Mesenchymal stem cells (MSC) are a potential alternative source of differentiated chondrocytes for cartilage tissue regeneration and repair of osteoarthritic (OA) joints. We investigated the effects of pentosan polysulfate (PPS) and polysulfated glycosaminoglycan (PSGAG) on chondrogenesis of canine bone marrow-derived mesenchymal stem cells (cBMSC) in alginate and micromass cultures (MMC). Chondrogenic differentiation medium (CDM) was supplemented with PPS or PSGAG at concentrations of 0 (positive control; PC), 1, 3 and 5 mu g/ml. 10% DMEM was used as negative control. Chondrocyte phenotype was analyzed by quantitative real-time PCR (qPCR) for alginate cultures and Alcian blue staining for proteoglycan (PG) synthesis for MMC. In alginate culture, PPS and PSGAG showed no significant effect on type II collagen, aggrecan and HIF-2 alpha mRNA expression. PPS had no significant effect on type I collagen whereas PSGAG significantly upregulated (P<0.05) it at all concentrations relative to other treatments. PPS demonstrated a dose-dependent inhibitory effect on type X collagen mRNA with significant inhibition observed at 5 mu g/ml compared to the NC. PSGAG showed an inverse effect on type X collagen with 1 mu g/ml significantly inhibiting its expression while increase in the concentration correspondingly increased type X collagen expression. In MMC, PPS significantly enhanced chondrogenesis and PG deposition whereas PSGAG inhibited chondrogenesis and promoted a fibrocartilage-like phenotype with reduced PG deposition. While PPS enhances chondrogenesis of cBMSC in MMC, the response of MSC to chondroinductive factors is culture system-dependent and varies significantly between alginate and MMC.