157
cells were positive for CD31, the development of cord-like structures was not uniformly seen throughout the constructs. Recent studies have also investigated the
impact of using hASCs in lieu of human dermal fibroblasts, together with NRVCMs
and HUVECs. ASCs have been found to be beneficial in ischemic settings like myocardial infarction. Studies that have injected hASCs into rats and pigs have found
paracrine secretion of multiple growth factors. Echocardiograms revealed a decrease
in infarct size, an increase in wall thickness, a decrease in end-systolic and end-diastolic volumes, an increase in left ventricle ejection fraction, and a left ventricle fractional shortening [102]. Therefore, within a monolayer in vitro system, NRVCM-hASC
and hASC-HUVEC co-cultures were first used to determine the appropriate tri-culture
ratios. In the tri-culture system, an NRVCM-hASC-HUVEC ratio of 10:2:1 resulted
in vessel development of 70 mm per 3240 mm
2
with an average of 454 junctions. The
electrophysiological properties were reported as 20 ± 2 cm/s; APD 80 and APD 30 of
122 ± 5 ms and 59 ± 4 ms, respectively; and maximum capture rate of 7.4 ± 0.6 Hz
[82]. From these studies, it could be seen that sequential seeding allowed cardiomyocytes to develop cell-cell connections and allow for improved electrophysiological
outcomes for cardiomyocytes. Although this allowed for the development of close
cell-cell connections, the cultures were 2D in nature.
Zhang et al. developed the AngioChip that enabled the formation of 3D tissues
with spatial and temporal control while seeding different cell populations (Fig. 6.3).
AngioChips were made from the micropatterned biodegradable elastomer
poly(octamethylene maleate (anhydride) citrate) (POMaC), and cells were encapsulated into collagen hydrogels. The tri-culture system was developed from NRVCMs
or hESC-CMs, hMSCs, and HUVECs. Uniquely, these devices allowed for the
development of channels to accommodate medium perfusion and to provide adequate nutrient transfer to cardiomyocytes. After optical mapping the resulting conduction velocity was 4.8 cm/s. In this system the channels were coated with vascular
cells to develop a channel for nutrient perfusion and ideal integration when implanted
in vivo. The AngioChip was surgically implanted and demonstrated “artery-toartery” anastomosis with host vasculature [138]. Employing similar principles for
spatial control, Gershlak et al. developed a decellularized spinach leaf system,
which served as an attractive biomaterial for cardiac tissue engineering. The native
venous architecture of the leaves provided intrinsic vasculature networks. The spinach leaf was reseeded with a tri-culture system consisting of hESC-CMs, hMSCs,
and HUVECs. After 10 days of culture, the tri-culture system resulted in 10% contractile strain. Contractile strength peaked at day 7 and was maintained until day 17.
After day 17, there was a decrease in contractile strength. The endothelial cells
(HUVECs) were reseeded, through the cannula, and appeared to line the spinach
leaf to form vessel like structures within the inherent plant structure [37].
Another promising strategy used cell clusters rather than individual cell suspensions (Fig. 6.3). Stevens et al. developed spheroid/cell cluster tri-culture systems
which were seeded with hESC-CMs, hDF, and HUVECs. In this system, there was
increased force of contraction over time, as well as increased force-length relationships in the presence of all three cell types. In addition, these clusters developed a
similar percentage of contraction in the patches as the control group, up to 3 Hz
6 Strategies for Tissue Engineering Vascularized Cardiac Patches to Treat Myocardial…
cells were positive for CD31, the development of cord-like structures was not uniformly seen throughout the constructs. Recent studies have also investigated the
impact of using hASCs in lieu of human dermal fibroblasts, together with NRVCMs
and HUVECs. ASCs have been found to be beneficial in ischemic settings like myocardial infarction. Studies that have injected hASCs into rats and pigs have found
paracrine secretion of multiple growth factors. Echocardiograms revealed a decrease
in infarct size, an increase in wall thickness, a decrease in end-systolic and end-diastolic volumes, an increase in left ventricle ejection fraction, and a left ventricle fractional shortening [102]. Therefore, within a monolayer in vitro system, NRVCM-hASC
and hASC-HUVEC co-cultures were first used to determine the appropriate tri-culture
ratios. In the tri-culture system, an NRVCM-hASC-HUVEC ratio of 10:2:1 resulted
in vessel development of 70 mm per 3240 mm
2
with an average of 454 junctions. The
electrophysiological properties were reported as 20 ± 2 cm/s; APD 80 and APD 30 of
122 ± 5 ms and 59 ± 4 ms, respectively; and maximum capture rate of 7.4 ± 0.6 Hz
[82]. From these studies, it could be seen that sequential seeding allowed cardiomyocytes to develop cell-cell connections and allow for improved electrophysiological
outcomes for cardiomyocytes. Although this allowed for the development of close
cell-cell connections, the cultures were 2D in nature.
Zhang et al. developed the AngioChip that enabled the formation of 3D tissues
with spatial and temporal control while seeding different cell populations (Fig. 6.3).
AngioChips were made from the micropatterned biodegradable elastomer
poly(octamethylene maleate (anhydride) citrate) (POMaC), and cells were encapsulated into collagen hydrogels. The tri-culture system was developed from NRVCMs
or hESC-CMs, hMSCs, and HUVECs. Uniquely, these devices allowed for the
development of channels to accommodate medium perfusion and to provide adequate nutrient transfer to cardiomyocytes. After optical mapping the resulting conduction velocity was 4.8 cm/s. In this system the channels were coated with vascular
cells to develop a channel for nutrient perfusion and ideal integration when implanted
in vivo. The AngioChip was surgically implanted and demonstrated “artery-toartery” anastomosis with host vasculature [138]. Employing similar principles for
spatial control, Gershlak et al. developed a decellularized spinach leaf system,
which served as an attractive biomaterial for cardiac tissue engineering. The native
venous architecture of the leaves provided intrinsic vasculature networks. The spinach leaf was reseeded with a tri-culture system consisting of hESC-CMs, hMSCs,
and HUVECs. After 10 days of culture, the tri-culture system resulted in 10% contractile strain. Contractile strength peaked at day 7 and was maintained until day 17.
After day 17, there was a decrease in contractile strength. The endothelial cells
(HUVECs) were reseeded, through the cannula, and appeared to line the spinach
leaf to form vessel like structures within the inherent plant structure [37].
Another promising strategy used cell clusters rather than individual cell suspensions (Fig. 6.3). Stevens et al. developed spheroid/cell cluster tri-culture systems
which were seeded with hESC-CMs, hDF, and HUVECs. In this system, there was
increased force of contraction over time, as well as increased force-length relationships in the presence of all three cell types. In addition, these clusters developed a
similar percentage of contraction in the patches as the control group, up to 3 Hz
6 Strategies for Tissue Engineering Vascularized Cardiac Patches to Treat Myocardial…