Induction of bone marrow mesenchymal stem cell chondrogenesis following short-term suspension culture

The objective of this study was to evaluate mesenchymal stem cell (MSC) chondrogenesis following incubation in chondrogenic suspension cultures from which single cells were obtained. MSCs were maintained in suspension over a nonadherent surface for 3 days, dissociated into a suspension, and then evaluated for chondrogenesis in agarose in the presence or absence of transforming growth factor beta (TGFβ). In a second experiment, MSCs from suspension culture were returned to monolayer expansion for 2 days prior to testing for chondrogenesis. In both cases, undifferentiated MSCs were evaluated as controls. Suspension culture alone did not stimulate chondrogenesis. Suspension followed by expansion stimulated a four- to ninefold increase in extracellular matrix (ECM) synthesis in TGFβ-free cultures, a finding that was attributed to an increase in viable MSCs that secreted a proteoglycan-rich ECM. Gene expression of aggrecan and type II collagen increased with suspension culture, but decreased with postsuspension expansion. Therefore, stimulation of ECM synthesis without additional TGFβ exposure could not be attributed to an enhancement of chondrogenesis with monolayer culture. ECM synthesis of suspension/expansion-conditioned MSCs without additional TGFβ exposure was less than samples maintained in TGFβ throughout the differentiation culture. Based on these findings, a better understanding of factors associated with early-stage chondrogenesis and MSC differentiation to a highly active phenotype may lead to improved methods for stimulating chondrogenesis during short-term culture.