Semester

Fall

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

College of Physical Activity and Sport Sciences

Department

Exercise Physiology

Committee Chair

Ming Pei

Abstract

Joint articular cartilage is vulnerable for degenerative disease after trauma due to the limited self-healing ability. Mesenchymal stem cells (MSCs) are considered a promising source for cartilage repair and regenerative medicine because of their excellent capacities of self-renewal and multipotent differentiation. Conventional in vitro expansion system, however, limits the utilization of MSCs for cartilage regeneration because in monolayer culture MSCs rapidly lose their self-renewal ability and differentiation potential. The lack of proper microenvironment in the current two-dimensional expansion system is attributed to the loss of self-renewal and multipotent differentiation in MSCs. Three-dimensional (3D) cell-deposited extracellular matrix (ECM) provides a novel strategy for in vitro MSC expansion, not only promoting cell proliferative rate but also improving chondrogenic potential. We generated 3D ECM deposited by synovium-derived stem cells (SDSCs) to reconstruct an in vitro 3D stem cell niche. First, MSCs were expanded on 3D ECM including SDSCs, adipose-derived stem cells (ADSCs) and bone marrow stem cells (BMSCs). The cell morphology of MSCs cultured on ECM changed to a thin and spindle-like shape and cell migration was directional along the fibrils of ECM. A dramatic increase in cell number and a greatly enhanced chondrogenic potential were observed, though surprisingly the ECM-treated MSCs did not display concomitantly improved adipogenic or osteogenic potentials. 3D ECM was also adopted to expand terminally differentiated cells including articular chondrocytes and nucleus pulposus (NP) cells, in order to improve the technique of autologous cell transplantation for cartilage repair and intervertebral disc regeneration. We found that ECM not only improved chondrocyte or NP cell expansion but also delayed dedifferentiation. Chondrocytes or NP cells expanded on ECM also acquired a strong redifferentiation capacity, particularly when treated with TGF-beta1 in a pellet culture system. Further, we compared the activity of several key signaling kinases and integrin expression in BMSCs cultured on ECM and conventional plastic flasks. 3D ECM increased the expression of SSEA-4, integrin alpha2, alpha4 and beta 5, and induced sustained activation of Src kinase, ERK1/2 and cyclin D1. Not only the expression but also the responsiveness of TGF-beta type II receptor was enhanced by 3D ECM in BMSCs. These observations suggested that 3D cell-deposited ECM promoted BMSC proliferative capacity through integrin/Src/ERK1/2 signaling pathway and enhanced chondrogenic potential by improving the responsiveness of TGF-beta receptors. Finally we generated two distinct types of ECM separately deposited by SDSCs and articular chondrocytes. SDSC-deposited ECM (S-ECM) contained fibrillar type I collagen, whereas chondrocyte-deposited ECM (C-ECM) is composed of fibrillar type I and II collagen with proteoglycans. S-ECM showed a superior influence on SDSCs to C-ECM in cell self-renewal and chondrogenic potential. The proliferative and chondrogenic abilities of chondrocytes were comparable on S-ECM and C-ECM. SDSC-deposited ECM was superior to chondrocyte-deposited ECM and functioned as a universal expansion system in vitro for MSCs and terminally differentiated cells to promote cell proliferative and chondrogenic potentials. In conclusion, we demonstrated that 3D cell-deposited ECM provides a proper microenvironment for in vitro expansion of MSCs and terminally differentiated cells, not only promoting self-renewal ability but also improving chondrogenic potential, and the novel 3D expansion system greatly improves cell-based cartilage and intervertebral disc regeneration.

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