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.

The many ways adherent cells respond to applied stretch

“…Like other synthetic polymers, PEG hydrogels are inert to matrix proteases, lack a fibrillar structure, and also have sub-micron pores. Consequently, encapsulated cells are confined by the surrounding polymer, which does not allow cell alignment or elongation in response to strain (Richardson et al 2013). We encapsulated U2OS osteosarcoma cells within collagen microspheres suspended within PEGDA hydrogel and investigated the morphological changes to the cells when the composite scaffold was subjected to cyclic uniaxial strain ( Figure 2).…”

Potential strain-dependent mechanisms defining matrix alignment in healing tendons

“…Third, while we simulated the effect of strain-dependent deposition, we assumed this cell response was a function of global strain and did not depend on each cell’s local orientation. Fourth, cell culture experiments have shown that mechanical loading can also modify cell proliferation (Sawaguchi et al 2010; Richardson et al 2013; Sun et al 2016). We did not include strain-induced proliferation in our model as past measurements in the supraspinatus tendon showed subtle decreases in cell density with increased levels of loading (Thomopoulos et al 2003; Galatz et al 2009).…”