SMALL DIAMETER RVOT CONDUITS WITH REGENERATIVE POTENTIAL FOR PEDIATRIC PATIENTS – PN-III-P2-2.1-PED-2019-2179, director proiect: Dan Simionescu
| Project Title | Small Diameter RVOT Conduits with Regenerative Potential for Pediatric Patients |
| Project Registration Code | PN-III-P2-2.1-PED-2019-2179 |
| Project Acronym | PEDIAVALVE |
| Project Scope and Objectives | The objectives of the project are: 1) To develop a technology for full decellularization and sterilization of small diameter RVOT conduits. Anatomical measurements of RVOT in several species of different ages, pointed out to ovine hearts as the perfect size and anatomy-matched tissue source (12-14 mm diameter, Figure 6). We will use existing technology developed in our lab to mount the ovine RVOT into purpose-designed perfusion systems for controlled decellularization. Quality control validation tests will be used to ensure consistency and to assess mechanical properties. 2) To test hemodynamics of the ovine acellular RVOT in purpose-designed bioreactor flow loops adapted with pressure, flow and imaging capabilities. These will be compared to native RVOT conduits harvested fresh from ovine sources. 3) To assess cytocompatibility of the conduit tissue components (muscle, cusps, sinus, artery) by seeding appropriate cells (endothelial cells, fibroblasts) onto the scaffolds and testing for cell viability, proliferation and migration. These tests will validate their potential to be safely used implants in future studies. 4) Mock “implantation” in ovine hearts to test surgical feasibility. This will be done in freshly collected ovine hearts. We will test suturability and ease of implantation as compared to freshly harvested ovine RVOTs. This test will attest to the surgical maneuverability of the new device. The use of decellularized tissues has passed the experimental stages and is considered safe for clinical use. According to one of the pioneers in the field, to date there are around 21 devices approved by the FDA for human use which consist of decellularized connective tissues (skin, dermis, intestinal submucosa, fascia lata, large nerves, dura mater, pericardium) from human, porcine, bovine and equine sources; these were implanted in more than 1 million patients with no signs of rejection and no adverse effects13, 14. The rationale for using acellular tissues is as follows: the major sources of antigens are cells and their MHC molecules and potentially some of the soluble extracellular matrix proteins. During decellularization, all cells and cell-derived molecules as well as all soluble proteins are completely removed by detergents and nucleases, leaving behind insoluble collagen and elastin fibers alongside other insoluble basement membrane proteins such as laminin and type IV collagen15. These highly insoluble proteins have not evolved considerably and are conserved among species (including mammals and even fish!) and thus are not antigenic when implanted across species. One classical example is bovine collagen preparations that have been implanted in human patients for decades for esthetical or restorative purposes without rejection or adverse effects16. We and others have tested our own acellular porcine aortic valves for immunogenicity by implantation of samples sub-dermally in rats17 and also as shunts in sheep18. In all instances we and others did not detect any immune reactions as expressed by presence of activated T-lymphocytes, infiltrated macrophages or IgG deposits19. Therefore, numerous published papers support the hypothesis that acellular xenogeneic tissues are not immunogenic. Moreover, numerous examples showed that the acellular tissues supported and promoted tissue regeneration by providing a tissue-mimetic scaffold with adequate 3D architecture and biological properties. To date, no acellular valves of any size exist on the market and this project offers a unique opportunity to develop and test such a product and to bring it closer to clinical application. We have developed the expertise and basic equipment to accomplish all four objectives. Since the RVOT is a complex 3D structure, we will use prototyping techniques for development of mounting and testing systems. These include 3D scanning of the RVOT, analysis and design in SolidWorks and 3D printing of adequate adapters and fittings. Achieving these objectives, providing evidence that the device fits anatomically and functionally and supporting evidence that the scaffold is sturdy, cell-friendly and implantable, will set the basis for projects whereby such devices will be implanted in juvenile sheep and followed for extended periods of time to assess for implant growth. These studies will be proposed in future grant applications. The long-term objective of this project is to prepare this device for controlled clinical trials. |
| Project Start Date | 01/05/2020 |
| Project End Date | 30/04/2022 |
| Project Duration | 24 months |
| Total budget value | 599.901,00 lei |
| Team: | Dan Teodor Simionescu -> PROJECT LEADER Ovidiu Cotoi -> Research member Lucian Hârceagă -> Research member Adrian Man -> Research member Adela Nechifor-Boila -> Research member Ionela Movileanu -> PhD student Terezia Octavia Preda -> Technician member Alexandru Chertes -> Technician member Hamida Al Hussein -> Technician member |
| Results | Deliverables Activity 1 will include an optimized method for efficient decellularization of the RVOT, with more than 95% reduction of DNA content, absence of any cell nuclei on histology sections, preserved matrix integrity as evidenced by histology, unaltered mechanical properties (<10% change in elasticity and Young’s modulus) and adequate suture holding strength (<10% lower than fresh). We also expect 100% sterility of the tissues.
Deliverables Acitivy 2: will include excellent hemodynamic performance of the valve with wide opening and minimal (<5%) regurgitation. Deliverables Activy 3: will include very low cytotoxicity of the acellular tissue segments (<10% cell death) at all time points. We also expect that cells will proliferate to a certain extent (20-30%) in the 2-week interval. We also anticipate that the acellular RVOT will pass the ISO 10993-5 tests. Deliverables Activity4: will include valuable input from cardiac surgeons and fine tuning of the device before it would be ready for future large animal testing and clinical trials. |
| Host institution | George Emil Palade University of Medicine, Pharmacy, Science and Technology of Targu Mures |