Paper 3 – In Vitro Studies of a Spontaneous Contracting Cardiac Patch Derived from Neonatal Rat Cardiac Cells and Fibrin
Overview: Cardiac patches are effective in covering large cardiac defects that cannot be easily repaired utilizing the patients own existing tissues. In a recent study by Tao et al., 2014, they present a cardiac patch created from fibrin gel seeded with neonatal rat cardiac cells to repair heart defects (Figure 1). The patch is designed to induce cell differentiation and proliferation, as well as withstand the contraction and relaxation pattern of the heart66.
Material Considerations
The scientists chose the neonatal rat cardiac cells as they have high regeneration and differentiation rates which are effective when implanting a foreign body biomaterial into an organism. Previously studies have shown that 1-day old neonatal mice hearts can regenerate after partial cardiac resection67, demonstrating their dynamic behavior. These cells are easily sourced from rat cardiac muscle and can be seeded effectively on fibrin gels. They utilized fibrin in this study due to its self-assembly (polymerization) properties and innate biocompatibility (naturally synthesized polymer by the body). The liver synthesizes fibrin from fibrinogen catalyzed by thrombin. This reaction can be performed utilizing the patient’s blood to limit foreign body response and implant rejection. See the page about fibrin glue for cardiac patches for a detailed mechanism of fibrin synthesis. Additionally, fibrin encourages even cell distribution and exceptional adhesion, making it an appropriate choice for application over large cardiac defects.
Patch Fabrication
Neonatal cardiac cells were taken from rats, isolated and purified by rinsing out existing blood cells and treatment with a dissociation solution before suspension in cell media. Fibrin gels were created on a plate treated with a dried polydimethylsiloxane elastomer (PDMS) polymer. They then added thrombin and fibrinogen to the plate to induce fibrin formation before added the purified cell culture (Figure 1). Overtime, the expectation is as the fibrin gel polymer develops, it will begin to detach itself from the PDMS layer in a thin patch-like layer which can be further analyzed.
Injected Neonatal Cells Beat Sponteaneously and Differentiate on the Fibrin Patch Scaffold
16 hours after injection, scientists manually counted cardiomyocytes which were observed in the fibrin gel, showing promising signs for further differentiation (Figure 2A,B and C). They also determined cardiomyocyte contraction rate via video analysis and found that higher cardiomyocyte density resulted in an increased rate of contraction (Figure 2E).
Copyright © 2014 John Wiley & Sons, Ltd.
Although a greater number of cardiac cells and overall patch cell density suggests positive proliferation trends and improved cell viability, they found that the ratio of types of cardiac cells in the sample is important for synchronizing tissue contractions. Ideally, there should be a balance between cardiomyocytes, cardiac fibroblasts and endothelial cells. However, cardiac fibroblasts and endothelial cells proliferate much faster. Thus, controlling final patch cell density (between 1 and 3 M) during the initial seeding and growth phases is important for maintaining the correct composition of cell types in the patch. To further explore the regulatory pathway between turning cardiomyocytes into fibroblasts, see our page about cardiomyocyte differentiation pathways in the cardiac mechanisms portion of this website.
The morphology of the injected cells was further studied on day 7, with a layer of cardiac cells and newly produced extracellular matrix forming on the fibrin gel scaffold (Figure 3).
To classify each cell type in the patch, they performed Masson trichome staining (Figure 3C) which helps visualize collagen structures and cardiac muscle tissue (dark red/pink area indicates cardiac tissue layer). Staining with von Willibrand Factor (vWF) indicated the presence of endothelial cells (red) (Figure 3D), while Ki67 staining identified areas of active nucleic division (white areas, Figure 3E) and collagen (purple in Figure 3F) within the newly grown patch tissue. In addition to classifying these cell types in the patch, they found areas with growing angiogenesis buds for forming new blood vessels. These buds have the potential to connect micro blood muscles and supply nutrients to strengthen the patch if implanted in vivo.
Conclusion
This paper demonstrates how fibrin polymers promotes endothelialization and growth of cardiac cells in an animal model. While this study was performed in vitro, further experimentation is required with in vivo models to assess how the patch interacts with surrounding body tissues and interacts with the cardiovascular system during the healing process. However, these findings confirm that cardiac cells interact positively with fibrin, justifying its potential use as a key biocompatible polymer in cardiac injury repair applications.
December 8, 2023 at 4:54 pm
When comparing to the other paper pages, I wished a bit that there was the same conclusion portion that tied back a bit to the main question. That being, I’m still quite impressed how for this and all of the other papers the main significant findings and considerations were distilled out into these very succinct summaries. This project really went extremely deep into all aspects that were explored! Congrats
December 8, 2023 at 8:05 pm
This is a super interesting paper! Seeing as they use neonatal rats as the model organism, would this application work for adult rats (and by extension, humans)? Regardless, it would be interesting to see this research applied to the field of neonatal cardiology one day. Also I agree with Joaquin that a conclusion for this paper like the first two papers would be nice.
Typo: “Injected Neonatal Cells Beat Sponteaneously and Differentaite on the Fibrin Patch Scaffold” -> spontaneously and differentiate
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This one was so interesting to read about! I would definitely be interested in reading a compare/contrast between this paper and the other paper on hydropatches that you guys covered. How similar/different are they in their construction, use, effectiveness? Is one a better option overall, or do they each have a specific benefit/strength? As Joaquin said in his comment, I think there is great opportunity here to draw connections between these papers and use these connections to reach impactful conclusions beyond what you have already covered
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As usual, concise summary and descriptions of experiment with results! Your use of infographics throughout the website has been really helpful. On this page, I was wondering what drawbacks might be present from using a 2014 paper. Your other two papers describe techniques explored in 2022 and 2023, and in such a fast-evolving field it might be good to qualify the findings of this paper with later developments.
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