1. Introduction

1. Consiglio Nazionale delle Ricerche - Convegno “Ricerca per le Biotecnologie nel Veneto, Azione Biotech II” – October, 16th 2007 Development of a new technology to produce of 3D functional tissue engineered hearth tissue through the integration of stem cells and 3D scaffold: Cardio-Patch Coordinator of local unit: N. Elvassore1, L. Vitiello2, P. De Coppi3, R. Busetto4, G. Gerosa5, M. Spina6, M. Dettin7, G. Fassina8 1DDipartimento di Principi e Impianti di Ingegneria Chimica , University of Padova, 2Dip.di Biologia, University of Padova, 3Dip.di Pediatria, University of Padova, 4Dip.di Scienze Veterinarie Cliniche, University of Padova, 5Dip.di Scienze Cardiologiche, Toraciche e Vascolari, University of Padova,5Dip. di Scienze Biomediche Sperimentali, University of Padova,6Dip. di Processi dell‘Ing. Chimica , University of Padova, 7Xeptagen 1. Introduction 2. Differentiation of stem cell toward cardiac lineage 3. Dynamic Culture and Electrical stimulation The overall goal of the cardio – patch project is to fabricate an in vitro cardiac tissue combining stem cells on 3D biocompatible scaffold. The engineered tissues can be arranged in a high-throughput fashion for biological or physiological studies as well as for pharmacological screening tests. The project is organized in 2 parts: 1) the aim of the first sub-project is to develop new methods and technologies to optimize the fabrication of in vitro tissue by the use of a dynamic system for stem cell culture. 2) the goal of the second sub-project is the fabrication of cardiac valve substitutes using decellularized scaffolds and autologous bone marrow stem cells. The 3D cultures face problems such as the inadequate mass transfer of nutrients and the limited gas species diffusion which, in vivo, are overcome by the functionality of the capillary net. The aim is to direct the cells to (re)establish the structure and function of the native tissue being repaired over clinically relevant thicknesses. In order to evaluate the clinical applicability of these methods, preliminary studies have been performed in vivo in small (rat) or in big animals (pig). The human AFS differentiation toward cardiac lineage (Figure 1) was studied performing 2D co-culture with neonatal cardiomyocyte and reproducing in a 2D in vitro culture the physical and mechanical properties of native cardiac muscle microenvironment. To engineer functional myocardium, we used a “biomimetic” approach that involves the cultivation of cardiac cell populations on scaffolds (designed to provide a structural and logistic template for tissue formation), using bioreactors (designed to provide environmental control and biophysical stimulation). Figure 1 Left)rCM and hAFS co-cultured immunostaining for cardiac troponin T (red) and DAPI (blu). Human cells are stained with CMFDA (green). Right) Semi quantitative evaluation of gene expression through PCR Figure 4. Bioreactor for stem cell expansion We produced an array of contractile independent, spatially and dimensionally controlled individual fibers, which reproduce the functionality and structure of cardiac tissue more closely than the traditional in vitro cultures (Figure 2). We develop and combine new scaffold biomaterial and dynamic perfusion culture methods in bioreactors (Figure 4-5). A B Figure 5 Bioreactor chamber scheme for the dynamic culture of stem cell Figure 2. Cardiomyocytes cultured on patterned PA hydrogel. A)BF image of cardiomyocytes after 4 days in culture; (B) cultured cardiomyocytes express troponin I (red) nuclei were counterstained with Hoechst (blue). (bars=100μm). Immunostaining for troponin I represented in the inset in picture B shows at higher magnification (40x) a developed contractile apparatus. (scale bar=75 μm) To induce synchronous contractions of cultured cardiac constructs, we apply electrical signals designed to mimic the synchronous contractions of cells in native heart (Figure 6). Tissue engineering approaches using 2D microtextured membranes improve both rCM and AFS alignment and co-culture organization after seeding (Figure 3). Figure 3. gfp+ rCM and hAFS co-cultured on 2D silicone microtextured membrane at 6 days. a): immunostaining for GFP (green) on cardiac gfp+ cells in co-culture with hAFS at 1000cells/cm2 density, magnification 20X; b) immunostaining for cardiac troponin T (red) on cells co-cultured, magn. 20x and c): merge of the two staining. Figure 6. Experimental setup for supra-threshold stimulation of cardiac myocytes. A. Electrical stimulation voltages are set using a computer program, output through 8-channel AO card, amplified, and interfaced to bioreactors. B. Petri dish with carbon rod electrodes. C. Close up view of scaffold positioned between electrodes and held in place with two stainless steel pins. 5. In vivo preliminary studies 4. Engineered Tissue Analysis 5. Engineered cardiac valves For better characterizing the influence of a perfusion bioreactor on these cultures, we developed protocols to perform flow cytometric analyses of cultures carried on collagen scaffolds (Figure 7). The potential of collagen scaffolds for attracting angiogenesis/arteriogenesis was studied in vivo by implantation on healthy or cryoinjured left ventricle of rats up to 60 days post-injury times. The clinical potential of decellurarized valves have been evaluated engrafting the aortic porcine root (Figure 11) in the pulmonary position of the recipient minipig (Figure 12). Coronaries are closed by suture. Figure 7. Examples of data collected by flow cytometry. Figure (A) represents a dot plot showing forward (FSC) vs. side light scatter (SSC). (B) represents a histogram of the cell population gated in (A) stained with PI for live/dead assay; (C) represents a dot plot showing the fluorescence of cells stained with PI after fixation; the area of the PI fluorescence peak is proportional to DNA content; the width of PI fluorescence peak is used to discriminate between single cells and aggregated cells with the same overall DNA content. In the heart, the collagen scaffolds were almost completely adsorbed in 60 days and became populated by new arterioles and capillaries in both intact and cryoinjured heart (Figure 9). Figure 11. Aortic porcine root isolated from a common breeding pig. The valve has been treated by decellularizing methodologies and used to be implanted in the pulmonary position into the recipient minipig. Figure 9. Typical aspect of arterioles (identified by the double color green + red) and capillaries (in red; some of them are indicated by red arrowheads) after 15 days implantation. Bars: 80 μm and 30 μm (insets). We have designed a method to measure the functionality of the cardiac engineered tissue. The high potential of the proposed device is in the possibility of an automated analysis of several samples during the culture (Figure 8). The vascularized scaffold can support in vivo hAFS and hCD133+ stem cell survival in a cryoinjured rat heart model (Figure 10). Figure 12. Surgery practice. The allogenic aortic valve is engrafted in the pulmonary position into the recipient animal. After 6 months, the 6 minipigs, engrafted with decellurarized aortic root , are alive. The ecocardiography shows only a negligible valve stenosis. Figure 10. Gross appearance and histological staining of rNU cryoinjured hearts with patch implantation 15 days after stem cell injection. a) and d): gross appearance of rNu hearts with respectively cmtmr+ hAFS and h CD133+ injection: C = cryoinjury area, P = collagen patch; b) and e): hematoxilin and eosin staining of rNu hearts with respectively cmtmr+ hAFS and h CD133+ injection, magnification 2,5x : LV = left ventricule; c) and f) Masson’s trichrome staining of rNu hearts with respectively cmtmr+ hAFS and h CD133+ injection magnification 2,5x: in blue the collagen patch and the cryoinjury area, in brown-red intact myocardium. Figure 8. Force transducer bioreactor (A) The bioreactor explosion. The bioreactor is constituted by a frame in polycarbonate (A1,A4) and by a central part in PDMS A2 (well 8x16 mm 20mm height) and glass A3. The devices are assembled from layers of PDMS and glass, attached via plasma treatment of both surfaces. (B) Side view of the bioreactor. The electrical stimulation is guarantee during the culture by 2 carbon rods (8mm length, 2mm diameter) set in the central well. The sensor is inserted in the 2mm diameter hole made in the top part of the polycarbonate frame. (C). Image of the assembled bioreactor. Figure 13. Evaluation after the surgery of the aortic valve engrafted in the pulmonary position. A turbulent flow can be observed inside the bioprosthesis.