Osteogenic differentiation of bone marrow mesenchymal stem cells on chitosan/gelatin scaffolds: gene expression profile and mechanical analysis

Abstract

Abstract In the present study we explore the extracellular matrix (ECM) produced by human bone marrow mesenchymal stem/stromal cells (BM-MSCs) induced to undergo osteogenic differentiation within porous chitosan/gelatin (CS:Gel) scaffolds by investigating their multiple gene expression profile and mechanical behavior. Initially, the efficiency of the BM-MSCs osteogenic differentiation within the constructs was confirmed by the significant rise in the expression of the osteogenesis associated genes DLX5, RUNX2, ALP and OSC. In line with these findings, OSC and Col1A1 protein expression was also detected in BM-MSCs on the CS:Gel scaffolds at day 14 of osteogenic differentiation. We then profiled, for the first time, the expression of 84 cell adhesion and ECM molecules using PCR arrays. The arrays, which were conducted at day 14 of osteogenic differentiation, demonstrated that 49 genes including collagens, integrins, laminins, ECM proteases, catenins, thrombospondins, ECM protease inhibitors and cell-cell adhesion molecules were differentially expressed in BM-MSCs seeded on scaffolds compared to tissue culture polystyrene control. Moreover, we performed dynamic mechanical analysis of the cell-loaded scaffolds on days 0, 7 and 14 to investigate the correlation between the biological results and the mechanical behavior of the constructs. Our data demonstrate a significant increase in the stiffness of the constructs with storage modulus values of 2 MPa on day 7, compared to 0.5 MPa on day 0, following a drop of the stiffness at 0.8 MPa on day 14, that may be attributed to the significant increase of specific ECM protease gene expression such as MMP1, MMP9, MMP11 and MMP16 at this time period.