Induction of chondrogenesis using human fibroblast-derived matrix and placenta-derived mesenchymal stem cells
- Induction of chondrogenesis using human fibroblast-derived matrix and placenta-derived mesenchymal stem cells
- 노용권; 김인걸; 두 핑; 하철원; 박귀덕
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- Formation of mature and functional articular cartilage is still challenging in cartilage tissue engineering. This study focuses on a chondrogenic potential of human placenta-derived mesenchymal stem cells (hPMSCs) combined with decellularied extracellular matrix. Human fibroblast-derived matrices (hFDM) were naturally obtained from in vitro-cultured human lung fibroblasts via a mild decellularization condition using detergents and enzymes. hFDM was then conjugated with heparin via EDC chemistry and hFDM-hep was immobilized with a transforming growth factor (TGF)-beta1. Toluidine blue O assay and Fourier transform infrared spectroscopy identified the heparin moieties on hFDM. Heparin-conjugated hFDM (hFDM-hep) and hPMSCs were encapsulated in collagen gel and cultured under chondrogenic medium for 4 weeks. Meanwhile, PKH26-labeled hPMSCs in 2% collagen gel were pre-conditioned in a chondrogenic media for 3 days and subsequently implanted in the back of nude mice for 4 weeks.
Collagen gel embedded with hFDM-hep and TGF-beta1 exhibited a sustained release of growth factor for 28 days in vitro. Chondrogenesis of hPMSCs is supported by accumulated GAG content and upregulated chondrogenic specific marker (Col II, aggrecan, SOX9) expression. In addition, combination of hPMSCs and hFDM-hep-TGF-beta1 within the collagen constructs showed a sign of chondrogenic phenotype via immunofluorescence of Col II and retained quite a few signals of PKH-26 positive hPMSCs. In histological examination, Safranin O staining was positive but von kossa staining was negative. These results suggest that TGF-beta1 immobilized hFDM can provide an appropriate microenvironment for chondrogenic differentiation of hPMSCs.
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