The development of a tissue-engineered alternative for current ligament grafts requires the creation of a fibrocartilaginous interface between the engineered ligament midsubstance and bone tissue. Therefore, the focus of this study was to examine the potential for cartilaginous extracellular matrix (ECM) formation by altering culture parameters for bovine anterior cruciate ligament (ACL) fibroblasts and marrow stromal cells (MSCs). Specifically, cells were cultured without chondrogenic media supplements on aggrecan-coated surfaces, tissue culture-treated control surfaces, and nonadhesive surfaces that promoted cell aggregation, and examined over 14 days. Aggrecan-coated surfaces promoted the aggregation of ACL fibroblasts and MSCs within 24 h after seeding. Aggrecan gene expression was significantly upregulated in cell aggregates, regardless of how cell clustering was induced, with as much as 10.9 +/- 1.2-fold upregulation in ACL fibroblasts and 9.7 +/-1.1-fold in MSCs after 3 days, compared to control surfaces. Dimethylmethylene blue (DMMB) results and immunostaining verified the presence of aggrecan in ACL fibroblast and MSC aggregates throughout the culture period. Results indicate that ACL fibroblasts retained the ability to alter their gene expression and produce aggrecan, though MSCs, in general, had a more consistent response to aggregation. These findings support the use of aggregate-inducing materials to encourage production of aggrecan and suggest that altering the degree of clustering could produce a range of phenotypes from a single cell source. As such, this represents a first step which may inform future approaches to producing tissue-engineered ligament grafts. Biotechnol. Bioeng. 2011;108: 151-162. (C) 2010 Wiley Periodicals, Inc.