Analysis of IGF-1 stimulated mesenchymal stem cells from human amniotic fluid differentiation into chondrocytes, cultured in micromass and on three-dimensional matrix

Abstract

It is well known that articular chondral and osteochondral lesions present a very low capability to regenerate. Therefore, the current clinical management is symptomatic or invasive, progressing very often to arthroplasties. Hence, the use of mesenchymal stem cells (MSC) for reconstruction of articular cartilage leads to a promising therapeutic alternative. Our laboratory has observed the chondrorgenic potential from human amniotic fluid (HAF) MSC on TGF-²3 stimuli (data under peer review). Thus, the aim of this study is to investigate the chondrogenic potential of MSC from human amniotic fluid in Micromass system (high-density cell culture) with IGF-1 and in a 3D co-culture of those cells differentiated in scaffolds of alginate combined with chitosan and pluronic. The amniotic fluid will be provided by the fetal medicine unit from pregnant women who have formal indication to undergo to an amniocentesis. This study was submitted to local ethics committee. The micromass will be performing using MSC cultured in monolayer. The control group will be the chondrocytes from adult human normal cartilage (to compare the gene expression and protein production). The chondrogenic potential will be verified by specific articular cartilage gene expression (SOX-9, type II, type IX, type XI e Type X collagens, and aggrecan) analyzed by real time PCR compared to the expression in adult cartilage. The protein production of type II collagen will be observed by Western Blotting. The micromass and the scaffoldswill be hystologically analysed using cartilage specific dyes. (AU)