The VALVE REGEN project. Developing an innovative tissue engineered heart valve, in vitro and in vivo testing – preliminary results
Authors: Movileanu I , Nistor D , Brinzaniuc K , Harpa M , Preda T , Cotoi O.S , Al Hussein H , Sierad L , Simionescu D 
Published: November 2020

Abstract

Background
Regenerative Medicine and Tissue Engineering are the grounds on which multidisciplinary teams aspire to obtain the perfect valvular substitute, which overcomes shortcomings of the present prostheses.

Purpose
Was to obtain a tissue engineered heart valve (TEHV) by repopulating with valvular resident cells – endothelial (EC) and fibroblasts (FB) a decellularized heart valve scaffold. Then their functionality and behavior was assessed in vitro and in vivo.

Methods
This study is part of a research grant approved by the Ethics Committee of the University. Six ovine pulmonary valves underwent a perfusion based decellularization protocol. Using a sequence of chemical and enzymatic treatment under a pressure gradient, cell removal was achieved and attested by histological investigations (DAPI nuclear staining –4',6-diamidino-2-phenylindol and haematoxylin-eosin) and DNA extraction. Ovine sub-dermal adipose tissue was harvested followed by stem cells isolation and culture. Using Endothelial Cell Growth Supplement and mechanical stimuli EC were differentiated and with Transforming Growth Factor-B1, FB were obtained. FB were internally seeded into cuspis bases using a 22 gauge needle and externally on the adventitia by using a rotator allowing a uniform distribution and seeding of cells. EC were seeded into leaflets pockets and intra-luminal also using the rotator. The repopulated valves were preconditionated in a bioreactor by gradually exposing them to the pulmonary hemodynamic regimen. By using a high speed camera, their behavior was examined when exposed to in vivo conditions. The in vivo testing was performed by surgical implantation in the gold model considered animal – the sheep. By transesophageal ultrasound (TEE US) and epicardic US, their intra-operatory function was evaluated. Post-procedure, evaluation was performed by periodic trans-thoracic (TTE US).

Results
Six TEHV were obtained. The decellularization histology assessment revealed acellular scaffolds and non-detectable nucleic material at the DNA extraction. Six adipose derived stem cells cultures were obtained and subsequently specialized towards EC and FB lines. The repopulation procedures underwent without incidents. During the bioreactor preconditioning, the TEHV showed complete opening and competent central coaptation. Leaflets presented physiological movement and absence of damage of valvular apparatus. The TEE US evaluation in vivo revealed normal valvular function without signs of stenosis or regurgitation. The periodic TTE US showed preserved valvular function.

Conclusions
Our preliminary results point out a manufactured TEHV with physiological behavior when tested in vitro and in vivo. Their interaction with a living body will be pointed out only in the explant phase, after histology analysis. The present results appear optimistic but only extended studies and follow-ups will certify their superiority in terms of performances and behavior.

Funding Acknowledgement
Type of funding source: Public grant(s) – EU funding. Main funding source(s): This paper was financed by a grant from the Competitiveness Operational Programme 2014-2020, Tissue engineering technologies for cardiac valve regeneration, valve-regen, id P_37_673, Mysmis code: 103431, contract 50/05.09.2016.