Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta 1 in monolayer and insulin-like growthfactor-I in a three-dimensional matrix

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (M SCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-di mensional cultures containing- insulin-like growth factor I (IGF-I). Bone m arrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibr in disks containing 0 or 100 ng of IGF-I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochem istry. oglycan production by [S-35]-sulfate incorporation, and disk DNA con tent by fluorometry. Mesenchymal cells in monolayer cultures treated with T GF-beta1 actively proliferated for the first 4 days, developed cellular rou nding, and formed cell clusters. Treated MSC cultures had a two-fold increa se in medium proteoglycan content. Pretreatment of MSCs with TGF-PI followe d by exposure of cells to IGF-l in three-dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, prot eoglycan and procollagen type II production by MSC's remained lower than pa rallel chondrocyte cultures. MSC pretreatment with TGF-beta1 without sequen tial IGF-I was less effective in initiating expression of markers of chondr ogenesis. This study indicates that although MSC differentiation was less t han complete when compared to mature chondrocytes, chondrogenesis was obser ved in IGF-I supplemented cultures, particularly when used in concert with TGF-beta1 pretreatment. (C) 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.