Tissue Engineering of the Mitral Valve Leaflets and Abdominal Aorta

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1 Medizinische Hochschhule Hannover Dr Morticelli L Supervisor: Dr Korossis S Niedersächsischen Zentrum für Biomedizintechnik und Implantatforschung The Tissue Engineering (TE) Concept Tissue Engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ (Ref.: Langer R. and Vacanti J. P. (1993). Science, 260 (5110) ). Scientific field focused on the reconstruction of TISSUES or whole ORGANS CELLS + extracellular matrix (ECM) Basic structures of organisms Extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells Tissue Engineering (TE) Approaches Ref.: Chan B. P. and Leong K. W. (2008). Eur Spine J, 17 (Suppl 4):S467 S479 In vitro TE: In vivo TE: CELLS + SCAFFOLD (cultured in a bioreactor) TISSUE ENGINEERED CONSTRUCT IN VIVO IMPLANTATION SCAFFOLD without CELLS

2 Example 1: Leaflets Guided Functional Re-engineering of the Mitral Valve Leaflets Morticelli L, Thomas D, Fisher J, Ingham E, Korossis S Background & Clinical Need The Human Heart The Mitral Valve The Mitral Valve (MV) is situated between the left atrium and the left ventricle of the heart; Normal MV Diseased MV It consists of the leaflets (anterior & posterior), the chordae tendineae which support the free edges of the leaflets and which anchor to the papillary muscles; MV function is to direct the blood flow from the left atrium directly towards the left ventricle. 100,000 operations each year in the developed countries for valve replacement or repair and 20,000 deaths due to MV diseases. Current strategies for MV reconstruction (repair and replacement) lackofin vivo regeneration Aim & Strategy of the Project Aim: Research and develop tissue-engineered leaflets for MV reconstruction using pericardial scaffold, seeded with porcine mesenchymal stem cells (pmscs) and biomechanically stimulated in vitro. pmscs multipotent cells (they can produce more than one type of specialized cells of the body, but not all types). Sample Isolation Decellularised Pericardial Scaffold pmscs Leaflet Equivalent Dynamic Culture In the bioreactor Recellularised Pericardial Scaffold

3 Cell Density Optimisation: pmscs Static Culture pmscs were cultured at different densities (2 10 4,1 10 5, cells/cm 2 ) to look at cell proliferation and penetration after 24 hours, 3 days and 1 week culture. The optimum seeding densities was cells/cm 2 and the optimum time point was 3 days. 24 hours 3 days 1 week 30 μm 200 μm 200 μm Sample Tissue Holder 200 μm Dynamic Bioreactor Conditioning: Live/dead staining Bioreactor stations Tissue holders 69-84% live cells; Cells started aligning along specific directions when cultured dynamically in the bioreactor 96-97% live cells Dynamic Bioreactor Conditioning: H & E Bioreactor stations Tissue holders Intact matrix and beginning of cell penetration

4 Example 2: Tissue Engineering of the Abdominal Aorta Allogeneic Aortic Transplantation in Rat Model after Decellularisation Morticelli L¹, Katsimpoulas M¹, Michalopoulos E, Gontika I, Chaniotakis I, Spyrliadis A, Stavropoulos-Giokas C, Kostakis A, Korossis S ¹Both authors contributed equally to this work Background & Clinical Need The abdominal aorta is the portion of the aorta which descends into the abdominal cavity; The abdominal aorta supplies oxygenated blood to all of the abdominal and pelvic organs and the legs; Abdominal aorta Abdominal aortic aneurysms cause 1-3% of all deaths among men aged years in developed countries: Current strategies for abdominal aorta reconstruction (endovascular repair) lack ofin vivo regeneration Aneurysm can be defined as a permanent and irreversible dilatation of a vessel which can lead to its rupture; Abdominal aortic aneurysm Aim & Strategy of the Project Aim: Implantation of the developed decellularised rat abdominal aortas in the rat animal model, in order to simulate the allogenic transplantation model. Scaffold Decellularisation Abdominal aorta isolation 15mm Length 0.100mm Thickness Mechanical & Biological Characterisation of Explants Decellularised Graft Syngenic Graft Allogenic Graft NATIVE AORTA IMPLANTED AORTA Implantation of decellularised/native grafts Genetically Identical Individuals (Wistar rats into Wistar) Individuals from the Same Specie, but with Different Genotype (DA rats into Wistar)

5 Mechanical Characterisation of Allografts, Syngenic Grafts and Decellularised Allografts Uniaxial tensile tests: loading to failure Force Force 6 samples loaded to failure per each group Statistical analysis Syngeneic Allogeneic Decellularised Syngeneic Allogeneic Decellularised Histology of Explanted Allografts, Syngenic Grafts and Decellularised Allografts Syngenic Grafts Collagen Fibres Elastic Fibres 100µm 100µm 100µm Histology of Explanted Allografts, Syngenic Grafts and Decellularised Allografts Decellularised Allografts E Good preservation of the histoarchitecture of tissue E Allogenic Grafts 100µm 100µm 100µm Not Prominent Mononuclear Cells Infiltration??? Mononuclear cells and Herytrocytes??? 100µm 100µm 100µm

6 Acknowledgements Engineering and Physical Sciences Research Council (EPRSC) Leeds Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPRSC Centre for Experimental Surgery Biomedical Research Foundation of the Academy of Athens (BRFAA) Thank you!