162
material. Also, stiffness is measured and reported differently throughout the literature, with some authors reporting elastic modulus and others choosing to measure
storage or complex moduli; these are related but they can be substantively different,
in the context of viscoelastic materials. Despite these irregularities, it is likely that
substrates optimized for endothelial proliferation and angiogenesis are much softer
than those optimized for cardiomyocyte maturation, as the reported stiffnesses of
angiogenic substrates ranged from under 100 Pa to about 6 kPa [2, 95, 107], while
cardiomyocyte differentiation and maturation are reported to thrive in matrices with
elastic moduli in the range of 10–50 kPa and have been shown to be actively suppressed on very soft (1–3 kPa) substrates [43, 71].
6.6.3 Alignment
Anisotropy and cellular alignment are prominent characteristics of cardiac physiology, and the alignment of cardiomyocytes is critical to normal electrophysiological
and contractile behavior. In comparing cultures of NRVCMs grown on a flat surface with random orientation to cultures grown on a grooved substrate with
increased alignment, Chung et al. found that the aligned cultures exhibited increased
conduction velocities [19]. Alignment of 3D NRVCM cultures was also shown to
increase the stimulated force of contraction [9]. Reproducing this organization has
been a major goal of the tissue engineering field, and cardiomyocyte alignment is
frequently used as a measurement of tissue maturity and predictor of functional
capacity.
This anisotropy is not limited to cardiomyocytes as it is also present in the vasculature of native myocardium, where blood vessels are highly aligned in nearparallel organization [54, 94]. There have been efforts to recapitulate this vessel-level
alignment to maximize biosimilarity, because, in theory, microvascular alignment
could improve nutrient delivery and promote the survival of cardiomyocytes in the
construct. An experiment in which pre-vascularized cardiac patches, formed with
either randomly oriented or uniaxially aligned vasculature, were grafted onto
infarcted myocardium in mice showed improved lumenal density and network
length among the aligned samples in vitro. It also showed that their in vivo effects
were indistinguishable, as they displayed similar total and perfused luminal densities, as well as similar improvements in scar tissue formation and functional outcomes [96]. These results raised questions regarding the importance of vascular
alignment to the function of engineered cardiac tissues. In another set of experiments, researchers combined separately generated layers of aligned microvessels
and aligned cardiomyocytes. They found that the combined patch exhibited much
greater force of contraction in vivo and significantly reduced scar formation in
mouse myocardial infarction models. Patches containing CMs only did not [101].
Without controlling for a patch with randomly oriented vasculature, the importance
of alignment is impossible to assess.
J. Morrissette-McAlmon et al.
material. Also, stiffness is measured and reported differently throughout the literature, with some authors reporting elastic modulus and others choosing to measure
storage or complex moduli; these are related but they can be substantively different,
in the context of viscoelastic materials. Despite these irregularities, it is likely that
substrates optimized for endothelial proliferation and angiogenesis are much softer
than those optimized for cardiomyocyte maturation, as the reported stiffnesses of
angiogenic substrates ranged from under 100 Pa to about 6 kPa [2, 95, 107], while
cardiomyocyte differentiation and maturation are reported to thrive in matrices with
elastic moduli in the range of 10–50 kPa and have been shown to be actively suppressed on very soft (1–3 kPa) substrates [43, 71].
6.6.3 Alignment
Anisotropy and cellular alignment are prominent characteristics of cardiac physiology, and the alignment of cardiomyocytes is critical to normal electrophysiological
and contractile behavior. In comparing cultures of NRVCMs grown on a flat surface with random orientation to cultures grown on a grooved substrate with
increased alignment, Chung et al. found that the aligned cultures exhibited increased
conduction velocities [19]. Alignment of 3D NRVCM cultures was also shown to
increase the stimulated force of contraction [9]. Reproducing this organization has
been a major goal of the tissue engineering field, and cardiomyocyte alignment is
frequently used as a measurement of tissue maturity and predictor of functional
capacity.
This anisotropy is not limited to cardiomyocytes as it is also present in the vasculature of native myocardium, where blood vessels are highly aligned in nearparallel organization [54, 94]. There have been efforts to recapitulate this vessel-level
alignment to maximize biosimilarity, because, in theory, microvascular alignment
could improve nutrient delivery and promote the survival of cardiomyocytes in the
construct. An experiment in which pre-vascularized cardiac patches, formed with
either randomly oriented or uniaxially aligned vasculature, were grafted onto
infarcted myocardium in mice showed improved lumenal density and network
length among the aligned samples in vitro. It also showed that their in vivo effects
were indistinguishable, as they displayed similar total and perfused luminal densities, as well as similar improvements in scar tissue formation and functional outcomes [96]. These results raised questions regarding the importance of vascular
alignment to the function of engineered cardiac tissues. In another set of experiments, researchers combined separately generated layers of aligned microvessels
and aligned cardiomyocytes. They found that the combined patch exhibited much
greater force of contraction in vivo and significantly reduced scar formation in
mouse myocardial infarction models. Patches containing CMs only did not [101].
Without controlling for a patch with randomly oriented vasculature, the importance
of alignment is impossible to assess.
J. Morrissette-McAlmon et al.