells but also a process of MSC isolation from marrow that enables the retrieval of a higher yield of cells than is typically isolated using traditional methods.