Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

Abstract

Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces

“… *Nanoscale stiffness of compressed collagen sheets determined by atomic force microscopy (AFM) was in part reported (Rothdiener et al, ).…”

“…Accordingly, the shapes of human bone‐marrow derived MSCs that adhered to different biomaterials with similar stiffnesses vs. similar biomaterial types with different stiffnesses were compared to determine whether specific baseline shapes could be generated by altering biomaterial substrate properties. For this purpose, compressed collagen sheets (Rothdiener et al, ) and uncoated and fibronectin‐coated silicone sheets were used. The MSC shape was described using a semi‐automated high‐throughput method for calculating quantitative shape descriptors.…”

Engineering the geometrical shape of mesenchymal stromal cells through defined cyclic stretch regimens

“…24. After overnight incubation at 4 °C, the samples were washed 3× with PBS, and incubated with secondary antibody (Goat anti-Rabbit IgG, sc-3739, Santa Cruz Biotechnology, dilution: 1:100) for 1 h at room temperature.…”

Oxygen and nutrient delivery in tissue engineering: Approaches to graft vascularization

“…Independent of their origin, these scaffolds are meant to provide a bioactive environment that may, to a certain extent, mimic the extracellular matrix (ECM), and thereby facilitate cell adhesion and activate signalling pathways for proliferation and (directed) differentiation. Cell-type specific responses due to the characteristics of the scaffold material, for example, substrate stiffness and bioactive groups, have been reported (Rothdiener et al, 2016;Seeger et al, 2015). Careful selection of such scaffolds is required in order to not interfere with vascularization in vitro.…”