Abstract
BackgroundMesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs). As an alternative, this study investigates the chondrogenic induction of MSCs via adenoviral gene-delivery of the transcription factor SOX9 alone or in combination with other inducers, and comparatively explores the levels of hypertrophy and end stage differentiation in a pellet culture system in vitro.MethodsFirst generation adenoviral vectors encoding SOX9, TGFB1 or IGF1 were used alone or in combination to transduce human bone marrow-derived MSCs at 5 × 102 infectious particles/cell. Thereafter cells were placed in aggregates and maintained for three weeks in chondrogenic medium. Transgene expression was determined at the protein level (ELISA/Western blot), and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.ResultsSOX9 cDNA was superior to that encoding TGFB1, the typical gold standard, as an inducer of chondrogenesis in primary MSCs as evidenced by improved lacuna formation, proteoglycan and collagen type II staining, increased levels of GAG synthesis, and expression of mRNAs associated with chondrogenesis. Moreover, SOX9 modified aggregates showed a markedly lower tendency to progress towards hypertrophy, as judged by expression of the hypertrophy markers alkaline phosphatase, and collagen type X at the mRNA and protein levels.ConclusionAdenoviral SOX9 gene transfer induces chondrogenic differentiation of human primary MSCs in pellet culture more effectively than TGFB1 gene transfer with lower levels of chondrocyte hypertrophy after 3 weeks of in vitro culture. Such technology might enable the formation of more stable hyaline cartilage repair tissues in vivo.
Highlights
Mesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs)
The aim of the present study was to explore the effects of adenoviral delivery of SRY - box 9 (SOX9) compared to TGFB1 alone (TGFB1), or IGF1 alone (IGF1) cDNA or combinations thereof, on chondrogenesis of primary MSCs and to investigate whether the levels and extent of chondrogenic hypertrophy are influenced by the choice of the transgene in an established in vitro aggregate culture model
Transgene expression by genetically modified MSCs in pellet culture Following transduction with Adenoviral vector (Ad).green fluorescent protein (GFP) or Ad.SOX9 vector and placement into pellet culture, expression of the GFP or sex-determining region Y-type high-mobility-group-box (SOX)/GFP transgene was observed by fluorescence microscopy
Summary
Mesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs). Chondrogenic hypertrophy of adult MSCs represents the stage of terminal chondrocyte differentiation and is undesired in cartilage regenerative approaches as apoptosis follows and formation of abnormal ECM components and mineralization of the tissue can occur [2]. This resembles processes within the growth plate, where the interplay of several factors, including BMPs, IHH and others, mediates the replacement of cartilage by bone via endochondral ossification. This observation corresponds to the related in vivo data, where MSCs and BMP2 induced tissue hypertrophy and osteophyte formation, when transplanted at orthotopic [11, 12] or ectopic [13, 14] sites
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