Differential effects of substrate modulus on human vascular endothelial, smooth muscle, and fibroblastic cells

Abstract

Regenerative medicine approaches offer attractive alternatives to standard vascular reconstruction; however, the biomaterials to be used must have optimal biochemical and mechanical properties. To evaluate the effects of biomaterial properties on vascular cells, heparinized poly(ethylene glycol) (PEG)-based hydrogels of three different moduli, 13.7, 5.2, and 0.3 kPa, containing fibronectin and growth factor were utilized to support the growth of three human vascular cell types. The cell types exhibited differences in attachment, proliferation, and gene expression profiles associated with the hydrogel modulus. Human vascular smooth muscle cells demonstrated preferential attachment on the highest-modulus hydrogel, adventitial fibroblasts demonstrated preferential growth on the highest-modulus hydrogel, and human umbilical vein endothelial cells demonstrated preferential growth on the lowest-modulus hydrogel investigated. Our studies suggest that the growth of multiple vascular cell types can be supported by PEG hydrogels and that different populations can be controlled by altering the mechanical properties of biomaterials.

Figure 1

Schematic of hydrogel formation.

Figure 2

Oscillatory rheology results illustrating the formation kinetics of hydrogels prepared with star PEGs. (A) Modulus (log scale) is given as a function of gelation time for 8wt% (●), 4wt% (■), 3wt% (▲) gels represented with closed symbols for storage modulus (G’) and open for loss modulus (G”). (B) The final moduli of the 8 wt%, 4 wt%, and 3 wt% hydrogels were determined to be 13.7 kPa, 5.2 kPa, and 0.3 kPa, respectively. Plots are an average of three repeats with standard deviation shown by error bars.

Figure 3

Cell morphology of AoAF (A), HUVEC (B), and T/G HA-vSMC (C) was assessed on TCPS and on 13.7 kPa, 5.2 kPa, and 0.3 kPa PEG-based hydrogels.

Figure 4

Cell Titer Blue results for NIH3T3, AoAF, HUVEC, and T/G HA-vSMCs grown on heparinized PEG-based hydrogels containing fibronectin and growth factor. Mean fluorescence units (+ std dev; n = 4) are given to indicate number of viable cells attached during the first two hours of culture (A) and the change in cell number over time (B). Asterisks indicate statistically significant differences by One Way ANOVA (p<0.01).

Figure 5

Hierarchical cluster analysis of gene expression data. Genes whose expression was significantly different on hydrogels (low modulus = 0.3 kPa, medium modulus = 5.2 kPa, high modulus = 13.7 kPa) versus TCPS are shown for AoAF (A), HUVEC (B), and T/G HA-vSMCs (C) cells in heat maps. Columns represent each sample and rows represent each gene. Red and green in cells reflect high and low expression levels, respectively, as indicated in the scale bar (log2-transformed scale).