TGFβ and osteoblast differentiation in a novel, serum‐free co‐culture system

Abstract

Our lab has previously described the production of large quantities of activated TGFβ coincident with chondrocyte hypertrophy. In this study, we investigated osteoblast differentiation in response to chondrocyte-produced TGFβ. Early (EH) and late (LH) hypertrophic chondrocyte populations were isolated from avian sterna and placed in serum-free alginate cultures. At day 5, the chondrocytes were transferred to serum-free co-culture with subconfluent monolayer of early mature osteoblasts (HOB-03-CE6; EMOB). At 24 hours, EMOB responded to EH and LH chondrocytes with a decrease in osterix gene expression, an osteoblast transcription factor. These data paralleled TGFβ-treated control EMOB monolayers and previous experiments with EH and LH conditioned media-treated EMOBs. Osteopontin gene expression, an extracellular matrix molecule, was increased in TGFβ-treated EMOB controls and EH and LH co-cultures, again, paralleling previous results with conditioned media-treatment. Smad2 and its phosphorylated form are the second messenger system for the TGFβ receptor. Immunocytochemistry revealed Smad2 staining in the cytoplasm of all cultured conditions. When compared to control monolayers of EMOBs, TGFβ-treated, EH and LH co-cultured EMOBs demonstrated a shift to predominantly nuclear localization for the phosphorylated Smad2. These data suggest that TGFβ produced by hypertrophic chondrocytes can alter osteoblast gene expression at the chondro-osseous border.