0391-3988/619-09 $15.00/0 Regenerative Medicine The International Journal of Artificial Organs / Vol. 30 / no. 7, 2007 / pp. 619-627 Differentiation of a fibrin gel encapsulated chondrogenic cell line E. V. DARE 1 , S. G. VASCOTTO 2 , D. J. CARLSSON 3 , M. T. HINCKE 1 , M. GRIFFITH 1 1 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario - Canada 2 University of Ottawa Eye Institute, Ottawa, Ontario - Canada 3 National Research Council Canada, Ottawa, Ontario - Canada © Wichtig Editore, 2007 ABSTRACT: Hyaline cartilage has very limited regenerative capacity following damage. Therefore engineered tissue substitutes have been the focus of much research. Our objective was to develop a fibrin-based scaffold as a cell delivery vehicle and template for hyaline cartilage regeneration, and compare its cellular properties against monolayer and pellet culture for chondrogenic cells. The chondrogenic precursor cell line, RCJ 3.1C5.18 (C5.18), was chosen as a test system for evaluating the effect of various culture conditions, including cell encapsulation, on articular chondrogenic cell differentiation. The C5.18 cells in monolayer showed elevated expression of collagen II, an articular chondrogenic marker, but also markers for fibrocartilage differentiation (collagen I and versican) when cultured with chondrogenic medium as compared to basic maintenance medium. Pellets of C5.18 cells cultured in chondrogenic medium were histologically more organized in structure than pellets cultured in control maintenance medium. The chondrogenic medium cultured pellets also secreted an extracellular matrix that was comprised of type II with very little type I collagen, indicating a trend towards a more hyaline-like cartilage. Moreover, when cultured in chondrogenic medium, fibrin-encapsulated C5.18 cells elaborated an extracellular matrix containing type II collagen, as well as aggrecan, which are both components of hyaline cartilage. This indicated a more articular-like chondrogenic differentiation for fibrin encapsulated C5.18 cells. The results of these experiments provide evidence that the C5.18 cell line can be used as a tool to evaluate potential scaffolds for articular cartilage tissue engineering. (Int J Artif Organs 2007; 30: 619-27) KEY WORDS: Fibrin, Hydrogel, Chondrogenic differentiation, Cartilage, Regeneration INTRODUCTION Hyaline articular cartilage is an avascular, alymphatic tissue that consists of chondrocytes encapsulated within a dense extracellular matrix (ECM). When damaged due to osteoarthritis, its regenerative potential is minimal and healing occurs with the formation of fibrocartilage (1). Fibrocartilage does not have the appropriate physical and mechanical properties to resist the forces that occur during normal wear and tear in daily articular joint function (2). Chondrocytes are essential for maintaining the function of articular hyaline cartilage (3). They secrete the ECM components, mainly type II collagen and aggrecan, as well as matrix metalloproteinases (MMPs) for matrix remodelling (4, 5). The transcription factor Sox9 regulates collagen II production and its expression is used as an early marker for chondrogenesis in hyaline cartilage formation (6, 7). During abnormal fibrocartilage development in repair, chondrocytes re-enter the cell cycle and proliferate, losing Sox9 expression, and actively transcribe collagen I and versican, rather than collagen II and aggrecan (8). Because of the poor capacity of hyaline cartilage for regeneration, cell and tissue engineering-based therapies for repair have been intensively researched. The rat calvaria-derived RCJ 3.1C5.18 (C5.18) cell line has key cartilage progenitor properties (9). This cell line differentiates and forms discrete three-dimensional (3D) cartilage nodules when grown in monolayer (10), thereby