Chondrocyte-like Colony Formation of Mesenchymal Cells by Dentin Extracts in Agarose Gel Culture

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Abstract
In this study, the effects of guanidine extracts from demineralized bovine dentin matrix on rat mesenchymal cells were investigated by use of an agarose gel culture. The dentin extracts were divided into water-soluble and -insoluble fractions. Rat mesenchymal cells obtained from the cultivation of skeletal muscle tissue and embedded in agarose gel were treated with these two fractions. After three weeks of cultivation, the treated cells formed colonies that were stained metachromatically with toluidine blue in a dose-dependent manner. The activity necessary to form chondrocyte-like colonies by the water-insoluble fraction was significantly higher than that by the water-soluble fraction. Each chromatographic fraction of the water-insoluble part of dentin extracts on tandem Sephacryl S-200 High-resolution columns was also investigated. Chondrocyte-like colony-forming activity was concentrated in a single fraction. However, the electrophoretic pattern of this fraction revealed that there were still some bands of molecular weight between 18 and 30 kDa. According to the Western blot analysis of this fraction, there was a band corresponding to purified transforming growth factor β (TGF-β) under the non-reducing condition. After reduction, this band disappeared and we found a band corresponding to a component of 13 kDa as well as TGF-β. These findings suggest that TGF-β is present not only in bone but also in the dentin matrix.