kartogenin and caprolactone

Cartilage engineering strategies using mesenchymal stem cells (MSCs) could provide preferable solutions to resolve long-segment tracheal defects. However, the drawbacks of widely used chondrogenic protocols containing TGF-β3, such as inefficiency and unstable cellular phenotype, are problematic. In our research, to optimize the chondrogenic differentiation of human umbilical cord MSCs (hUCMSCs), kartogenin (KGN) preconditioning was performed prior to TGF-β3 induction. hUCMSCs were preconditioned with 1 μM of KGN for 3 d, sequentially pelleted, and incubated with TGF-β3 for 28 d. Then, the expression of chondrogenesis- and ossification-related genes was evaluated by immunohistochemistry and RT-PCR. The underlying mechanism governing the beneficial effects of KGN preconditioning was explored by phosphorylated kinase screening and validated in vitro and in vivo using JNK inhibitor (SP600125) and β-catenin activator (SKL2001). After KGN preconditioning, expression of fibroblast growth factor receptor 3, a marker of precartilaginous stem cells, was up-regulated in hUCMSCs. Furthermore, the KGN-preconditioned hUCMSCs efficiently differentiated into chondrocytes with elevated chondrogenic gene ( SOX9, aggrecan, and collagen II) expression and reduced expression of ossific genes (collagen X and MMP13) compared with hUCMSCs treated with TGF-β3 only. Phosphokinase screening indicated that the beneficial effects of KGN preconditioning are directly related to an up-regulation of JNK phosphorylation and a suppression of β-catenin levels. Blocking and activating tests revealed that the prochondrogenic effects of KGN preconditioning was achieved mainly by activating the JNK/Runt-related transcription factor (RUNX)1 pathway, and antiossific effects were imparted by suppressing the β-catenin/RUNX2 pathway. Eventually, tracheal patches, based on KGN-preconditioned hUCMSCs and TGF-β3 encapsulated electrospun poly( l-lactic acid-co-ε-caprolactone)/collagen nanofilms, were successfully used for restoring tracheal defects in rabbit models. In summary, KGN preconditioning likely improves the chondrogenic differentiation of hUCMSCs by committing them to a precartilaginous stage with enhanced JNK phosphorylation and suppressed β-catenin. This novel protocol consisting of KGN preconditioning and subsequent TGF-β3 induction might be preferable for cartilage engineering strategies using MSCs.-Jing, H., Zhang, X., Gao, M., Luo, K., Fu, W., Yin, M., Wang, W., Zhu, Z., Zheng, J., He, X

Topics: Anilides; Animals; beta Catenin; Caproates; Cartilage; Cell Differentiation; Cells, Cultured; Chondrocytes; Chondrogenesis; Collagen; Core Binding Factor Alpha 1 Subunit; Humans; Lactones; Male; MAP Kinase Signaling System; Mesenchymal Stem Cells; Mice; Mice, Nude; Phthalic Acids; Rabbits; Signal Transduction; Tissue Engineering; Transforming Growth Factor beta3; Umbilical Cord; Up-Regulation

Tracheal stenosis is one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, we fabricated coaxial electrospun fibers using poly(L-lactic acid-co-caprolactone) and collagen solution as shell fluid and kartogenin solution as core fluid. Scanning electron microscope and transmission electron microscope images demonstrated that nanofibers had uniform and smooth structure. The kartogenin released from the scaffolds in a sustained and stable manner for about 2 months. The bioactivity of released kartogenin was evaluated by its effect on maintain the synthesis of type II collagen and glycosaminoglycans by chondrocytes. The proliferation and morphology analyses of mesenchymal stems cells derived from bone marrow of rabbits indicated the good biocompatibility of the fabricated nanofibrous scaffold. Meanwhile, the chondrogenic differentiation of bone marrow mesenchymal stem cells cultured on core-shell nanofibrous scaffold was evaluated by real-time polymerase chain reaction. The results suggested that the core-shell nanofibrous scaffold with kartogenin could promote the chondrogenic differentiation ability of bone marrow mesenchymal stem cells. Overall, the core-shell nanofibrous scaffold could be an effective delivery system for kartogenin and served as a promising tissue engineered scaffold for tracheal cartilage regeneration.

Topics: Anilides; Animals; Caproates; Cells, Cultured; Chondrocytes; Chondrogenesis; Collagen; Drug Delivery Systems; Lactones; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Nanofibers; Phthalic Acids; Polyesters; Rabbits; Regeneration; Tissue Engineering; Tissue Scaffolds; Trachea