CD105 Expression Correlates with the Ability of Passaged Human Articular Chondrocytes to form Cartilage Tissue In Vitro
Bianchi, Vanessa (1, 2), Parsons, Michael (2), Backstein, David (3), Kandel, Rita (1, 2)
(1) Institute of Biomaterials and Biomedical Engineering, University of Toronto
(2) Lunenfeld-Tanenbaum Research Institute
(3) Division of Orthopaedic Surgery, Mt. Sinai Hospital
Introduction: Passaged human chondrocytes will form hyaline-like cartilage when cultured with transforming growth factor beta (TGFβ). As accessibility to healthy cartilage is limited, cells must be obtained from osteoarthritic cartilage (OAC). Studies in animal models have demonstrated loss of the TGFβ receptor ALK5 in severe OAC. It is not known if the expression levels of ALK5, and its co-receptors endoglin (CD105) and TGFβ receptor 3 (TGFβRIII) change in human OAC in a grade dependent manner. Thus the purpose of this study was to assess the effect of OAC grade on in vitro chondrogenesis by human chondrocytes.
Methods: OAC was harvested from joints resected for arthroplasty from areas showing either minimal or severe osteoarthritic changes, graded using the OARSI scale and enzymatically digested to isolate chondrocytes. Primary cells (P0) were analyzed directly or passaged twice (P2). Image flow cytometry was used to assess expression of ALK5, TGFβRIII or CD105. Fluorescent activated cell sorting (FACS) was used to separate CD105+ and CD105- subpopulations. To assess tissue formation cells were seeded on membrane inserts (2x106 cells) and grown for 3 weeks in chondrogenic media containing TGFβ3 (10ng/ml). Tissues were evaluated histologically, immunohistochemically (IHC), or biochemically.
Results: Grading confirmed a significant difference between OAC with minimal and severe changes. No grade specific differences in the levels of ALK5, TGFβRIII, or CD105 were detected. P0 and P2 cells showed similar amounts of ALK5. Few P0 cells expressed TGFβRIII. This did not change with passaging. Cells expressing CD105 and the density of CD105 receptor/cell was significantly greater in P2 than P0 cells. With passaging FACS sorted CD105- populations became CD105+. P0 chondrocytes did not form cartilage tissue in the presence of TGFβ3. In contrast, P2 cells were able to form cartilage tissues. There were no significant differences in the proteoglycan or collagen contents. IHC showed that the tissues that developed were hyaline-like.
Conclusions: OAC grade did not affect cartilage formation. Although P0 cells express levels of ALK5 (the TGFβ receptor considered to regulate chondrogenesis) similar to P2 cells, only P2 cells form cartilage. Clearly additional co-receptors and/or factors are needed. With passage these cells change to largely CD105 expressing cells. Further studies are required to determine if high levels of CD105 regulate chondrogenesis by P2 cells.