161
6.6.2 Effect of Substrate Stiffness, Guidance, and Alignment
Cues
Of the many characteristics of the extracellular matrix, mechanical stiffness has
been identified as particularly important to cellular differentiation and behavior.
The role of matrix stiffness in supporting cardiomyocytes is poorly understood, and
a wide range of elasticities has been reported as optimal for their culture. In one of
the earliest studies on this topic, Jacot et al. reported that NRVCMs grown on
10 kPa polyacrylamide (PA) displayed improved markers of functional maturity,
such as contractile force generation and sarcoplasmic calcium stores [53]. Similarly,
Engler and colleagues found that embryonic cardiomyocytes isolated from rats and
seeded on collagen-coated polyacrylamide matrices displayed much greater functional maturity on matrices with elastic moduli in the range of normal rat myocardium (~11 kPa), as opposed to stiffer matrices falling in the range of infarcted
myocardium (~34 kPa) [28]. However, shortly thereafter, it was reported that
NRVCMs cultured on very soft, 3 kPa PA scaffolds developed more electrophysiologically mature phenotypes but that other measures of functional maturity, such
as cellular elongation and contractile force generation, were optimized on stiffer
50 kPa scaffolds [8]. The question of optimal substrate stiffness is further complicated by evidence that cardiomyocytes require different levels of substrate stiffness
at different stages of differentiation. In 2010, Young and Engler found that a hyaluronic acid matrix, engineered to stiffen over time, increased expression of maturity markers in NRVCMs nearly threefold over standard scaffolds [136]. Another
recent report suggested that CMs derived from iPSCs display improved initial differentiation, on protein-coated tissue culture plastic (elastic modulus ~2 GPa), but
that they developed more mature beating behavior and contractile function on
20 kPa PA scaffolds [43].
Stiffness is also an important consideration in vascularization, as evidenced by
experiments showing that angiogenesis in hydrogels seeded with HUVECs is
strongly affected by the elastic modulus of the hydrogel. In a key report, Sieminski
and colleagues showed that the softer collagen gels (Young’s modulus = 6 kPa)
developed more robust vascular networks than the stiffer gels (10 kPa) they tested
[107]. This report also suggested that the relative magnitudes of the matrix stiffness,
and the force of endothelial cell traction, regulate angiogenesis. They found that
human blood outgrowth endothelial cells (HBOEC), which exert more traction
force than HUVECs, formed thin, branched vasculature with thick walls in floating
10 kPa gels, while HUVECs formed vasculature with thin walls and larger lumens.
However, if the stiffness was decreased to 6 kPa, both cell types formed narrow,
thick-walled structures, while, if the apparent stiffness was increased by mechanically restraining the gel, both cell types formed thin-walled vasculature with large
lumens.
Determining the ideal elasticity for vascularization of cardiac tissues is further
complicated by inconsistencies in the literature. Wide varieties of matrix materials
have been utilized, but it is very possible that optimal stiffness is different for each
6 Strategies for Tissue Engineering Vascularized Cardiac Patches to Treat Myocardial…
6.6.2 Effect of Substrate Stiffness, Guidance, and Alignment
Cues
Of the many characteristics of the extracellular matrix, mechanical stiffness has
been identified as particularly important to cellular differentiation and behavior.
The role of matrix stiffness in supporting cardiomyocytes is poorly understood, and
a wide range of elasticities has been reported as optimal for their culture. In one of
the earliest studies on this topic, Jacot et al. reported that NRVCMs grown on
10 kPa polyacrylamide (PA) displayed improved markers of functional maturity,
such as contractile force generation and sarcoplasmic calcium stores [53]. Similarly,
Engler and colleagues found that embryonic cardiomyocytes isolated from rats and
seeded on collagen-coated polyacrylamide matrices displayed much greater functional maturity on matrices with elastic moduli in the range of normal rat myocardium (~11 kPa), as opposed to stiffer matrices falling in the range of infarcted
myocardium (~34 kPa) [28]. However, shortly thereafter, it was reported that
NRVCMs cultured on very soft, 3 kPa PA scaffolds developed more electrophysiologically mature phenotypes but that other measures of functional maturity, such
as cellular elongation and contractile force generation, were optimized on stiffer
50 kPa scaffolds [8]. The question of optimal substrate stiffness is further complicated by evidence that cardiomyocytes require different levels of substrate stiffness
at different stages of differentiation. In 2010, Young and Engler found that a hyaluronic acid matrix, engineered to stiffen over time, increased expression of maturity markers in NRVCMs nearly threefold over standard scaffolds [136]. Another
recent report suggested that CMs derived from iPSCs display improved initial differentiation, on protein-coated tissue culture plastic (elastic modulus ~2 GPa), but
that they developed more mature beating behavior and contractile function on
20 kPa PA scaffolds [43].
Stiffness is also an important consideration in vascularization, as evidenced by
experiments showing that angiogenesis in hydrogels seeded with HUVECs is
strongly affected by the elastic modulus of the hydrogel. In a key report, Sieminski
and colleagues showed that the softer collagen gels (Young’s modulus = 6 kPa)
developed more robust vascular networks than the stiffer gels (10 kPa) they tested
[107]. This report also suggested that the relative magnitudes of the matrix stiffness,
and the force of endothelial cell traction, regulate angiogenesis. They found that
human blood outgrowth endothelial cells (HBOEC), which exert more traction
force than HUVECs, formed thin, branched vasculature with thick walls in floating
10 kPa gels, while HUVECs formed vasculature with thin walls and larger lumens.
However, if the stiffness was decreased to 6 kPa, both cell types formed narrow,
thick-walled structures, while, if the apparent stiffness was increased by mechanically restraining the gel, both cell types formed thin-walled vasculature with large
lumens.
Determining the ideal elasticity for vascularization of cardiac tissues is further
complicated by inconsistencies in the literature. Wide varieties of matrix materials
have been utilized, but it is very possible that optimal stiffness is different for each
6 Strategies for Tissue Engineering Vascularized Cardiac Patches to Treat Myocardial…