Inhibition of ERK promotes collagen gel compaction and fibrillogenesis to amplify the osteogenesis of human mesenchymal stem cells in three-dimensional collagen I culture

Tissue morphogenesis remains one of the least understood problems in cell and developmental biology. There is a disconnect between the mechanisms that apply to two-dimensional (2D) cultures and those seen in vivo. Three-dimensional (3D) culture presents a complex stimulus triggering cellular responses that are only partially understood. We compared 2D and 3D cultures of human mesenchymal stem cells in the presence of mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, to determine the role of extracellular signal-related kinase (ERK) in collagen-induced differentiation. 3D collagen I culture enhanced and accelerated the osteogenic differentiation of human mesenchymal stem cells (hMSC). Contrary to 2D results, the addition of PD98059 induced a significant amplification of osteogenic gene expression and matrix mineralization in 3D cultures. The inhibition of ERK altered cell-mediated compaction, proliferation, and resulted in the development of distinct tissue microstructure. Therefore, we suggest that the ability to reorganize collagen in 3D is an important step in ERK-mediated osteogenic differentiation. This work aims to propose a correlation between osteogenic differentiation and hMSC-directed collagen I remodeling. We present a potential mechanistic link (ERK) through which the three dimensionality of an engineered tissue acts to differentially induce and maintain cellular phenotype during tissue development.