Altered phenotypic gene expression of 10T1/2 mesenchymal cells in nonuniformly stretched PEGDA hydrogels

Abstract

Disease-related phenotype modulation of many cell types has been shown to be closely related to mechanical loading conditions; for example, vascular smooth muscle cell (SMC) phenotype shift from a mature, contractile state to a proliferative, synthetic state contributes to the formation of neointimal tissue during atherosclerosis and restenosis development and is related to SMC mechanical loading in vivo. The majority of past in vitro cell-stretching experiments have employed simplistic (uniform, uniaxial or biaxial) stretching environments to elucidate mechanobiological pathways involved in phenotypic shifts. However, the in vivo mechanics of the vascular wall consists of highly nonuniform stretch. Here we subjected 10T1/2 murine mesenchymal cells (an SMC precursor) to two- and three-dimensional nonuniform stretch environments. After 24 h of stretch, cells on an elastomeric membrane demonstrated varied proliferation [assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation] depending on location upon the membrane, with maximal proliferation occurring in a region of high, uniaxial stretch. Cells subjected to a nonuniform stretching regimen within three-dimensional polyethylene glycol diacrylate (PEGDA) hydrogel constructs demonstrated marked changes in mRNA expression of several phenotype-related proteins, indicating a sort of "hybrid" phenotype with contractile and synthetic markers being both upregulated and downregulated. Furthermore, expression levels of mRNAs were significantly different between various locations within the stretched gel. With the proliferation results, these data exhibit the capability of nonuniform stretching devices to induce heterogeneous cell responses, potentially indicative of spatial distributions of disease-related behaviors in vivo.