Carbon dioxide helps to prepare aortic valve prostheses

Carbon dioxide helps to prepare aortic valve prostheses

Aortic valve transplantation is needed for numerous patients. Transplants of biological origin may cause undesirable immune response and calcification. Pre-treatment of animal valves can lower the risks, but the aid of supercritical carbon dioxide provides a much better result.

Millions of people across the globe suffer from various cardiac conditions, including congenital heart diseases and other illnesses with a valvular anomaly. Thousands of patients receive aortic valve replacements, but this approach has severe drawbacks. For example, biological valves are usually made of animal tissues pretreated with chemicals to minimise the risk of the immune response. Such implants are prone to calcification and may require additional surgery. Mechanical valves, on the other hand, make the patients undergo a life-long anticoagulant therapy that poses a risk of haemorrhage. The current efforts in this research field are aimed at tissue engineering which could be used in the production of stable bioprostheses without related complications. Scientists from Sechenov University and their colleagues have performed a study where supercritical carbon dioxide (CO2) was used in the preparation of aortic valve bioprostheses. The results have been reported in the journal Molecules.

The key strategy of making a suitable bioprosthesis without a high level of immunogenicity is tissue decellularisation. It is the processing of biotissue which removes cells but keeps the extracellular matrix. This way, the structure of the tissue and its hemodynamic function can be preserved, improving the prognosis for the patients.

The current decellularisation technologies are based on detergents (such as sodium dodecyl sulphate or sodium deoxycholate), enzymes, and alkali. In addition, repetitive freezing–thawing and high pressure may be used. Unfortunately, none of these methods is perfect. Supercritical CO2 was introduced as a decellularisation agent quite recently. The compound, often used in ‘green chemistry’ strategies, has a low environmental impact and is widely available. Sechenov researchers analysed the application of supercritical CO2 in tissue decellularisation in combination with other current treatments.

The experiments were based on the ovine aortic root model. The donor animal (sheep) was chosen because of its immunological resemblance to humans and the tissue’s tendency to calcify. The scientists assessed the quality of decellularisation by histology and evaluated the biomechanical properties of the heart valve with uniaxial tensile tests.

With supercritical CO2 and ethanol as a co-solvent, the decellularisation time of 3 hours gave an effect, although it was not entirely satisfactory. The high pressure helped remove smooth muscle cells only partially, while it also led to the condensation of collagen fibres.

The combination of carbon dioxide and alkali resulted in substantial elimination of all cell types from the aortic walls and valve leaflets. However, the scientists detected pores and dense collagen and elastin fibres in the tissue after the treatment — no matter what pressure was applied in the procedure.

The best results were observed with the protocols that included pre-conditioning in a detergent solution followed by supercritical CO2-assisted processing. The pressure of 15 MPa already gave a good level of decellularisation, whereas 25 MPa produced outstanding results. The tissue was almost devoid of residual nuclear material or membrane fragments of fibroblasts and smooth myocytes. The researchers also found that the cytotoxicity of the aortic valves treated with detergents and supercritical carbon dioxide was significantly lower compared to what without supercritical carbon dioxide. This makes the detergent–CO2 protocol advantageous for clinical applications.

‘Humanity aims to achieve active longevity, and it makes scientists develop new techniques which could restore the functions of damaged organs and tissues. We have particular hopes for “green chemistry” targeted removal of foreign cells from donor tissues, or decellularisation’, said Ekaterina Grebenik, Leading Research Fellow at the Institute for Regenerative Medicine (Sechenov University) and one of the authors of the study. ‘In this paper, we presented a comprehensive analysis of the decellularisation methods used for the aortic valve — based on combinations of detergents, alkali, or ethanol with supercritical CO2. We have shown that supercritical CO2 facilitates the removal of cells and toxic substances from tissues, however, it also stiffens the tissues. The effectiveness of decellularisation with detergents is higher than with ethanol, while tissue stiffening is less prominent compared to the effect of alkali treatment. Our results indicate the prospects of supercritical fluid technologies in tissue engineering. We hope to use these findings in the development of aortic valve allografts’.

The research was carried out by the Institute for Regenerative Medicine (Sechenov University) in partnership with Kurnakov Institute of General and Inorganic Chemistry (Russian Academy of Sciences, Moscow), Semenov Federal Research Centre for Chemical Physics (Russian Academy of Sciences, Moscow), Institute of General Pathology and Pathophysiology (Moscow), Scientific Research Institute of Eye Diseases (Moscow), and Lomonosov Moscow State University (Department of Chemistry and Department of Biology).

Read more: Gafarova ER, Grebenik EA, Lazhko AE, et al. Evaluation of Supercritical CO2-Assisted Protocols in a Model of Ovine Aortic Root Decellularization. Molecules (2020).

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