Yan Su1,2, Janet M Denbeigh1, Emily T Camilleri3, Scott M Riester1, Joshua A Parry1, Eric R Wagner1, Michael J Yaszemski1,4, Allan B Dietz5, Simon M Cool6, Andre J van Wijnen1,3,7, Sanjeev Kakar1. 1. Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN. 2. Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China. 3. Masonic Cancer Center, University of Minnesota, Minneapolis MN. 4. Department of Biomedical Engineering and Physiology, Mayo Clinic College of Medicine, Rochester, MN. 5. Department of Laboratory Medicine & Pathology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN. 6. Institute of Medical Biology, Agency for Science, Technology and Research (ASTAR), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore; Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074, Singapore. 7. Department of Biochemistry & Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN.
Abstract
PURPOSE: The poor healing potential of intra-articular ligament injuries drives a need for the development of novel, viable 'neo-ligament' alternatives. Ex vivo approaches combining stem cell engineering, 3-dimensional biocompatible scaffold design and enhancement of biological and biomechanical functionality via the introduction of key growth factors and morphogens, represent a promising solution to ligament regeneration. METHODS: We investigated growth, differentiation and extracellular matrix (ECM) protein production of human adipose-derived mesenchymal stem/stromal cells (MSCs), cultured in 5% human platelet lysate (PL) and seeded on three-dimensional polycaprolactone (PCL) scaffolds, in response to the connective-tissue related ligands fibroblast growth factor 2 (basic) (FGF2) and growth and differentiation factor-5 (GDF5). Phenotypic alterations of MSCs under different biological conditions were examined using cell viability assays, real time qPCR analysis of total RNA, as well as immunofluorescence microscopy. RESULTS: Phenotypic conversion of MSCs into ECM producing fibroblastic cells proceeds spontaneously in the presence of human platelet lysate. Administration of FGF2 and/or GDF5 enhances production of mRNAs for several ECM proteins including Collagen types I and III, as well as Tenomodulin (e.g., COL1A1, TNMD), but not Tenascin-C (TNC). Differences in the in situ deposition of ECM proteins Collagen type III and Tenascin-C were validated by immunofluorescence microscopy. SUMMARY: Treatment of MSCs with FGF2 and GDF5 was not synergistic and occasionally antagonistic for ECM production. Our results suggest that GDF5 alone enhances the conversion of MSCs to fibroblastic cells possessing a phenotype consistent with that of connective-tissue fibroblasts.
PURPOSE: The poor healing potential of intra-articular ligament injuries drives a need for the development of novel, viable 'neo-ligament' alternatives. Ex vivo approaches combining stem cell engineering, 3-dimensional biocompatible scaffold design and enhancement of biological and biomechanical functionality via the introduction of key growth factors and morphogens, represent a promising solution to ligament regeneration. METHODS: We investigated growth, differentiation and extracellular matrix (ECM) protein production of human adipose-derived mesenchymal stem/stromal cells (MSCs), cultured in 5% human platelet lysate (PL) and seeded on three-dimensional polycaprolactone (PCL) scaffolds, in response to the connective-tissue related ligands fibroblast growth factor 2 (basic) (FGF2) and growth and differentiation factor-5 (GDF5). Phenotypic alterations of MSCs under different biological conditions were examined using cell viability assays, real time qPCR analysis of total RNA, as well as immunofluorescence microscopy. RESULTS: Phenotypic conversion of MSCs into ECM producing fibroblastic cells proceeds spontaneously in the presence of human platelet lysate. Administration of FGF2 and/or GDF5 enhances production of mRNAs for several ECM proteins including Collagen types I and III, as well as Tenomodulin (e.g., COL1A1, TNMD), but not Tenascin-C (TNC). Differences in the in situ deposition of ECM proteins Collagen type III and Tenascin-C were validated by immunofluorescence microscopy. SUMMARY: Treatment of MSCs with FGF2 and GDF5 was not synergistic and occasionally antagonistic for ECM production. Our results suggest that GDF5 alone enhances the conversion of MSCs to fibroblastic cells possessing a phenotype consistent with that of connective-tissue fibroblasts.
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