Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart
Harald C Ott, Thomas S Matthiesen, Saik-Kia Goh, Lauren D Black, Stefan M Kren, Theoden I Netoff & Doris A Taylor. Nature Medicine: Volume 14, Number 2, 2008
Summary:
The topic of designing or recreating portions or even an entire bioartificial heart is an ongoing, refining process by many researchers around the world since there is very relevant need. In the United States alone, the paper mentions that around 5 million people live with heart failure and around 550,000 new cases are reported annually. This paper talks about the complete dececullarization of a cadaveric heart and repopulating it with neonatal cardiac cells or endothelials. By decellularizing via direct immersion, the entire ECM scaffold of the heart remains and acts as an acellular scaffold for the growth and development of new cardiac tissue in specific regions, like ventricles, or the entire heart.
In the process of creating a decellularized cardiac scaffold, 3 detergents were tested, including Sodium dodecyl sulfate (SDS), Triton-X 100, and Polyethylene Glycol (PEG). Using antegrade coronary perfusion for 12 hours, the tissues were tested for amount of remaining intact cells, DNA content remaining, and the overall ECM structure of the cardiac scaffold. Using thin-section staining, most cells were visible in the Triton-X100 and PEG detergent samples, but SDS treated samples showed complete decellularization. Immunoflurescent staining with DAPI also showed no nuclei in the decellularized tissue as well, and there appeared to be no change in the amount of glycosaminoglycan content. After washing the SDS treated samples with Triton-X100, no SDS content could be detected, thus resulting in a purely glycosaminoglycan cardiac scaffold without detectable endothelial or smooth muscle cells.
When conducting stress-strain calculations on the resulting dececullarized scaffold, it showed that the samples were very highly anisotropic and stiffer in the circumferential than tangential directions. They also showed that after adjusting for thickness, the decellularized samples showed no difference at 40% strain when compared to cadaveric heart tissue. This shows that despite treating with detergents, the structural integrity of the resulting sample also remains intact and ready for recellularization.
For the process of recellularization, the decellularized scaffolds were placed in a bioreactor with fresh, neonatal cardiac cells. The medium flow was pulsatile with antegrade left heart perfusion. After 8 days, there was around 33.8+/-3.4% recellularization with 1mm thickness of confluency. An important achievement was that after 8 days, the seeded cells where forming tissue that was able to contract with electrical stimulus and respond to drugs.
Significance:
The process growing cardiac tissue has been a difficult task despite significant research in this area. Instead of using artificial scaffolds for growing the tissue, decellularizing cadaveric heart while retaining an almost perfect scaffold allows for receding with endothelial cells that can be grown for a specific region of the heart to be replaced for a patient, and even potentially for the growth of an entire organ. Using this process of decellularization to retain scaffolds can also be extended to other organs like lungs, liver, or kidneys, and use of stem cells or progenitor cells can be used for custom developed tissues in vitro.
5 comments:
It's amazing to me that no one thought of this earlier. One of the hardest parts of tissue engineering is getting the matrix/platform right, and this research shows that the simplest and potentially best solution was just "nature's platform." After growing the heart, however, why didn't they implant it in a mouse to see if it would actually work? Once this research expands and if transplantable organs become possible, it seems like the only limiting factor would be the amount of cadaveric organs. Hopefully there's a significantly greater amount of cadaveric organs than regular ones.
I reviewed a paper using similar methods to engineer pulmonary heart valves, so of course their methods were much simpler than decellularizing an entire heart. They simply used EDTA and trypsin to acellularize the conduit before seeding it. This paper addresses the major concern of structural integrity following the perfusion treatment of the heart. I wonder if the paper mentions the conditions of perfusion? For example, do they discuss the velocity at which they perfuse and how similar it is to the velocity at which blood normally flows through the heart? I don’t know if this is at all relevant, but I venture to think that if we expose the heart to less extreme conditions in processing, it will survive with greater structural integrity. Also, I wonder if it is adequate to solely analyze stress-strain for the cadaveric heart tissue as a measure of structural integrity? It would seem that there a more factors complicating the integrity of the tissue and that the researchers might have to explore other methods of determining the effects of the decellularization process on the tissue. Despite needing much work to continue testing their methods, their research is an exciting advance in the engineering of a bioartificial heart. Research in this arena is widely needed as we see a continued rise in heart disease in the American population and around the world.
In the recellularization process of an entire heart based on its ECM scaffold, structural integrity is undoubtedly a critical point if it were to perform as its role as a mechanically pulsatile tissue. In the process of reseeding the decellularized heart, the engineers documented having a perfusion rate of 20ml/min for the atrial flow and 6ml/min for coronary flow at 37oC for 24 hours. The scaffold was seeded with 200 uL of neonatal cardiomyocytes, fibroblasts, smooth muscle cells, and endothelial cells, thus delivering 75e6 cells. This was performed 50 times for sufficient reseeding. Without other factors such as electrovoltaic changes during reseeding, the cells could attach and begin growth on the preexisting scaffold. In short, the seeding conditions were such that the researchers were able to simulate flow through the decellularized heart that that would allow attachment of new endothelial and cariomyocite cells initially, followed by similar conditions as that of a developing heart until it achieved around 25% function of a 16-week fetal human heart.
As to characterize solely on stress-strain for structural integrity, I agree that there could be further tests that could be done to check for structural integrity. For example, the researchers could also try profiling the pulsatile flow and characterize weaknesses in the recellularized structure. By analyzing the longitudinal and circumferential stress-strains, it still gives a relatively good idea of the overall structural integrity of the newly seeded scaffold.
James, you raise an interesting point of why the heart wasn’t transplanted in a live animal to check for in vivo success with structural integrity and applicability. The researchers mentioned in their paper that their scope for this experiment was to achieve 3 milestones: 1) provide structural scaffold for growth of whole heart tissue 2) seeding and growth of cells in this scaffold and 3) achieve nascent pumping function in tissue. Having achieved these 3, it would seem logical that the probably are working optimizing this technique and development of tissue suitable for transplant.
I am also intrigued by the efficacy of this simple but very promising technique. I did have one main question, however, in terms of the seeded cells developing into tissue that responded to electrical impulse and specific drugs--could you describe in a bit more detail what types of drugs were incorporated and also, did the authors employ any other, more traditional, tests for surface marker characteristics of cardiomyocytes
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