Extracellular macrostructure anisotropy improves cardiac tissue-like construct function and phenotypic cellular maturation

Regenerative cardiac tissue is a promising field of study with translational potential as a therapeutic option for myocardial repair after injury, however, poor electrical and contractile function has limited translational utility. Emerging research suggests scaffolds that recapitulate the structure of the native myocardium improve physiological function. Engineered cardiac constructs with anisotropic extracellular architecture demonstrate improved tissue contractility, signaling synchronicity, and cellular organization when compared to constructs with reduced architectural order. The complexity of scaffold fabrication, however, limits isolated variation of individual structural and mechanical characteristics. Thus, the isolated impact of scaffold macroarchitecture on tissue function is poorly understood. Here, we produce isotropic and aligned collagen scaffolds seeded with embryonic stem cell derived cardiomyocytes (hESC-CM) while conserving all confounding physio-mechanical features to independently assess the effects of macroarchitecture on tissue function. We quantified spatiotemporal tissue function through calcium signaling and contractile strain. We further examined intercellular organization and intracellular development. Aligned tissue constructs facilitated improved signaling synchronicity and directional contractility as well as dictated uniform cellular alignment. Cells on aligned constructs also displayed phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macrostructure on tissue function and inform the design of optimized cardiac tissue for regenerative and model medical systems.


Introduction
Mature cardiac tissue has a limited capacity for autologous regeneration.Therefore, regenerative medicine therapies have focused on the development of engineered tissue to repair or replace damaged myocardium.Regenerative cardiac patches, which are usually seeded with stem cell derived cardiomyocytes, have emerged as a promising solution because they encourage ventricular wall thickening, reduce cardiac wall stresses, and improve ventricular function thereby circumventing many of the limitations of purely cell-based studies including poor engraftment, survival, and erosion [1][2][3][4][5][6][7][8][9].The introduction of 3-D biomaterial scaffolds to cardiac tissue engineering has elucidated the predominant role of the ECM, not only as a passive architectural element but as a critical modulator of tissue morphology and behavior [8,[10][11][12].
The functional advantages of scaffold anisotropy in cardiac tissue engineering have been attributed to microarchitectural features as most studies have focused on microenvironment variation.Through techniques such as micro-patterning or electrospun fiber arrangement, studies have shown a significant impact on tissue behavior and cellular maturation based on nano-scale features [10][11][12][22][23][24].Anisotropic microstructures have been shown to enhance conduction velocity, signaling synchronicity, and cellular proliferation [24][25][26].The extracellular microenvironment on a nm scale has been shown to directly influence cardiomyocyte size and shape as well as the intracellular organization of contractile machinery [22,23].For example, the use of micropatterning was shown to coordinate the orientation of multiple cells, and thus intercellular sarcomere orientation, resulting in enhanced calcium handling and contractility [20].Li et al. further demonstrated that hiPSC-derived cardiomyocytes seeded onto aligned PLGA microfilaments (500-2000 nm Ø) demonstrated improved electrical signal propagation and improved electrical engraftment on in vivo rat ventricles [12].Similarly, Macqueen et al. constructed miniaturized ventricles with hiPSC cardiomyocytes on PCL/gelatin nanofibers oriented concentrically [27].These constructs displayed synchronous directional calcium signal propagation as well as a coordinated cyclic contraction pattern.Furthermore, disruption of the construct architecture via hole punch resulted in global signaling dysfunction [27].
Recent studies have incorporated macrostructural anisotropy into 3-D engineered cardiac tissue in order to replicate the helicoid macrostructure of the native myocardium and enhance gross tissue function on a millimeter scale [8,15,28,29].Gonnerman et al. used ice-templated collagen-glycosaminoglycan scaffolds, where isotropic and aligned structures were constructed using different freezing protocols resulting in conditions with differing pore sizes, microporosity, cross-bridging density, and strut wall thickness [8].While there was improved cellular alignment, spontaneous contraction, and phenotype specific gene expression on aligned constructs, these findings were confounded by variable microenvironments [8].Similarly, Rao et al., showed that fibronectin coated polydimethylsiloxane scaffolds with parallel grooves (10 μm wide, 4 μm deep) increased cellular alignment, sarcomere organization, and improved Ca 2+ cycling in induced pluripotent stem cellderived cardiomyocytes compared to non-grooved controls [29].Associated modifications in gene expression were not observed.While this study did conserve mechanical properties between conditions, the microenvironment was not conserved.Additionally, cells were seeded as a single layer on the structure, limiting the assessment of a threedimensional environment.Fleischer et al. seeded cardiomyocytes onto a modular assembly of grooved (~100 μm wide) amorphous electrospun albumen scaffolds.These structures facilitated cellular self-assembly into aligned elongated bundles and further demonstrated directional spatiotemporal calcium transience parallel to the scaffold grooves, however, no isotropic macroarchitectural comparison was described [15].These studies have shown that both extracellular micro-and macroarchitecture effectively modulate engineered cardiac tissue function.However, due to the technical complexity of scaffold fabrication, and the innate interdependence between micro-and macrostructure, researchers have not isolated the functional effects of 3-D macroarchitectural organization in engineered cardiac tissue [8,15,29].
In this work, we aim to isolate the functional and phenotypic effects of macroarchitectural order on engineered cardiac tissue.We used a 3-D ice-templated collagen scaffold with unidirectional pore alignment and leveraged the inherent planar asymmetry to produce thin patches that were dominated by either isotropic or anisotropic pore macrostructures.The microenvironment that comprises ice-templated collagen scaffolds has been shown to encourage cell migration and attachment via both the inherent binding motifs present on the collagen macromolecule as well as the mechanical surface texture and microporosity obtained through the ice-templating process [30][31][32][33][34][35][36][37][38].The use of a single parent structure ensures that all confounding microstructural and physio-mechanical features, such as strut wall thickness, permeability, and surface roughness are conserved across conditions.Isotropic and anisotropic scaffolds seeded with human embryonic stem cell derived cardiomyocytes (hESCs-CM) were assessed for hierarchical tissue function at multiple length scales.We compared global tissue function via contractile strain dynamics, tissue deformation, and spatiotemporal calcium signal transience.At a cellular level, cell orientation, sarcomere morphology, and early gap junction development were also compared.Finally, phenotypic maturation was assessed via molecular analysis of cardiac genetic biomarkers.

Collagen slurry preparation
A 1 w.t.% suspension of insoluble type I bovine dermal collagen (Devro) was prepared in 0.05 M acetic acid solution (Sigma-Aldrich UK).The mixture was left at 4 • C to swell for 24 h and homogenized in a blender at 22,000 rpms for 6 min.Gas was removed from the solution using a vacuum chamber (VirTis SP Scientific Wizard 2.0); the pressure was ramped from 750 torr to 2000 mtorr in 10 min.The slurry was allowed to habituate to room temperature (25 • C).

Directional ice-templating
Collagen slurry (9 ml) was loaded into a cylindrical polycarbonate mold (30 mm height, 20 mm internal diameter, 40 mm external diameter) with a copper base (2 mm thickness).The mold was placed onto a PID temperature controlled cold finger cooled with liquid nitrogen and programmed to hold at − 10 • C for 1 min followed by cooling at a rate of 0.2 • C min − 1 .The top of the mold was exposed to the ambient environment.
After solidification, scaffolds were dried in a freeze drier (VirTis SP Scientific Wizard 2.0) at 0 • C under a vacuum of less than 100 mtorr for 20 h.

Cross linking
Cross-linking was carried out using a ratio of 5:2:20 EDC:NHS:COOH groups in collagen to cross link at 5 % of the standard (5:2:1) [39,40].Cross-linking reagent quantities were determined according to scaffold weight.Reagents were dissolved in 95 % ethanol and scaffolds were soaked for 2 h.Scaffolds were washed (5 × 5 min) with deionized water.
After cross linking, scaffolds were freeze dried (VirTis SP Scientific Wizard 2.0) with a cooling rate of 0.2 • C min − 1 to a primary freezing temperature of − 20 • C. Drying occurred at 0 • C under a vacuum of less than 100 mtorr for 20 h.

Scaffold slicing
Scaffolds were punched with an 8 mm biopsy punch and sliced with a straight razor to a thickness of 500-700 μm.Aligned structures were cut such that the circular face of the scaffold was parallel to the longitudinal plane of structural alignment.Isotropic scaffolds were cut such that the circular face of the scaffold was parallel to the transverse plane of structural alignment as shown in Fig. 1a.

Scaffold imaging
Scanning electron microscopy (SEM) micrographs were taken of scaffolds prior to cross linking.Collagen scaffolds were sputter coated with gold for 2 min at a current of 20 mA.All micrographs were taken using a JEOL 820 SEM, with a tungsten source, operated at 10 kV.
X-ray micro-computed tomography (μCT) images (Skyscan 1172) were taken of each scaffold with a voltage of 25 kV, current of 138 mA, and a pixel size of 5.46 mm.Reconstructions of μCT images were performed with NRecon software by Skyscan.

Scaffold analysis
Reconstructions were divided into nine volumes of interest (2.5 × 2.5 × 6.5 mm 3 ) dispersed across the bottom, middle, and top of the structure.Pore size analysis was applied to each transverse slice within the regions of interest.ImageJ software was used to binarize and watershed transverse slices and particle analysis was employed to compile pore size data [41].The pore sizes were analyzed and visualized in MATLAB R2020a.
Fast Fourier Transform analysis was used to assess pore alignment according to the method laid out by Ayres et al. [42].2-D fast Fourier transform (FFT) analysis was performed, and radial sums of the resultant transform were collected in ImageJ.Pixel intensity for each radial direction was normalized to a minimum value of 0 and plotted in MATLAB R2020a.The order parameter for alignment was termed S FFT and utilized to compare between samples.

Cardiac cell selection
Differentiated cardiac cells were metabolically selected via lactate selection.Media was removed from beating cardiomyocytes on day 14.The wells were washed with PBS and TryPLE (Life Technologies) (500 ml per well in a 12 well plate) was added.Plates were incubated at 37 • C for 8-12 min until dissociated.CDM BSA and DNase (DNase I Solution (1 mg ml − 1 ) cat. 7900 Stemcell Technologies) diluted to 1:500 stock (1 mg ml − 1 ) was added, 1 ml per well.Cells were collected in a falcon tube and centrifuged (3 min at 1200 rpm).Cells were resuspended in CDM BSA to a concentration of 1 × 10 6 cell ml − 1 .Rock inhibitor (Y-27632 cat.11573560 Millipore) (1:1000) was added.Cells were plated in a 6-well plate (2 × 10 6 cells well − 1 ) and incubated at 37 • C for 8-12 h.Media was removed from wells and lactate media (DMEM without Glucose/pyruvate with non-essential amino acid (cat.554084Gibco) (1:100 from stock solution) and Sodium lactate (cat.L7022-10G SIGMA) (1:250 from 1 M stock, 4 mM final concentration) was added (2 ml well − 1 ).Cells were incubated in lactate media for 96 h, media was refreshed after 48 h.
The lactate selected hESC-derived cardiomyocytes were pelleted via centrifugation and resuspended in Fixation/Solubilization solution (BD Cytofix/Cytoperm Fixation/Permeabilization Kit, Biosciences) for 20 min at 4 • C. Cells were then pelleted by centrifugation and resuspended in 1× BD Perm/Wash Buffer containing anti-Cardiac Troponin-T APC antibody or Isotype control (Miltenyi Biotech) and incubated for 2 h at • C. Cells were washed three times in 1× BD Perm/Wash Buffer and then resuspended in phosphate buffered saline containing 0.1 % BSA and 2 mM EDTA.Data was acquired on BD LSRFortessa™ Flow Cytometer and analyzed with FlowJo™ v9.

Scaffold cellularization
Scaffolds described in Section 2.1.4were sterilized in 70 % EtOH for 30 min.The EtOH was removed by PBS washing 3 × 5 min prior to scaffold conditioning with cell culture media (CDM BSA) for 1 h in preparation for cell seeding.Cardiomyocytes were dissociated using TrypLE (Life Technologies) and seeded at a density of 2 × 10 6 cells per scaffold in CDM BSA supplemented with ROCK inhibitor 1 μM.

Analysis of construct performance 2.7.1. Viability
PrestoBlue Cell Viability Reagent (Thermo Scientific) was added to culture media according to the manufacturer's instructions after 7 days of culture.Cells were incubated with the dye for 4 h.Media was then sampled and fluorescence at 560 nm was analyzed using VICTOR Multilabel Plate Reader (Perkin Elmer).Media containing PrestoBlue incubated in empty wells was used as background control.

Strain analysis
Bright field videos were recorded on an Axiovert inverted microscope (Zeiss) using a Sony LEGRIA camera.Strain analysis of bright field video samples was performed via Ncorr digital image correlation software run on MatlabR2020a.The scaffold structure under bright field provided a reliable speckle pattern with sufficient contrast for analysis.A subset radius of 30 pixels and spacing of 5 pixels was used with the high strain option enabled.The reference image was redefined while the scaffold was relaxed after each beat to avoid error due to global translation.Principal strain calculations were performed with Matla-bR2020a.Principal angle characterization was performed using circular analysis of diametrically bimodal circular distributions [44].

Calcium dynamic analysis
On day 7 after seeding, Fluo-4 AM (10 μg ml − 1 , Life Technologies) was added to the cell culture media for 30 min at 37 • C. Scaffolds were then transferred in Tyrode's buffer and videos were recorded either with no stimulation or while pacing at frequencies of 1 and 1.5 Hz using c-PACE EM pace (IONOPTIX).Videos were recorded on an Axiovert inverted microscope (Zeiss) using a Sony LEGRIA camera.
Video analysis was performed in MatlabR2020a.Fluorescence intensity was normalized and mean intensity was plotted against time.Both intensity peak frequencies and Fast Fourier Transform analysis were used to calculate the pulse rate.For samples that did not exhibit spatial deformation during calcium fluorescence, pulse rates were also calculated for each pixel to indicate global signaling uniformity.Individual pulse times were recorded for each pixel and the temporal signaling uniformity in space was visualized through isochrones in MatlabR2020a.

Immunocytochemistry
Cell-seeded constructs were washed once in PBS and then fixed for 1 h with 4 % PFA.The cells were subsequently permeabilized with 0.1 % Triton (Sigma), and 0.5 % BSA (Sigma) in PBS for 15 min before blocking with 3 % BSA (Sigma) in PBS for 1 h.Incubation with primary antibody (diluted accordingly) was then performed.Constructs were then washed in PBS and incubated overnight with the appropriate secondary antibody, or phalloidin where appropriate.Constructs were then washed and stained with DAPI (Sigma, 1 μg ml − 1 ) for 1 h prior to imaging.Micrographs were obtained using an SP-5 confocal microscope (LEICA).Primary (I) and secondary (II) antibodies are listed in Table 1.

Cell density
Dapi stained nuclei were counted with particle analysis in ImageJ.
The cell density for 200 μm 2 regions of interest was calculated in Mat-labR2020a for each scaffold.

Cellular alignment
F-actin staining was used to characterize cellular spreading and cytoskeletal alignment.The F-actin orientation and coherence of cardiomyocytes after 7 days of culture were measured for 50 μm 2 sections (27 measurements were taken per scaffold) with the OrientationJ plugin for ImageJ.The intra-scaffold orientation variance was calculated for each individual scaffold.

Sarcomere development
Representative sarcomere chains were isolated (N = 6 per construct) from confocal images showing α-actinin.Sampled regions of interest were evenly distributed across a 6000 μm 2 surface sample of each construct.Sample images of isolated chains were cropped such that the sarcomere band spanned the height of the image.Banding intensity was characterized [45,46].Fluorescence intensity was normalized by the minimum fluorescence (f 0 ) such that, The mean fluorescence intensity signal was plotted along the length of the sarcomere chain and the relative prominence of each intensity peak was measured in MatLabR2020a to calculate sarcomere intensity.Sarcomere width was defined as the signal wavelength.

Connexin-43 organization
Immunofluorescence staining of connexin-43 was used to visualize connexin-43 protein expression and organization through fluorescence microscopy.Coalescence of connexin-43 was considered indicative of early gap junction formation and focal connexin-43 staining densities were counted with particle analysis in ImageJ.The early gap junction density per nucleus was calculated in MatlabR2020a for each scaffold.

RNA extraction, retrotranscription and RT-qPCR
RNA was extracted using GenElut Mammalian Total RNA Miniprep Kit (Sigma) according to the manufacturer's instructions.RNA (100 ng) was subsequently retrotranscribed to complementary DNA (cDNA) using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific).RT-qPCR was performed using Fast SYBR Green Master Mix on a 7500 Real-Time PCR System using GAPDH as a housekeeping gene.All primers were designed to span an intron-exon junction and are listed in Table 2.
The relative expression of mRNA was obtained using the DCt method.

Statistics
Experiments were executed three times in triplicate.A t-test with a 95 % confidence interval was used to determine statistical significance.Error bars represent standard error throughout.N-numbers are reported throughout.

Generation of anisotropic and isotropic collagen scaffolds by directional ice-templating
Directionally freeze-cast scaffolds had a mean pore size of 120 ± 9 μm prior to cross linking and sectioning.The scaffold pore structure was comprised of an inherent structural asymmetry (Fig. 1a-d) as shown by scanning electron microscopy (SEM) and micro-computed tomography (μCT), demonstrating different scaffold macrostructures and alignment across the transverse and longitudinal planes of the scaffolds.On the transverse plane, the pore structure was composed of homogenous, isotropic, circular pores, while on the longitudinal plane, the pore structure was composed of unidirectionally aligned pores (Fig. 1b & c).Fast Fourier transform analysis [42] of each planar surface showed significantly increased alignment in the longitudinal plane (S FFT = 0.66 ± 0.08 and S FFT = 0.18 ± 0.06 normalized intensity units on transverse and longitudinal planes respectively) (Fig. 1d).Scaffolds sliced along the longitudinal plane of the parent scaffold were characterized by aligned pores (aligned scaffolds) whereas those sliced along the transverse plane were characterized by non-aligned circular pores (isotropic scaffolds) (Fig. 1a).

Tissue contractility
A full field spatial-temporal assessment of construct deformation using optical strain analysis was performed (Supplementary Video 1).Scaffold architecture was found to dramatically influence deformation profiles and, thus, resultant principal strains (ε 1 and ε 2 ).Principal strain dynamics for both conditions occurred concurrently during contraction, however, isotropic constructs produced strains with equal and opposite magnitudes, indicating no net surface area change during contraction (Fig. 2c).The average maximal contractile strain (ε 1 ) was -0.018 ± 0.008 with a maximal inotropic strain rate of ~0.1 s − 1 and lusitropic strain rate of ~0.05 s − 1 (Fig. 2d).Spatial analysis of each principal strain at peak contraction showed large variability in magnitude and direction across the construct surface (Fig. 2e & f, Supplementary Video 1a-c).
Analysis of aligned constructs showed an average maximal contractile strain magnitude, ε 1 (0.15 ± 0.04), that was ten times greater than the orthogonal component, ε 2 (0.014 ± 0.023), indicating a net negative change in surface area (Fig. 2g).The ε 1 of aligned structures was similarly greater than the principal strains of isotropic constructs (Fig. 2k & l).A comparable relationship was observed for strain rate (Fig. 2h).Spatial analysis of peak contraction for aligned constructs demonstrated principal strain coordination across the whole field of analysis (Fig. 2i & j).Furthermore, structural anisotropy was found to direct deformation such that ε 1 was oriented parallel to scaffold alignment (Fig. 2j; Supplementary Video 1d-f).Scaffold alignment dramatically impacted construct contractility such that aligned scaffolds facilitated increased contractility indicated by contractile principal strain magnitudes (Fig. 2k & l) and reduced directional variance (circular variance of principal angles of 0.16 ± 0.10 for aligned and 0.40 ± 0.10 for isotropic; p = 0.041) (Fig. 2m).
The directional deformation dynamics produced by aligned constructs were consistent with previously reported deformation profiles of native cardiac tissue in vivo, where ε 1 is shown to be 2.5 times greater than ε 2 [47].The shape of the strain rate profile produced by aligned constructs was consistent with that of previously reported native myocardium, with the exception of the intermediate peak due to isovolumetric contraction (SRa) [47].SRs and SRe were found to be -0.04 and 0.025, approximately 44 % and 83 % of physiologically recorded values respectively (Fig. 2n) [47].

Calcium transient analysis
Time-dependent calcium fluorescence showed periodic fluorescence for both structural conditions (visualized by Fluo-4 AM) (Supplementary Videos 2a & b).Fast Fourier Transform analysis of each signal showed that engineered constructs with aligned architecture had a significantly faster pulse rate than isotropic structures (0.55 ± 0.09 Hz for aligned and 0.33 ± 0.03 Hz isotropic; p = 0.019) (Fig. 3a-c).(Fig. 3d-g; Supplementary Video 2a & b).Spatial analysis of calcium fluorescence in isotropic constructs showed uneven pulse rate distributions and high spatial variance (0.02 ± 0.01) across all samples.In contrast, aligned constructs resulted in largely uniform spatial pulse rate distributions and reproducibly small spatial variance (0.0010 ± 0.0006) (Fig. 3h).Similarly, the spatial distribution of the time of peak florescence within a single pulse (Fig. 3i-l) was larger for isotropic constructs (0.06 ± 0.11) relative to aligned constructs, which resulted in predominantly concurrent signaling across the tissue surface (0.0005 ± 0.0002) (Fig. 3m).
The calcium handling capacity of isotropic constructs under paced conditions at 1 and 1.5 Hz was evaluated.Isotropic structures, irrespective of pacing frequency, displayed reduced regularity in calcium cycling when compared to anisotropic constructs (Fig. 4a-f).Anisotropic constructs were found to conform to the external pacing frequency at both 1 and 1.5 Hz, while isotropic constructs did not conform to the 1.5 Hz pacing frequency as shown in Fig. 4f (1.47 ± 0.09 vs 1.17 ± 0.22 Hz in aligned and isotropic scaffolds respectively; p = 0.089).Additionally, calcium fluorescence waveform analysis demonstrated a shorter time to peak fluorescence and time to 90 % decay for aligned constructs under both pacing conditions compared to those of isotropic constructs as shown in Fig. 4g-k.

Cellular alignment
Quantitative Fourier analysis of immunofluorescence micrographs with Phalloidin staining of actin cytoskeletal structure showed reduced preferential orientation on the face of isotropic scaffolds (Fig. 5a-d), whereas cells seeded onto aligned constructs conformed to the extracellular macrostructure and exhibited a more uniform orientation on the face of the construct (Fig. 5e-h).Aligned structures demonstrated increased cellular coherence (0.24 ± 0.03 for aligned and 0.16 ± 0.04 isotropic; p = 0.008) relative to isotropic structures, indicating increased length-to-width ratios for cells on aligned structures (Fig. 5i &  j).A reduction of orientation variance (230 ± 150 for aligned and 2.5 × 10 3 ± 1.6 × 10 3 for isotropic; p = 0.012) was also observed, indicating increased directional alignment of the long axis of the cells seeded onto aligned constructs (Fig. 5i & k).In cross section, on the y-z plane of the construct (Fig. 6a), there was similarly no preferential cell orientation on isotropic scaffolds (Fig. 6b & c).Aligned constructs in cross section, however, again demonstrated cellular elongation along the axis of the pore alignment (Fig. 6d & e).

Phenotypic gene expression
Molecular analysis of seeded cardiomyocytes via qPCR showed significantly larger MYH7/MYH6 expression ratios for cells seeded onto aligned constructs compared with isotropic constructs (Fig. 7m).Additionally, cells on aligned constructs trended toward greater ryanodine receptor (RYR2) expression and larger TNNI3/TNNI1 expression ratios compared to those on isotropic structures (Fig. 7l & n).

Discussion
Technical advances in scaffold fabrication have enabled increased control and specificity over scaffold structure and facilitated a substantial shift toward biomimetic macroarchitecture in engineered cardiac constructs [9,[13][14][15]28].A substantial body of work has demonstrated that anisotropy at the nanometer scale has a significant and beneficial impact on engineered cardiac tissue behavior and cellular maturation [10][11][12][22][23][24]26,48].However, despite widespread acceptance, the functional benefits of macroarchitectural anisotropy have not been systematically studied, due, in part, to the interdependent relationship between micro-and macroarchitecture during scaffold fabrication [8,10,14,29].
Our study independently varied macrostructure in engineered cardiac tissue by deriving both comparison groups via perpendicular subdivision of unidirectionally aligned ice-templated collagen scaffolds with pore sizes optimized for cardiomyocytes [49].Prior studies have demonstrated the strut walls of ice-templated collagen scaffolds are characterized by micropores and fibrillar bridges at the μm scale and by collagen fibers within the lamellae at the nm scale [50][51][52][53][54][55].In prior studies, a small degree of anisotropy has been identified with regard to the collagen fiber orientation within the lamellae wall, all other microenvironmental features have been found to be amorphous [53,54].In the present work, the microenvironment is equivalent between experimental conditions because each construct is derived from scaffolds produced under identical conditions and the macrostructure is varied via orthogonal sectioning.Through directional freeze casting, the macrostructural pore orientation can be directed without compromising the microenvironment [13,[56][57][58].Therefore, slicing our experimental scaffolds on orthogonal planes resulted in a varied degree of macrostructural order while maintaining the cellular microenvironment.We chose to seed our scaffolds with H9 hESCs as they have well-established usage in translational medical science [59].Cellular populations and distribution were equal between scaffold conditions enabling differences in tissue function to be attributed to scaffold macroarchitecture.
Through a direct comparison, we demonstrated that scaffold macrostructural order improves the biomimetic signaling and contractile functionality of engineered cardiac constructs.Contractility is an emerging parameter that describes the dynamic pulsatile deformation of engineered cardiac tissue [60,61].Adaptive reference-digital image correlation (AR-DIC) combined with strain analysis provides a spatiotemporal measurement of engineered tissue deformation without the confounding error of imposing a physical force gauge onto the structure [60].Here, we used AR-DIC and demonstrated that long-range macrostructural alignment improved the contractile synchronicity of  engineered cardiac tissue and facilitated directional contraction with tissue densification (net reduction in volume); characteristics not seen in the isotropic construct deformation patterns (Fig. 2).Optical digital image correlation has also been used to characterize in vivo myocardial contraction during open heart surgery [47].The directional contraction observed in the aligned constructs presented in this work closely matches the directional deformation profile of in vivo cardiac tissue, which is characterized by the presence of a dominant principal strain, strain rate magnitude that is greater than its orthogonal component, and a coordinated strain orientation across the construct surface.Analysis of native myocardial strain rates over time has resulted in the identification of three key features: a global minimum during systole (SRs), a global maximum (with reduced absolute magnitude) during diastole (SRe) and a local maximum during isovolumetric contraction (SRa) [47].While both the isotropic and aligned constructs demonstrated similar total strain rate profiles with features consistent with SRs and SRe, the aligned constructs generated deformation patterns that were more similar to native myocardial contractions with faster, more physiologically relevant strain rates (Fig. 2n).The SRa was not identified for either condition as our construct geometry prohibits isovolumetric contraction.The increased contractility observed in the aligned constructs was likely due to uniform cellular orientation and advanced sarcomeric maturation.Aligned scaffolds facilitated increased cellular coherence and reduced cellular orientation variance relative to isotropic scaffolds (Figs. 5 & 6) [20][21][22].This finding is consistent with prior literature where it has been shown that uniform cellular elongation and intercellular alignment facilitate parallel sarcomere shortening and a summative contractile effect across the whole construct [20,62,63].Phenotypic maturity has also been shown to increase contractile force [14,64,65].Cells seeded onto aligned constructs have longer sarcomere subunits and increased banding prominence, indicating more advanced sarcomere development and cardiomyocyte maturation [61,66].Furthermore, genetic expression of sarcomere components for cells seeded onto aligned constructs indicated a higher level of phenotypic maturation.There was increased expression of genes linked to a more adult phenotype, β-myosin (MYH7) and troponin-I 3 (TNNI3), relative to fetal type genes α-myosin (MYH6) and troponin-I 1 (TNNI1) [61,67].
In vivo, contractile synchronicity is facilitated by efficient calcium signal transience across the myocardium [20,28].Calcium fluorescence patterns serve as a surrogate for action potential propagation and describe excitation-contraction coupling kinetics [20,27].Using spatiotemporal calcium fluorescence analysis of spontaneous signaling patterns we showed that aligned cardiac tissue constructs facilitated faster and more synchronous calcium cycling (Fig. 3).Heat maps of anisotropic scaffold pulse rate and time of peak intensity showed little spatial variance relative to isotropic conditions (Fig. 3).The spatial signaling heterogeneities seen in heat maps of isotropic scaffolds can, in part, be attributed to structural interruption of cellular confluence.It has been shown that physical interruption of cellular continuity induces derangement of spatiotemporal signal transience [27].While pore sizes were optimized for cardiomyocyte migration and survival, when organized isotopically the pores may induce structural discontinuities that are large enough to disrupt signal transmission.The distribution of connexin-43, the primary protein in gap junctions, is also an important factor that impacts transmural signal conduction [21].Additionally, the formation, prevalence, and organization of focal connexin-43 densities have been associated with tissue maturation [21,43,[68][69][70].Aligned constructs had an increased prevalence of focal connexin-43 staining densities consistent with early gap junction structures (Fig. 7).
Under full field pacing conditions calcium cycling kinetics occur synchronously.Calcium fluorescence waveform analysis of paced constructs showed that aligned constructs had more efficient calcium cycling, with a faster time to peak and time to 90 % decay than isotropic constructs.Aligned constructs were also more responsive to highfrequency pacing, whereas isotropic constructs did not conform to pacing at higher frequencies (Fig. 4).The calcium release and reuptake rates of cardiomyocytes have been shown to correlate with cellular maturity.The mechanism behind the functional changes in calcium handling during maturation is multifactorial and has been attributed to sarcoplasmic reticulum calcium handling protein expression, L-type channel expression, and gap junction formation [21].Specifically, recent studies have identified sarcoplasmic reticulum-dependent calcium transience to correlate with the rates of calcium fluorescence upstroke and decay [71,72].Here, we found cells seeded onto aligned scaffolds trended toward increased expression of the ryanodine receptor (RYR), a sarcoplasmic reticulum protein that may contribute to the improved calcium handling kinetics observed in aligned constructs [73,74].
Our results indicate that macrostructural extracellular order may positively impact the electromechanical function of engineered cardiac tissue, as well as the phenotypic development of the seeded cardiomyocytes.It is difficult to propose a direct mechanism by which longrange scaffold alignment influences gene expression.We propose an indirect mechanism by which phenotypic cellular maturity is enhanced based on the findings of Bouchard et al. who showed that the application of increasing external electromechanical stimulation hastened the maturation process of pluripotent stem cell-derived cardiomyocytes [61].We hypothesize that long-range anisotropic architecture facilitates a more coordinated contractile behavior via globally organized cellular orientation.This enhanced and uniform contraction is effectively an auto-loading system in which the contractile force facilitated by structural alignment serves to cyclically stimulate the tissue construct and enhance the rate of cellular maturation.Theoretically, as cardiomyocyte maturity increases, so too does the magnitude of the contractile force, creating an autologous loop that parallels the work done by Bouchard and colleagues [61].The effect would be reduced for the isotropic structures due to a lack of long-range cellular order.

Conclusions
Biomimetic scaffold microenvironment has been shown to positively regulate cellular behavior and phenotypic function in engineered cardiac tissue by improving contractility, signaling synchronicity, and cellular organization [8,[10][11][12]14,15,[21][22][23][24]26,48].The complexity of scaffold fabrication, however, has limited the isolated assessment of extracellular macrostructure and its impact on engineered cardiac tissue function.In this work, we used the planer asymmetry of unidirectionally aligned scaffolds to isolate macroarchitectural order between experimental conditions while conserving scaffold microstructure and other physio-mechanical properties.
Our results show that anisotropic extracellular structure enhances the functional biomimetic capacity of engineered heart tissue at multiple length scales.Specifically, aligned macrostructure facilitates improved signaling synchronicity and directional contractility as well as dictating more uniform cellular alignment.Cells on aligned constructs also displayed phenotypic and genetic markers of increased maturation.While further study is still needed to fully deconvolute the relationship between tissue structure and cellular maturation, our study informs the design of engineered myocardium for regenerative medicine and disease modeling applications.

Statement of significance
Cardiac tissue engineering is a promising field of study with translational potential as a therapeutic option for myocardial repair after injury.Despite its success in preclinical studies, the translational utility of engineered cardiac tissue has been limited by relatively low contractility and immaturity.The architecture of engineered cardiac tissues has been identified as an important modulator of maturation and contractility.However, the isolated impact of scaffold macroarchitecture on tissue function is poorly understood due to the complexity of scaffold fabrication limiting the isolated variation of individual structural and mechanical characteristics.Here, we utilize the planar asymmetry of directionally freeze-cast scaffolds to conserve the local physio-mechanical and micro-architectural scaffold features and investigate the isolated effects of pore macrostructure on engineered tissue function.We demonstrate that macroarchitectural anisotropy alone improves directional contractility, signaling synchronicity, cellular organization, and phenotypic maturity.Our results represent one step forward in deconvolving the relationship between form and function.They advance the current understanding of optimized cardiac tissue design and assist ongoing efforts to bridge the gap between the lab and the clinic.

Fig. 1 .
Fig. 1.Engineered cardiac tissue (a) slicing schematic with biopsy punches and corresponding slices from the L longitudinal plane and T transverse plane (b & c) SEM micrographs of scaffold structure in the (b) longitudinal plane and the (c) transverse plane (scale bars 500 μm) (d) normalized FFT ((f/f 0 ) − 1) alignment at each radial orientation, insert shows the maximal alignment for each slicing plane (N = 18) (e) a representative distribution of Troponin-T positive cells measured via flow cytometry.Blue indicates IgG antibody control; red indicates cells stained with an anti-Troponin-T antibody following lactate selection.(f & g) Engineered construct viability and cellular density measured through (f) Fluorescence intensity of Almar Blue (N = 3) and (g) nuclei density for each construct architecture (N = 8).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .
Fig. 2. Live bright field imaging of constructs in the relaxed and contracted state for (a) isotropic and (b) aligned constructs.Strain dynamics for (c-f) isotropic constructs and (g-j) aligned constructs.(c & g) Principal component strain in time ± standard deviation.(d & h) Mean strain rate for each principal component strain.(e & i) Spatial color map for ε 1 at maximum strain.(f & j) Spatial color map for ε 2 at maximum strain.(k) Maximum ε 1 for all conditions (l) Maximum ε 2 for all conditions.(m) Variance of principal strain direction for all conditions.(n) Total construct strain rate for comparison with physiological strain rate.All scale bars are 1 mm; error bars represent standard error; N = 3.

Fig. 3 .Fig. 4 .
Fig. 3. Live Fluo-4 AM calcium staining was performed on immature cardiomyocytes derived from H9 hESCs after 7 days of culture, video recordings of fluorescence dynamics were used to assess the temporal and spatial signaling uniformity.(a & b) Mean fluorescence intensity in time for cardiomyocytes on isotropic and aligned scaffolds, respectively.(c) Pulse rate for all isotropic and aligned samples (aligned N = 5; isotropic N = 8).(d-g) Pulse rate in space and associated histogram for (d & e) isotropic constructs and (f & g) aligned constructs.(h) Spatial variance of the pulse rate for all isotropic (N = 5) and aligned (N = 3) constructs.(i-l) Time of peak fluorescence in space within a single pulse indicated by the green boxes in a & b, and associated histograms for (i & j) isotropic constructs and (k & l) aligned constructs.(m) Spatial variance of time of peak fluorescence within a pulse for all isotropic (N = 5) and aligned (N = 3) constructs; Scale bars represent 0.2 mm; error bars represent standard error.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .
Fig. 5. Cellular orientation of hESC-CM stained with phalloidin (red) and DAPI (blue) after 7 days on the face of (a-d) isotropic scaffolds and (e-h) aligned scaffolds; scale bars represent 100 μm.(a & e) Composite image.(b & f) Isolated Phalloidin channel showing actin organization.(c & g) Actin orientation colormap resulting from Fourier transform orientation analysis over a moving pixel average of 2 pixels, color bar indicates the correlating orientations.(d & h) Polar histograms of the actin orientation of cardiomyocytes within a single scaffold.(i) Schematic of orientation variance and coherence measurements.(j) Average actin orientation coherence for all isotropic and aligned samples.(k) Average actin orientation variance for all isotropic and aligned samples; all error bars represent standard error; N = 5. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 .
Fig. 6.Cross sectional cellular orientation of hESC-CM after 7 days.(a) Schematic of cutting planes.(b-e) Representative immunofluorescence micrographs of cardiomyocytes on the y-z plane of (b & c) isotropic scaffolds and (d & e) aligned scaffolds; scale bars represent 30 μm. (b & d) Cells stained with phalloidin (red) and DAPI (blue).(c & e) Cells stained for sarcomeric α-actinin (red) and DAPI (blue).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7 .
Fig. 7. Intra-cellular structures and gene expression.(a-f) hESC-CM stained for sarcomeric α-actinin (red) after 7 days on (a & b) isotropic scaffolds and (c & d) aligned scaffolds; scale bars represent 20 μm.(b & d) Representative quantification of sarcomere organization through relative intensity peak prominence along a single sarcomere chain (N = 6 chains per construct).(e) Average relative intensity peak prominence (sarcomere intensity) on aligned (N = 4) and isotropic (N = 3) scaffolds.(f) Sarcomere length for cells on aligned (N = 4) and isotropic (N = 3) scaffolds.(g-j) hESC-CM stained for Dapi (blue) Troponin (Green) and Connexin (red) after 7 days on (g & h) isotropic scaffolds and (i & j) aligned scaffolds; scale bars represent 50 μm.(k) Early gap junction density for isotropic (N = 5) and aligned (N = 4) samples.(l & m) qPCR quantification of relative expression of (l) RYR and (m & n) MYH7 to MYH6 and TTNI3 to TTNI1 expression ratios (N = 3).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1
Primary and secondary antibodies.