916. Genetically Modified Mesenchymal Stem Cells Expressing Transforming Growth Factor-β1 Develop a Cartilage Phenotype

Abstract

INTRODUCTIONAcute cartilage injury and subsequent osteoarthritis are a debilitating and common syndrome in the aging population. Transplantation of MSCs to joints has been successfully used in small animal models. However, in extensive defects the application of MSCs improved defect filling with only fibrous tissue. Enhancing chondrogenesis of the MSCs by TGF-β1 gene transduction before transplantation may provide the impetus for better quality cartilaginous repair.MATERIALS AND METHODSAadenovirus vector encoding equine TGF-β1 (AdTGF-β1) was used to transfect MSCs. Monolayer cultures were transduced with AdTGF-β1, AdGFP, or no virus. Monolayers were pelleted after 24 hours, and 15 pellets harvested at 2, 7, 14, and 21 days. Chondrogenesis was determined by qPCR assessment of TGF-β1, aggrecan, coll IIA, and coll IIB gene expression. Medium was assayed for PG and TGF-β1. Confirmation of pellet chondrogenesis was made by HandE architecture, collagen II elaboration on immunohistochemistry, and toluidine blue PG staining.RESULTSPCR indicated AdTGFβ1 transfected MSCs expressed TGF-β1 RNA (Fig1Fig1), which induced markers of chondrogenic differentiationincluding aggrecan, and collagen types IIA and IIB (Figs 2Figs 2). Aggrecan expression was significantly enhanced on day 2. Immuno analysis detected collagen type II as early as 2 days after beginning aggregate cultures. This phenomenon continued through day 21, with intense collagen type II as the predominant collagen on day 21 (Fig 2Fig 2).Fig. 1View Large Image | View Hi-Res Image | Download PowerPoint SlideFig. 2View Large Image | View Hi-Res Image | Download PowerPoint SlideCollagen type I immuno analysis showed collagen type I deposition throughout all time periods, with increases under the influence of TGF-β1.DISCUSSIONSignificant increases in TGF-β1 expression were induced in AdTGF-β1 transduced MSC cells. Chondrogenic response was reflected in collagen type IIA and IIB expression which were significantly increased on day 7, 14 and 21. Longer term cultures showed a preponderance of collagen type IIB expression. Combined with morphologic evidence of strong chondrogenic condensation, these data provide molecular, histological and immunohistochemical evidence for substantial in vitro chondrogenic differentiation of equinebone-marrow derived MSCs. Based on these results AdTGF-β1 treated MSCs may be a robust source of chondrocytes for use in cartilage repair.