A tissue-engineered scale model of the heart ventricle

In the paper

“animal models and isolated heart preparations are evaluated using pre-clinical and clinical measurement modalities such as catheterization3, echocardiography4, and magnetic resonance imaging5.”

What the sources say

• Partly supported

  The Introduction describes catheterization to measure cardiac function in mouse and rat animal models, including intact beating hearts. It does not establish the cited technique's use in isolated heart preparations. Citation 3 is attached to catheterization, so the separate echocardiography and MRI citations are not assessed here. (LLM, conf=0.85)

  cites [3] Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S. & Kass, D. A. Measurement of cardiac function using pressure–volume conductance catheter technique in mice and rats. Nat. Protoc. 3, 1422–1434 (2008). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [4] Ram, R., Mickelsen, D. M., Theodoropoulos, C. & Blaxall, B. C. New approaches in small animal echocardiography: imaging the sounds of silence. Am. J. Physiol. Heart Circ. Physiol. 301, H1765–1780 (2011). source
• ?Not checked — no full text of the sourcecites [5] Bakermans, A. J. et al. Small animal cardiovascular MR imaging and spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 88–89, 1–47 (2015). source

In the paper

“Data obtained using these methods allow direct comparison to patient data, but differences in genetics, physiology and disease etiology limit the utility of animal models for developing therapeutic interventions6,7.”

What the sources say

• ?Not checked — no full text of the sourcecites [6] Chandrasekera, P. C. & Pippin, J. J. The human subject: an integrative animal model for 21st century heart failure research. Am. J. Transl. Res. 7, 1636–1647 (2015).
• Partly supported

  The Introduction says animal models may not reproduce the varied causes of human heart failure and that these differences limit translation to clinical therapies. The Conclusions also identify species and physiological differences as barriers. Our copy does not establish that data obtained using the citing paper’s methods allow direct comparison with patient data, or specifically identify genetic differences between animal models and humans as a barrier. (LLM, conf=0.83)

  cites [7] Gloschat, C. R. et al. Arrhythmogenic and metabolic remodelling of failing human heart. J. Physiol. 594, 3963–3980 (2016). sourceExperimental — accuracy not yet measured

In the paper

“The myocardial ECM is fibrillar, anisotropic, and provides nanotopographical cues that guide cardiomyocyte alignment and assembly44, ultimately forming a helicoid structure that optimizes ejection fraction during ventricular contraction45.”

What the sources say

• Supported

  Citation 44 is attached to the claim about myocardial ECM and cardiomyocyte alignment and assembly, not the subsequent helicoid and ejection-fraction claim attributed to citation 45. The Results describe aligned ECM fibrils whose orientation correlates with adjacent myocardial cells and suggest that the ECM provides nanotopographic cues guiding alignment; they also report that nanoscale patterns guide cell monolayers into an aligned architecture. (LLM, conf=0.91)

  cites [44] Kim, D. H. et al. Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs. Proc. Natl Acad. Sci. USA 107, 565–570 (2010). source
• ?Not checked — no full text of the sourcecites [45] Savadjiev, P. et al. Heart wall myofibers are arranged in minimal surfaces to optimize organ function. Proc. Natl Acad. Sci. USA 109, 9248–9253 (2012). source

In the paper

“Engineered cardiac organoid chambers comprised of cardiomyocytes embedded in isotropic hydrogels showed significant promise for obtaining intra-ventricular pressure measurements26–29 but the lack of a scaffold to guide cell assembly hampers the formation of organized tissues that recapitulate the laminar architecture30,31 of the native myocardium.”

What the sources say

• ?Not checked — no full text of the sourcecites [26] Lee, E. J., Kim do, E., Azeloglu, E. U. & Costa, K. D. Engineered cardiac organoid chambers: toward a functional biological model ventricle. Tissue Eng. Part A 14, 215–225 (2008). source
• ?Not checked — no full text of the sourcecites [27] Gonen-Wadmany, M., Gepstein, L. & Seliktar, D. Controlling the cellular organization of tissue-engineered cardiac constructs. Ann. N. Y. Acad. Sci. 1015, 299–311 (2004). source
• ?Not checked — no full text of the sourcecites [28] Yildirim, Y. et al. Development of a biological ventricular assist device: preliminary data from a small animal model. Circulation 116, I-16–I-23 (2007). source
• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [29] Li, R. A. et al. Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells. Biomaterials 163, 116–127 (2018). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [30] Costa, K. D., Takayama, Y., McCulloch, A. D. & Covell, J. W. Laminar fiber architecture and three-dimensional systolic mechanics in canine ventricular myocardium. Am. J. Physiol. 276, H595–607 (1999). source
• ?Not checked — no full text of the sourcecites [31] Arts, T., Costa, K. D., Covell, J. W. & McCulloch, A. D. Relating myocardial laminar architecture to shear strain and muscle fiber orientation. Am. J. Physiol. Heart Circ. Physiol. 280, H2222–2229 (2001). source

In the paper

“We selected polycaprolactone (PCL)/gelatin nanofibers as our material of choice because they are biocompatible and biodegradable, they promote cell adhesion, provide sufficient structural integrity for ventricle culture and catheterization, and can be produced by a variety of nanofiber production systems that include electrospinning and force-spinning methods40.”

What the sources say

• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [40] Capulli, A. K., MacQueen, L. A., Sheehy, S. P. & Parker, K. K. Fibrous scaffolds for building hearts and heart parts. Adv. Drug Deliv. Rev. 96, 83–102 (2016). sourceExperimental — accuracy not yet measured

In the paper

“In the native heart, overlapping myocardial fibers are arranged into distinct laminae four to six myocytes thick and separated from adjacent laminae by an extracellular collagen network42.”

What the sources say

• Partly supported

  The Discussion (3-D perimysial collagen organization; page unavailable) describes branching myocardial layers grouped by perimysial collagen and reports them as approximately four cells thick, not four to six. The Results (page unavailable) show collagen between adjacent layers, but the paper also says laminar structure is not evident in the outer 10–20% of the rat left ventricle, so the claim is too general. (LLM, conf=0.86)

  cites [42] Pope, A. J., Sands, G. B., Smaill, B. H. & LeGrice, I. J. Three-dimensional transmural organization of perimysial collagen in the heart. Am. J. Physiol. Heart C 295, 1243–1252 (2008). sourceExperimental — accuracy not yet measured

In the paper

“Cardiomyocyte spontaneous beat rates in our model ventricles were similar to other in vitro assays based on NRVM49 or hiPSC-CM16, showing moderate positive chronotropy (~40% beat rate)10,18.”

What the sources say

• ?Not checked — no full text of the sourcecites [49] Meiry, G. et al. Evolution of action potential propagation and repolarization in cultured neonatal rat ventricular myocytes. J. Cardiovasc. Electrophysiol. 12, 1269–1277 (2001). source
• Supported

  The citation to this paper is attached to the hiPSC-CM assay, not the later ~40% positive-chronotropy claim, which has separate citations. The Results (Functional Characterization of hiPSC-EHTs) describe spontaneously beating hiPSC-derived heart tissues and report a baseline rate of 47 ± 6 bpm, supporting its use as an example of that assay. (LLM, conf=0.91)

  cites [16] Mannhardt, I. et al. Human engineered heart tissue: analysis of contractile force. Stem Cell Rep. 7, 29–42 (2016). source
• ?Not checked — no full text of the sourcecites [10] Eder, A., Vollert, I., Hansen, A. & Eschenhagen, T. Human engineered heart tissue as a model system for drug testing. Adv. Drug Deliv. Rev. 96, 214–224 (2016). source

In the paper

“The moderate positive chronotropy and reduction of stroke work observed at high isoproterenol doses are characteristic of immature tissues exhibiting poorly developed calcium handling properties8, suggesting that maturation protocols14,21,22,73 may be required to obtain positive force-frequency relationships, as observed in healthy adult human ventricular myocardium74.”

What the sources say

• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [8] Karakikes, I., Ameen, M., Termglinchan, V. & Wu, J. C. Human induced pluripotent stem cell-derived cardiomyocytes: insights into molecular, cellular, and functional phenotypes. Circ. Res. 117, 80–88 (2015). sourceExperimental — accuracy not yet measured
• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [14] Nunes, S. S. et al. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat. Methods 10, 781–787 (2013). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [74] Endoh, M. Force–frequency relationship in intact mammalian ventricular myocardium: physiological and pathophysiological relevance. Eur. J. Pharmacol. 500, 73–86 (2004). source

In the paper

“Contractile work in our ventricle models was measured using conductance catheters and was smaller than reported for cardiac organoid chambers based on NRVM26 or pluripotent hES2 human embryonic stem cell-derived cardiomyocytes29, where volume changes were indirectly assessed from video recordings.”

What the sources say

• ?Not checked — no full text of the sourcecites [26] Lee, E. J., Kim do, E., Azeloglu, E. U. & Costa, K. D. Engineered cardiac organoid chambers: toward a functional biological model ventricle. Tissue Eng. Part A 14, 215–225 (2008). source
• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [29] Li, R. A. et al. Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells. Biomaterials 163, 116–127 (2018). source

In the paper

“For example, the tensile elastic modulus of our scaffold in the direction of fiber alignment (EL ~ 500 kPa) was higher than healthy ventricular ECM (E ~350 kPa)68 and may have limited cardiomyocyte shortening during contraction.”

What the sources say

• Partly supported

  The Results report tensile testing of healthy ventricular ECM and its elastic modulus, supporting the use of this paper as a comparator. But the available text does not give the cited value of approximately 350 kPa (searched: “350” and “kPa”). The scaffold modulus and possible effect on cardiomyocyte shortening are the citing authors’ own comparison and inference, not findings attributed to this source. (LLM, conf=0.83)

  cites [68] Quinn, K. P. et al. Optical metrics of the extracellular matrix predict compositional and mechanical changes after myocardial infarction. Sci. Rep. 6, 35823 (2016). sourceExperimental — accuracy not yet measured

In the paper

“Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 μL (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle)”

In the paper

“Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function.”

In the paper

“In all cases, spontaneous calcium activity showed stable excitation patterns with plane waves (healthy) or pinned spiral waves (injured) repeating at a near-constant rate (between 4 Hz and 5Hz) for the duration of experiments (~2 hours).”

In the paper

“Pressure/volume loops measured before and after isoproterenol exposure showed an isoproterenol-induced reduction in stroke work, concomitant with an increase in beat frequency (Fig. 3b,c).”

In the paper

“Plane wave propagation was observed prior to injury (Fig. 5a, top), whereas injured ventricles generated spiral waves that were pinned to the controlled anatomical defects (Fig. 5a, bottom panels, and Supplementary Movie 12).”

In the paper

“The HBR includes both intra- and extra- ventricular flow loops, separate chambers for optional valve inserts, and additional access ports for catheters (Fig. 4a, Supplementary Fig. S8).”

In the paper

“Mean calcium wavefront propagation velocity from apex to base was 9.33 cm/sec or 5.2 cm/sec for NRVM or hiPSC-CM ventricles, respectively.”

In the paper

“The resulting tissue-engineered ventricle chambers were sutured to tubing or bioreactor components through which catheter sensors were introduced and stable contraction of both NRVM and hiPSC-CM ventricles permitted time-dependent pressure and volume measurements.”

In the paper

“In our scaffold, gelatin was used to enhance cell and biomolecule adhesion during cell seeding, and the polymer backbone (PCL) provided structural features (microporosity, nanotopography, anisotropy) that resisted degradation during culture.”

In the paper

“Calcium fluorescence intensity measured near the rotor poles of a single-hole injury model showed consistent phase difference and a rotation rate of ~ 5 Hz (Fig. 5b, Supplementary Figure S12, and Supplementary Movie 13).”

In the paper

“Using small animal echocardiography equipment, we recorded both unassisted natural spontaneous ventricle contraction (Fig. 4c, left, and Supplementary Movie 10) and HBR-assisted contraction (Fig. 4c, right, and Supplementary Movie 11).”

In the paper

“Measured differences in chamber pressure were ~50 μmHg (rat or human) and the volume change was ~5 μL (rat) or 1 μL (human).”

In the paper

“Synchronous, coordinated ventricle contraction developed spontaneously after 3–5 days of culture (Supplementary Movies 3,4) and persisted for the duration of experimental procedures (Supplementary Movie 5), conducted 14 days after seeding.”

In the paper

“Cyclic pressure applied to the extra-ventricular loop drives intra-ventricular fluid flow via assisted ventricle contraction, and unidirectional flow can be achieved using commercially available valves or with custom valve inserts in the HBR.”

In the paper

“Ventricle beat rates showed a positive chronotropic response over the concentration range in both the NRVM and hiPSC-CM ventricles (Fig. 3c), as expected for healthy NRVM59, hiPSC-CM60, engineered hiPSC-CM cardiac tissues16, and human patients61.”

What the sources say

• ?Not checked — no full text of the sourcecites [59] Simpson, P. & Savion, S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, and chronotropic response to isoproterenol. Circ. Res. 50, 101–116 (1982). source
• ?Not checked — no full text of the sourcecites [60] Moretti, A. et al. Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N. Engl. J. Med. 363, 1397–1409 (2010). source
• ?Not checked — no full text of the sourcecites [61] Koglin, J., Bohm, M., Vonscheidt, W., Stablein, A. & Erdmann, E. Antiadrenergic effect of carbachol but not of adenosine on contractility in the intact human ventricle in-vivo. J. Am. Coll. Cardiol. 23, 678–683 (1994). source

In the paper

“The spontaneous beat rate of NRVM ventricles (~130 ±15 bpm) was higher than hiPSC-CM ventricles (~85 ± 15 bpm), and both increased by ~40% following exposure to 10−4 M isoproterenol.”

In the paper

“These features provided sufficient structural integrity for ventricle culture and they guided cardiomyocyte assembly”

In the paper

“Thus, ejection fractions were ~1% (rat) or ~0.2% (human) and stroke work was WS ~ 0.25 mmHg × μL (rat) or WS ~ 0.05 mmHg × μL (human).”

In the paper

“Whereas regular beat frequencies were observed in calcium-containing M199 or Tyrode’s solution, exposure to calcium-free PBS led to broad beat rate distributions (Supplementary Fig. S7B).”

In the paper

“We also cultured a non-proliferative hiPSC-CM line (Cor.4U) in model ventricle scaffolds for 6 months, demonstrating that tissue anisotropy was preserved (Supplementary Figure S14A) and stable pressure-volume loops could be recorded (Supplementary Figure S14 B).”

In the paper

“When cultured in differentiation media, the resulting hiPSC-CM showed spontaneous contraction and early stages of sarcomeric assembly within 8–12 days of culture (Supplementary Figure S13A, Day 12), and anisotropic sarcomeric assembly with early stages of z-disk alignment after 1 month in culture (Supplementary Figure S13A, Day 30).”

In the paper

“Both types of model cardiac chambers generated similarly shaped elliptical PV-loops (Fig. 3b, Supplementary Fig. S14), which are expected from ventricles contracting in the absence of valves and show similarities to PV loops obtained from patients with severe valvular insufficiencies78.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [78] Klabunde, R. E. Cardiovascular Physiology Concepts 2nd edn (Lippincott Williams & Wilkins/Wolters Kluwer, Philadelphia, 2012).

In the paper

“The EF of our tissue-engineered rat ventricles was ~1% (1/50 EF of healthy rats), suggesting that discontinuities in cardiomyocyte coverage limited tissue-engineered ventricle contractile strength.”

In the paper

“We observed a non-linear conductance/volume catheter response (Supplementary Fig. 4B.i), as expected for these systems82.”

What the sources say

• ?Not checked — no full text of the sourcecites [82] Pearce, J. A., Porterfield, J. E., Larson, E. R., Valvano, J. W. & Feldman, M. D. Accuracy considerations in catheter based estimation of left ventricular volume. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2010, 3556–3558 (2010). source

In the paper

“Both showed evidence of spontaneously generated calcium plane wave propagation but one had greater homogeneity; this favorable sample is reported in our revised manuscript.”

In the paper

“The cell density in our ventricles, estimated by counting immunostained (DAPI) NRVM ventricle surfaces and cross-sections (Supplementary Fig. S3), was ~90,000 cells/mm3 for a total cell number ~2.3 × 106 in a 26 mm3 scaffold volume, which is approximately 10-fold less than in 21-day-old or 3-month-old rat hearts75.”

What the sources say

• ?Not checked — no full text of the sourcecites [75] Bai, S. L., Campbell, S. E., Moore, J. A., Morales, M. C. & Gerdes, A. M. Influence of age, growth, and sex on cardiac myocyte size and number in rats. Anat. Rec. 226, 207–212 (1990). source

In the paper

“but direct comparison with natural ventricle pressure and volume dynamics requires three-dimensional (3D) contractile cardiac chambers.”

In the paper

“These cells are assembled into functional engineered heart tissues2,9,10, including muscular thin films11,12 or 3D muscle strips13–20, to model contractile pathophysiologies11, promote hiPSC-CM maturation9,14,15,21, and produce drug responses that are increasingly comparable to human patients16,18,22.”

What the sources say

• ?Not checked — no full text of the sourcecites [2] Tzatzalos, E., Abilez, O. J., Shukla, P. & Wu, J. C. Engineered heart tissues and induced pluripotent stem cells: macro- and microstructures for disease modeling, drug screening, and translational studies. Adv. Drug Deliv. Rev. 96, 234–244 (2016). source
• ?Not checked — no full text of the sourcecites [9] Feric, N. T. & Radisic, M. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv. Drug Deliv. Rev. 96, 110–134 (2016). source
• ?Not checked — no full text of the sourcecites [10] Eder, A., Vollert, I., Hansen, A. & Eschenhagen, T. Human engineered heart tissue as a model system for drug testing. Adv. Drug Deliv. Rev. 96, 214–224 (2016). source

In the paper

“These and other limitations associated with animal models, for example high maintenance costs and low experimental throughput, motivate the development of alternative in vitro cardiology assays based on human cells.”

In the paper

“These properties allowed us to fabricate ventricle scaffolds that were both structurally stable and permissive to chamber contraction by cardiomyocyte shortening.”

In the paper

“Echocardiography performed in the absence of extra-ventricular tissues can improve image clarity compared with in vivo studies, underscoring the advantage of organ modularity in our HBR.”

In the paper

“In this configuration, cultured ventricles can be exposed to externally-controlled physiological or pathological pressure variations and PV loops acquired with or without valves (Fig. 4b)”

In the paper

“We previously described anisotropic PCL/gelatin nanofiber production by pull spinning and their use to guide skeletal muscle tissue assembly46.”

What the sources say

• ?Not checked — no full text of the sourcecites [46] Deravi, L. F. et al. Design and fabrication of fibrous nanomaterials using pull spinning. Macromol. Mater. Eng. 302, 1600404 (2017). source

In the paper

“Scaffold engineering should account for the fact that the myocardium is a composite in which the dominant stiffness shifts from myocytes to the ECM with increasing strain67, emphasizing the ECM’s role maintaining structural integrity.”

What the sources say

• ?Not checked — no full text of the sourcecites [67] Chaturvedi, R. R. et al. Passive stiffness of myocardium from congenital heart disease and implications for diastole. Circulation 121, 979–988 (2010). source

In the paper

“These results have implications for preclinical cardiology and regenerative medicine research where human organ models are sought to improve translation of therapeutic strategies.”

In the paper

“A fundamental aspect of myocardial function is the response to adrenergic agonists, which has been studied extensively in both rodents and humans, making β-adrenergic response a good test of the ability of our engineered ventricles to recapitulate normal cardiac pharmacology57,58.”

What the sources say

• ?Not checked — no full text of the sourcecites [57] Barrett, A. M. & Carter, J. Comparative chronotropic activity of beta-adrenoceptive antagonists. Br. J. Pharmacol. 40, 373–381 (1970). source
• ?Not checked — no full text of the sourcecites [58] Brito-Martins, M., Harding, S. E. & Ali, N. N. Beta(1)- and beta(2)-adrenoceptor responses in cardiomyocytes derived from human embryonic stem cells: comparison with failing and non-failing adult human heart. Br. J. Pharmacol. 153, 751–759 (2008). source

In the paper

“while gelatin was chosen as an RGD-containing biopolymer derived from denatured collagen that improves scaffold binding to cells and biomolecules such as fibronectin47,48.”

What the sources say

• ?Not checked — no full text of the sourcecites [47] Ruoslahti, E. RGD and other recognition sequences for integrins. Annu Rev. Cell Dev. Biol. 12, 697–715 (1996). source
• ?Not checked — no full text of the sourcecites [48] Katagiri, Y., Brew, S. A. & Ingham, K. C. All six modules of the gelatin-binding domain of fibronectin are required for full affinity. J. Biol. Chem. 278, 11897–11902 (2003). source

In the paper

“These preliminary results suggest that our scaffolds support functional myocardial tissue production using relatively small numbers of seeded hiPSC that can subsequently be cultured for extended time periods.”

In the paper

“Our use of conductance catheters was advantageous for direct volume readouts but electrical pacing during catheterization was not recommended by the catheter manufacturer and therefore not performed in these studies.”

In the paper

“All procedures were approved by the Harvard Animal Care and Use Committee”

In the paper

“Our work suggests that including a nanofibrous component to printable inks would be advantageous for directing cardiomyocyte shape and assembly in printed scaffolds or tissues.”