Presented on 24.04.20 for the 3DbioNet online journal club Annabelle Fricker from the Department of Materials Science and Engineering, Faculty of Engineering, University of Sheffield, supervised by Professor Ipsita Roy.

This paper reports the production of 3D scaffolds for cardiac tissue engineering, with a focus on the design providing anisotropic properties that closely resemble those of the native right ventricular (RV) myocardium. In the native RV myocardium, cardiac muscle fibres are surrounded and coupled by endomysial collagen sheaths that are bundled within a honeycomb-like network of undulated perimysial collagen fibres. This structure produces directionally dependent electrical and mechanical properties that are together termed as cardiac anisotropy. Previous 3D scaffolds had been structurally and mechanically incompatible with the formation of a fully biomimetic tissue, and therefore this paper aimed to mitigate this problem. The aims of the study were to:

1. Match the anisotropic in-plane mechanical response of native myocardium within the physiological regime
2. Provide low in-plane resistance to contraction, and
3. Provide an inherent structural capacity to guide cardiomyocyte orientation in the absence of external stimuli Figure 1 shows the orientation of cardiac muscle and the stress-strain plots representing the circumferential and longitudinal axes of the heart. The researchers used the material PGS as it is an elastomeric material, making it suitable for a cardiac application, and it degrades via surface erosion, hence degrading gradually whilst maintaining its overall structure. Initial PGS curing conditions of 16 h at 160°C were chosen based on previous PGS soft lithography studies. The honeycomb structure was made by excimer laser microablation of a PGS membrane with a thickness of about 250 µm. This thickness was chosen to mitigate mass transport limitations based on previous modelling analysis of oxygen diffusion through solid PGS and tissue engineered myocardium. The excimer laser microablation processing technique yielded well-defined features which were comparable to the original design (Figure 2). The researchers compared the effective stiffness anisotropy ratio of the scaffold to both the adult rat right and left ventricular myocardia. The results showed that the scaffold bore closer mechanical properties to the RV myocardium, and therefore they chose to approximate the scaffold with the RV myocardium with the reasoning that RV myocardial grafts, with the potential to grow, regenerate, and remodel, could ultimately lead to new treatments for congenital heart defects, which typically affect the right ventricle, as well as for right ventricular infarctions that frequently complicate left ventricular infarctions and are associated with substantial first-year mortality. They next sought to get a better match between the effective stiffness of accordion-like honeycomb scaffolds with that of the adult RV myocardium, by reducing the PGS curing time. Utilizing the standard curve (Figure 2d), they predicted that 7.5 h curing would most closely match the adult RV myocardium. To assess longer term mechanical stability, accordion-like honeycomb scaffolds made of 7.5h cured PGS were placed in a fatigue bath and subjected to in vitro cyclic stretch, mimicking physiological loading. Specimens were subjected to cyclic loading at 1 Hz and peak strain of 0.1 (i.e., 10%) for 1 week in buffered saline at room temperature. Compared to adult rat RV myocardium, fatigued scaffolds exhibited similar mechanical properties. Next, scaffolds made of 7.5h cured PGS were autoclave-sterilized, seeded with cells isolated from neonatal rat ventricles, and cultured in vitro for 1 week. Compared to adult rat RV myocardium, accordion-like honeycomb scaffolds with cultured heart cells exhibited a lower effective stiffness in the preferred direction (PD), EPD, and higher strain-to-failure, εf, values. Together, these data suggested that in vitro culture of the heart cells reduced EPD, and the group hypothesized that this is due to a combination of scaffold bioresorption and extracellular matrix deposition. These effects need to be considered in future studies if the scaffold mechanical properties are to be matched to adult rat RV myocardium post-culture. To compare grafts based on accordion-like honeycomb scaffolds of varying effective stiffnesses, neonatal rat heart cells were seeded on scaffolds made of PGS cured for 7.5, 12, and 16 h (Figure 3). The cells filled the pores of the scaffold, were grossly aligned in parallel to the PD, and showed some cross-striations albeit less developed than those in the adult rat RV myocardium. Electrophysiological assessment demonstrated synchronous contractions in all scaffold groups, with all grafts capable of being paced by electrical field stimulation up to at least 2 Hz. Excitation thresholds (ET) were found not to depend significantly on PGS curing time. Hence, these data sets were pooled to yield a directional dependence of ET for a given scaffold orientation, with respect to the electric field - ET was 11 ± 4% lower when measured with the scaffold oriented parallel versus perpendicular to the applied electric field. Separate experiments were undertaken to compare accordion-like structures shown in a, square (200 × 200 µm, shown in b), and rectangular (400 × 200 µm; shown in c) honeycomb scaffolds fabricated from 16 h cured PGS. These scaffolds exhibited different degrees of mechanical anisotropy (Figure 4). These results confirmed that of the scaffolds tested, the accordion-like honeycomb scaffold exhibited anisotropic mechanical properties most closely resembling that of adult rat RV myocardium. To assess whether the electrical excitation threshold was influenced by scaffold microstructure, all three structure designs were seeded with cardiac fibroblasts followed by neonatal rat heart cells enriched for cardiomyocytes and cultured in vitro for 1 week. As few as 4 days after seeding, spontaneous contractions were observed in all groups. After 1 week of culture, the majority of pores were filled by neonatal rat heart cells and the grafts could be synchronously paced at up to 4 Hz. The excitation threshold (ET) depended significantly on the orientation of the scaffold PD with respect to the electric field. Anisotropic accordion-like and rectangular honeycomb scaffolds respectively had significantly lower excitation threshold values when the scaffold PD was aligned parallel to the electric field. The scaffolds demonstrated utility by:
4. Closely matched mechanical properties compared to native adult rat right ventricular myocardium, with stiffness controlled by the polymer curing time
5. Heart cell contractility inducible by electric field stimulation with directionally-dependent electrical excitation thresholds (p<0.05)
6. Greater heart cell alignment (p<0.0001) than isotropic control scaffolds A problem in this research is that the thickness of the scaffolds (~250um) is too low and does not address reconstruction of the full-thickness of the myocardium. As a first step toward addressing these limitations, the researchers produced prototype bilaminar honeycomb scaffolds of greater thickness (~400 µm) with interconnected 3D square pore networks. In a preliminary study, unseparated neonatal rat heart cells were cultured for 1 week on bilaminar scaffolds. Cellular penetration of and interconnectivity between the top-to-bottom and lateral pores were observed (Figure 5). These findings further suggest that combined excimer laser microablation and lamination might be useful in fabricating honeycomb scaffolds of greater thickness, with the caveat that thicker scaffolds would likely require perfusion to maintain cardiomyocyte viability. Future studies would also need to focus on programming the excimer laser to generate the more complex arrays of troughs required for the assembly of a bilaminar accordion-like honeycomb scaffold. To conclude, this paper shows that accordion-like honeycomb scaffolds can overcome principal structural-mechanical limitations of scaffolds for myocardial tissue engineering by promoting the formation of grafts with preferentially aligned neonatal rat heart cells and mechanical properties more closely resembling that of adult rat RV myocardium. This could pave the way toward further integration of tissue-specialized scaffolds into advanced tissue engineering strategies.

Full reference: Engelmayr, G., Cheng, M., Bettinger, C. et al. Accordion-like honeycombs for tissue engineering of cardiac anisotropy. Nature Mater 7, 1003–1010 (2008). https://doi.org/10.1038/nmat2316

Discussion notes: What was the thickness of the scaffold and was this sufficient for in vivo study?

• The thickness was 250m, and this had been decided through previous research into the mass transport and oxygen diffusion limitations of solid PGS. In some additional preliminary work they introduced another layer of the scaffold to increase the thickness to 400m and showed that cells could integrate throughout the scaffold, however there are limitations of perfusion and therefore maintaining the viability of the graft. This paper is from 2009, how much progress has there been in the area of cardiac patches?
• Certainly, the processing techniques available have improved and become more extensive. For example, with techniques such as 3D printing and melt electrospinning we can now relatively easily produce scaffolds with complex structures. Continued focus has been put on the co-culturing of cells in grafts, including the addition of vascular cell types which wasn’t carried out in this research project. This is to solve the issue of graft vascularisation and therefore the maintenance of viability of thicker grafts. However, there are still many issues that remain to be overcome. What types of cells did they use and would you choose an alternative?
• They used primarily neonatal rat heart cells, as their research compared the properties of their graft to that of native rat right ventricular myocardium. Improvements have been made in the field of stem cells, and now use of induced pluripotent stem cells (iPSCs) and from human sources, helping to bridge the gap between animal models and human patients. Why do we need the mechanical properties to match the property of the heart?
• In order for the cardiomyocytes on a cardiac patch to be viable it is important for the scaffold to have the right mechanical properties, matching that of the native myocardium. The heart is continuously contracting and relaxing and therefore a patch needs to be elastomeric and able to stretch in the same way that the heart muscle does. These mechanical properties also enable the cells seeded onto them to grow into the correct morphology, for example the elongation of cardiomyocytes. Does the patch need to be bioresorbable?
• Bioresorbability is good when the aim of treatment is to repopulate an area with living cells. In the case of a cardiac patch, a scaffold is used to deliver cells to the site of interest and to keep them in position so that they can integrate with the host tissue. Therefore, over time the scaffold material should resorb, ultimately leaving behind the regenerated tissue.