Preclinical testing of tissue-engineered heart valves re-endothelialized under simulated physiological conditions

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Angenommen vom Senat der Medizinischen Hochschule Hannover am 12.05.2009

Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover

Präsident: Professor Dr. Dieter Bitter-Suermann

Betreuer der Arbeit: Professor Dr. Axel Haverich

Referent: Professorin Dr. med. Denise Hilfiker-Kleiner

Korreferent: PD Dr. med. Kambiz Norozi

Tag der mündlichen Prüfung: 12.05.2009

Promotionsausschussmitglieder:

Professor Dr. Hermann Haller

Professor Dr. Klaus Otto

Professor Dr. Christoph Klein

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Meine Mutter

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Aus der Abteilung Herz-, Thorax-, Transplantation und Gefäßchirurgie der Medizinischen Hochschule Hannover

(Leiter: Prof. Dr. med. Axel Haverich)

Preclinical Testing of Tissue-Engineered Heart Valves Re-Endothelialized under Simulated Physiological Conditions

Dissertation zur Erlangung des Doktorgrades der Medizin in der

Medizinischen Hochschule Hannover

Vorgelegt von Igor Tudorache

aus Chisinau, Moldawien

Hannover 2008

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3. Literaturverzeichnis... 13

4. Lebenslauf... 15

5. Danksagungen... 22

6. Erklärung nach § 2 Abs. 2 Nr. 5 und 6 PromO... 23

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ISSN: 1524-4539

Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX

DOI: 10.1161/CIRCULATIONAHA.105.001206 2006;114;559-565

Circulation

Haverich

Ringes-Lichtenberg, Matthias Karck, Gudrun Brandes, Andres Hilfiker and Axel Tudorache, Heidi Goerler, Joon-Keun Park, Denise Hilfiker-Kleiner, Stefanie

Artur Lichtenberg, Igor Tudorache, Serghei Cebotari, Mark Suprunov, Greta Simulated Physiological Conditions

Preclinical Testing of Tissue-Engineered Heart Valves Re-Endothelialized Under

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Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-␣-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n⫽5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n⫽7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-␣-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types.

Conclusion—In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.(Circulation. 2006;

114[suppl I]:I-559–I-565.)

Key Words:tissue engineering 䡲 surgery 䡲 valves

S

ince the first successful prosthetic treatment of heart valve disease in the early 1960s, scientists and surgeons have sought for the ideal prosthesis for valve replacement.^1,2 Tissue engineering (TE) may help to construct viable valve substitutes that feature lifetime durability and growth potential.³Various concepts using polymer biodegradable or biological allogeneic and xenogeneic decellularized scaffold were already developed.^{4 – 8}

Detergent decellularization of allogeneic biological valve tissue provides a promising approach for the valve scaffold production.⁴ Because of higher immune histocompatibility, comparable anatomic geometry, and structure, allogeneic

scaffolds might be used successfully for TE despite the limited availability of human material.^5,9

The need for repopulation of valve scaffolds either in vitro or in vivo and the choice of cells is, however, still controversially discussed.^{3–5,8 –11}Efficient tissue regeneration has been obtained by repopulation of biological decellularized scaffolds with interstitial cells in vivo.^8,10Interestingly, an extensive re-endothelialization of such valves after implantation in animals was apparently absent.¹⁰ The importance of endothelium for anti- thrombogenic response and modulation of underlying interstitial cells are well-known^3,5,12,13and might be a significant factors for the long-term function of TE constructs.

From the Departments of Thoracic and Cardiovascular Surgery (A.L., I.G., S.C., M.S., G.T., H.G., S.R.-L., M.K., A.H., A.Haverich), Nephrology (J.-K.P.), Cardiology and Angiology (D.H.-K.), and Laboratory for Cell Biology and Electron Microscopy (G.B.), Medical School Hannover, Hannover, Germany.

A.L. and I.T. contributed equally to this work

Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.

Correspondence to Artur Lichtenberg, Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail lichtenberg.artur@mh-hannover.de

Circulationis available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.105.001206 I-559

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Hemodynamic forces act on the endothelium by shear- stress that can modulate endothelial cell (EC) structure and function.^4,13–16 In vitro adaptation of re-seeded ECs on shear-stress forces could improve its structure and function.⁴ Here, we present preclinical in vivo testing of detergent decellularized allogeneic pulmonary valve conduits (PV) re-endothelialized under simulated physiological conditions in a valve bioreactor system in comparison with only decellularized non-reseeded PV.

Materials and Methods

All animal experiments and surgical procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animalsas published by the US National Institutes of Health (NIH Publication 85-23, revised 1996) and were approved by the local animal care committees. The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.

Decellularization

Decellularization and re-endothelialization of PV were performed as described (see online supplement).⁴

Cell Source and Culture

Isolation and cultivation of ECs from juvenile ovine jugular veins were described (see online supplement).⁴

Re-Endothelialization Under Simulated Physiological Conditions

Cell reseeding was performed in a dynamic bioreactor system with pulsatile circulation described previously (see online Figure I).⁴

Implantation of PV Conduits

Re-endothelialized PV (EPV) and decellularized PV (DPV) (n⫽7 each) were implanted into merino lambs (age 10 to 12 weeks, 20 to 25 kg, obtained from the local breeder [Lower Saxony]) in orthotopic position as described (see online supplement).^7,17Donor of ECs and recipients of EPV were identical (autologous conditions).

Echocardiography

Echocardiography was performed immediately after surgical implantation and subsequently at 4 and 12 weeks postoperatively using a Sonos 2500 (Philips) as previously described.⁷

Explantation of Pulmonary Valve Conduits

Animals were heparinized, then euthanized (1 mL/kg body weight pentobarbital intravenous; Provet). The PV were excised under aseptic conditions, macroscopically examined, and photographically documented. Specimens of valvular leaflets, pulmonary artery wall, and anastomotic suture rings were formalin fixed and paraffin- embedded, or embedded in freezing medium (Jung) and stored at

⫺80°C. For scanning electron microscopy (SEM), samples of PV were fixed in 2.5% glutaraldehyde (Polyscience) in 0.1 mol/L sodium cacodylate buffer (Merck) as described.⁴Additionally, both lungs were investigated macroscopically for signs of pulmonary embolism.

Histology and Immunohistochemistry

For histological analysis formalin-fixed tissues were stained with hematoxylin-eosin (H&E), Movat-pentachrome, or von Kossa.

Immunohistochemistry were essentially performed as described (online supplement).^4,7,18,19

SEM

SEM was performed as described (see online supplement).⁴

Statistics

All data are reported as mean⫾SD. The unpaired Studentttest was used for analyses. Statistical significance was defined asP⬍0.05.

The SPSS statistical software package 11.0 for Windows (SPSS) was used for statistical analysis.

Results

Morphology of Decellularized and

Re-Endothelialized Pulmonary Valve Conduits After detergent treatment of PV followed by DNase I diges- tion, DPV samples showed⬍5% of residual DNA compared with native tissue samples.⁴Histology, immunohistochemistry and SEM revealed an efficiently preserved 3-dimensional network of the scaffold including collagens, elastic fibers, and glycosaminoglycans with complete maintenance of the basement membrane (BM) all along of the inner surface of the pulmonary wall and on both sides of the leaflet (Figure 1A, 1C, 1E, 1F, 1G).

After re-endothelialization the luminal surface of PV was covered with a confluent cell layer (Figure 2A, 2B). Reseeded cells expressed von Willebrand factor (vWF) and endothelial nitric oxide synthase (eNOS) (Figure 2C, 2D), demonstrating an endothelial origin. No interstitial cells were detected on the conduit surface or inside the scaffold (data not shown).

Postoperative Period

All animals survived the operative procedure. As a result of valve endocarditis, the loss of a DPV and an EPV animal occurred after 25 and 39 days, respectively (Figure 3A).

These animals were excluded from further analysis. DPV and Figure 1.Matrix structure integrity after detergent decellularization. Movat-pentachrome staining shows preservation of collagens (yellow), elastic fibers (red) and glycosaminoglycans (blue) in DPV (A) compared with native PV (B). SEM of a transversal sec- tion of DPV leaflet demonstrates the presence of the BM (arrow) on the luminal surface, but no cells (C), whereas a cell layer (arrow) is present on the native leaflet (D). Immunohistochemis- try demonstrates maintenance of collagen-I (E), collagen-IV (F), and laminin (G) in the valve leaflet of a DPV. DPV n⫽5. Bars in A, B⫽100␮m; in C, D⫽10␮m; in E, F, G⫽1 mm.

I-560 Circulation July 4, 2006

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EPV were electively explanted 1 (n⫽3 each) or 3 months after operative procedure (n⫽3 each).

Echocardiography and Transvalvular Pressure Gradient

Echocardiographic parameters of all studied PV were similar (Table 1). In situ, all implanted valves (DPV and EPV) showed no significant insufficiency or stenosis and no gross morphological changes. The effective orifice valve area in- creased slightly in both groups after 3 months; however, these differences failed to reach statistical significance (Table 1).

In all animals no significant increase of transvalvular pressure gradient (TPG) was observed as compared with measurements immediately after implantation.

Macroscopic Evaluation

No signs of augmented inflammation or adhesions were found in animals in the area around the conduits 1 or 3 months after implantation (EPV, Figure 3B; DPV data not shown). Three months after implantation, all DPV showed moderate thrombotic structures on the leaflets and in the valve sinus (Figure 3C). In one DPV explanted after 3 months, moderate leaflet thickening and sclerosis were observed (Figure 3D).

In contrast, all EPV showed a smooth surface of the excellent maintained conduit wall and fine and translucent leaflets without vegetations, fenestrations, calcification, swelling, or thrombi after 1 (Figure 3E) and 3 months (Figure 3F) and were comparable with native tissue (data not shown).

The subvalvular area appeared normal and was free of macroscopic calcification (Figure 3E, 3F).

Histological, Immunohistochemical, and SEM Analysis of Explanted EPV and DPV

EPV explanted after 1 or 3 months showed a homogeneous confluent cell monolayer with typical cobblestone-like EC

morphology overlying the BM of both leaflet sides and the pulmonary wall (Figure 4A, 4B). These cells expressed eNOS and vWF (Figure 4C, 4D), indicating an endothelial character.

In contrast, 1 month after implantation no ECs were detected on the surface of DPV (online Figure IIb), and 3 months after implantation, inhomogeneous clusters of cells with EC morphology partially covered the BM of the DPV. In addition, a fibrin net and conglomerates of thrombocytes were observed on DPV (Figure 4E, 4F).

Interstitial cells were found in the wall of both DPV and EPV 1 month after implantation, whereas the leaflets were mostly free of interstitial cells (EPV, Figure 5A, 5B; DPV, online Figure IIa, IIb). The higher density of cells in the outer regions of the wall is suggestive to an invasive migration of these cells from the out side and not from the vessel lumen (Figure 5A). Cells in the valve interstitium expressed SM-␣- actin indicative for a myofibroblast identity (EPV, Figure 5C;

DPV, online Figure IIc). The positive procollagen-I immu- nostaining suggested that newly invaded interstitial cells synthesized new matrix components (EPV, Figure 5D; DPV, online Figure IId). Three months after implantation, the conduit walls were repopulated with homogenously distributed interstitial cells, whereas the leaflets were repopulated only partially and predominantly in the proximal leaflet segments (EPV, Figure 5E, 5F; DPV, online Figure IIe, IIf).

Figure 3.Macroscopic morphology of explanted valves. Infec- tive valve endocarditis in an EPV animal (39 days) (A). External view on an EPV in situ (arrows) (3 months) without graft dilata- tion, extensive adhesion, or inflammation around the graft (B).

DPV (3 months) with thrombotic formations (arrow) on the leaflet and in the valve sinus (C) and leaflet sclerosis (arrow) (D). Main- tained translucent leaflets (arrows) with no signs of structural deterioration in the wall and subvalvular area of EPV explanted after 1 (E) or 3 (F) months.

Figure 2.Complete re-endothelialization of the decellularized valve matrix under simulated physiological conditions. H&E staining shows reseeded cells (arrows, dark purple) on both sides of the leaflet (A). SEM image shows a confluent EC layer with typical cobblestone-like morphology (B). Cell monolayer of an in vitro reseeded leaflet stains positive for eNOS (arrows, dark brown) (C) and vWF (arrows, dark brown) (D). EPV n⫽5.

Bar in A⫽100␮m; B⫽10␮m; C, D⫽1 mm.

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In DPV neointima hyperplasia was significantly higher than in EPV and more distinct in the sinus area. Neointimal cells in the sinus of DPV, identified by SM-␣-actin staining as myofibroblasts (Figure 6A), showed a high synthesis of procollagen-I (data not shown). For comparison, in the sinus area of EPV only few neointimal cells were detected (Figure 6B).

Macroscopically evident moderate thrombotic formations on the luminal surface of DPV (Figure 3C, higher magnification) are present in the valve sinus of H&E-stained sections with first signs of calcification (Figure 7A, 7B). No thrombotic formations (Figure 7C, higher magnification) or calcification (data not shown) were observed in the valve sinus of EPV. No signs of calcification were found inside the valve scaffold in DPV or EPV (Figure 7D, 7E).

Lung Examination

Lung arteries in all elective sacrificed animals were free of embolisms (data not shown).

Discussion

This study demonstrates for the first time to our knowledge that in vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improved the EC coverage of the matrix and avoided thrombotic formations and neointimal

hyperplasia within 3 months after implantation in a large animal model.

The maintenance of ECM components directly involved in cell-matrix interaction is a crucial aspect for effective re-endothelialization after decellularization of PV. It has been shown that the BM serves as an adhesion platform for EC through endothelial adhesion ligands such as collagen IV, laminin, and perlecan, which is prerequisite for stable EC–

matrix connections.²⁰We have shown that decellularization of PV with detergents allowed an adequate preservation of main ECM structures including collagens, elastins, and glycosaminoglycans.⁴ Moreover, this method proved to be highly efficient for removal of native cells, the main source of immunogenicity. Future investigations should determine the clinical relevance of potential retained antigens and possible immunogenic response induced by the decellularized construct.

It seems that the absence of an endothelium on decellularized matrices may predispose the unprotected matrix surface to thrombosis and intimal hyperplasia with subsequent graft failure.^5,21These negative effects might depend on the methods of decellularization and may affect the quality of the retention of the BM. We demonstrate that detergent DPV with preserved BM show re-endothelialization in a sheep model with moderate thrombotic formations and neointimal hyperplasia. The origin of EC on the re-endothelialized DPV is not clear but we suspect the involvement of circulating EC and/or endothelial progenitor cells.

Echocardiographic and Hemodynamic Evaluation

Studied Groups EPV DPV

1-month follow-up (n⫽3) (n⫽3)

Insufficiency after implantation 0.0⫾0.0 0.3⫾0.6

Previous explantation 0.0⫾0.0 0.3⫾0.6

Effective orifice area (cm) after implantation

1.3⫾0.1 1.2⫾0.2

Morphology after implantation 0.0⫾0.0 0.0⫾0.0

TPG (mm Hg) after implantation 9.3⫾1.5 9.3⫾1.5

3-month follow-up (n⫽3) (n⫽3)

Insufficiency after implantation 0.3⫾0.6 0.3⫾0.6

Effective orifice area (cm) after implantation

1.2⫾0.1 1.1⫾0.1

Morphology after implantation 0.0⫾0.0 0.0⫾0.0

TPG (mm Hg) after implantation 8.7⫾1.5 8.3⫾2.3

TPG indicates transvalvular systolic pressure gradient.

PV insufficiency: absent⫽0, trivial⫽1, moderate⫽2, and severe⫽3. PV morphology: normal⫽0, mild thickening without structural abnormalities⫽1, mild structural abnormality without functional loss⫽2, structural deformation with functional loss⫽3, and severe valve deformation with complete loss of function⫽4.

Continuity equation method was used for calculation of the valve effective orifice area. No statistical significant differences were found between DPV and the EPV.

Figure 4.Confluent cell monolayer on the surface of EPV but not DPV leaflets. SEM images show a confluent EC monolayer on an EPV leaflet 1 (A) or 3 (B) months after implantation. Fluo- rescence microscopy on EPV leaflets (3 months) shows eNOS (arrows, green), BM (anti-perlecan, red), and nuclei (DAPI, blue) (C). Immunohistochemistry shows vWF (arrows, brown staining;

nuclei are stained purple) (D). SEM images of DPV leaflets (3 months) show inhomogenously distributed cells overlying the BM partially covered with a fibrin net (arrows) (E) and aggrega- tions of adherent thrombocytes (arrows) (F). Bars in A, B⫽20␮m; C, D⫽50␮m; E⫽10␮m; F⫽5␮m.

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In contrast to explanted DPV, explanted EPV were devoid of thrombotic formations and neointimal hyperplasia and were covered with an endothelial monolayer expressing eNOS and vWF, suggestive for functional endothelium. This finding suggests that the in vitro gen- erated EC monolayer may stay intact on implantation and seems to be superior to DPV. In this regard, shear-stress application in vitro is needed for maintenance of EC activity especially for cell adhesion.14,16,22,23Our previous in vitro analysis showed that EC maintained under static conditions are washed off when high flows were applied.⁴ In the present study, we therefore used valves accustomed to physiological flow conditions in vitro,⁴ and show that these valves have excellent functionality in vivo. However,

we have not compared EPV with other methods of intro- ducing EC on valve conduits in our in vivo model. In addition, we were not able to define the origin of EC on EPV after explantation and therefore it might also be possible that the in vitro re-endothelialization has modified the decellularized matrix in such a way that an in vivo repopulation with EC occurred more efficiently then in DPV. The precise mechanism(s) of in vivo re-endothelialization in DPV and EPV will be addressed in future experiments.

Despite the higher incidence of thrombotic formation and neointima formation in DPV 3 months after implantation, both study groups (EPV and DPV) revealed excellent valve function with a low transvalvular gradient and only a slight increase of the effective orifice area, with no signs of significant valve regurgitation or stenosis. Cer- tainly, only long-term experiments in animals and subsequent clinical evaluation will uncover a potential superi- ority in respect to function of EPV compared with DPV.

The biological base for growth is the interstitial repopulation of decellularized matrices with cells. Current investigations showed that on implantation of acellular valve tissue almost complete repopulation with myofibroblasts and fibroblasts occurred within 1 year.^10,24This process is reported to be likely cell migration-related inside the matrix.^8,10

Three months after implantation interstitial cells were homogenously distributed in all areas of the DPV and EPV wall and the valve sinus tissue with a distinct increase of cell numbers compared with grafts 1 month after implantation, supporting the notion that the migration process proceeded continuously and occurs apparently from out- side by beginning in the adventitia toward the luminal surface. These observations suggest that our decellularization method allowed efficient interstitial cell repopulation Figure 5.Repopulation of valve conduit and leaflet interstitium

in EPV. Representative H&E staining shows interstitial cells in the conduit wall (arrows, outer region) of an EPV (1 month) (A).

Fluorescence microscopy of a leaflet shows a monolayer of eNOS positive EC (green) with nuclei (DAPI, blue) on the surface, but no nuclear staining (absence of cells) in the matrix of an EPV (1 month) (B). Immunohistochemistry identifies sm-␣- actin–positive myofibroblasts (brown) (C), and anti-procollagen-I staining indicates newly synthesized matrix collagens (spotted brown) (D) in the valve conduit of an EPV (1 month). H&E staining shows evenly distributed interstitial cells in the conduit wall (arrows, outer region) (E) and partial repopulation of the leaflet interstitium (F) of an EPV (3 months). Bars in A, C, D, E⫽200␮m; B, F⫽100␮m.

Figure 6.Differences in neointima formation between DPV and EPV (3 months). Immunohistochemistry with anti-sm-␣- actin (brown) shows extensive neointimal hyperplasia of myofibroblasts in the valve sinus (double red arrow) on the primary matrix surface (black arrows) of DPV (A). No neointimal formation was found on the primary matrix surface of an EPV (black arrows), whereas SM-␣-actin staining marks interstitial cells in the recellularized EPV matrix (B). Bar in a⫽100␮m;

b⫽200␮m.

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and reseeding had no influence on interstitial cell repopulation of valves. The leaflets as a result of anatomically greater distance from the external valve layers were repopulated predominantly in the proximal segment and therefore after three months still not complete. Conforming to the long-term observations of other groups, we believe that decellularized valve leaflets may be evenly repopulated after a longer period time.^8,10

Re-endothelialization and interstitial invasion may depend on the regeneration capacity of the individual recipient, and therefore dependent on age and health status. Regenerative abilities of elderly animals seem to be lower than of juvenile sheep. This could influence the migrative and proliferative function of cells and their capacity for matrix repopulation. In this respect, the use of lambs represents a study limitation. In future studies we will therefore perform similar experiments in elderly, fully grown sheep. Moreover, long-term studies are required to validate the benefits of the presented decellularization method as compared with other alternative approaches.

In conclusion, here we present a protocol for the tissue engineering of endothelialized heart valve prostheses that show excellent valve function in vivo with a low transvalvular gradient, only a slight increase of the effective orifice area, no signs of significant valve regurgitation or stenosis, and no thrombotic structures or hyperplasia.

Source of Funding

This work was supported by Fo¨rdergemeinschaft Deutsche Kinderherzzentren.

Disclosures

None.

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Figure 7.Thrombotic formation and calcification in DPV. H&E staining shows a thrombus in the valve sinus (A) and calcification indi- cated by von Kossa staining inside the thrombus (B) of a DPV (3 months). In the valve sinus of EPV, no thrombotic formations were observed (C). DPV (E) and EPV (F) showed no signs of calcifications inside the valve scaffold. Bars in A, B, C⫽500␮m; D, E⫽200␮m.

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2. Zusammenfassung

Herz-Kreislauferkrankungen stellen die häufigste Todesursache weltweit dar. Pathologische Veränderungen der Herzklappen sind dabei eine bedeutende Untergruppe mit weltweit geschätzten 250.000 Herzklappenoperationen pro Jahr, davon etwa 80.000 in Deutschland. Zur Verfügung stehen derzeit mechanische und biologische Klappenprothesen. Nachteil der mechanischen Prothesen ist die lebenslang erforderliche Antikoagulation. Es liegt eine lineare Inzidenz von Thromboembolien und schweren Blutungen bei jeweils 1 bis 3 % je nach Klappentyp vor. Die biologischen Prothesen haben trotz besserer hämodynamischer Parameter den Nachteil, dass sie mit der Zeit, abhängig vom Patientenalter, degenerieren. Nach 15 Jahren sind bei den Zwanzig- bis Vierzigjährigen noch 30 % der Klappen frei von strukturellen Veränderungen, bei den über Siebzigjährigen noch 90 %. Diese Klappen bestehen aus porcinen Xenografts oder bovinen perikardialen Xenografts. Mit zunehmendem Alter der Patienten steigt die Rate der antikoagulationsbedingten Komplikationen an, deswegen werden mechanische Prothesen überwiegend bei jüngeren Patienten verwendet. Bei älteren Patienten (> 65 Jahre) werden in der Regel biologische Prothesen implantiert. Für Frauen im gebärfähigen Alter stellt die Antikoagulation eine Kontraindikation für eine Schwangerschaft dar. Aufgrund der erhaltenden synthetischen Komponente sind beide Prothesenarten mit einem Infektionsrisiko behaftet.

Allografts sind eine Alternative zu mechanischen oder biologischen Prothesen und haben einige Vorteile diesen gegenüber. Homovitale und kryopräservierte Allografts bestehen aus lebensfähigem Gewebe, welches relativ resistent gegenüber Infektionen ist und physiologische hämodynamische Flusseigenschaften besitzt. Andererseits löst die Zellviabilität Immunreaktionen aus, die möglicherweise zur späteren Degeneration und Verkalkung der Klappe führen. Antibiotika-sterilisierte humane Allografts haben eine begrenzte Haltbarkeit wegen des Fehlens von lebenden Zellen innerhalb der Matrix. Aufgrund von fehlender Wachstumsfähigkeit sind alle bis jetzt dargestellten Grafts für den pädiatrischen Herzklappenersatz nur beschränkt anwendbar und müssen daher in regelmäßigen Abständen ausgetauscht werden. Die dadurch erforderlichen Re-Operationen stellen ein erhöhtes Letalitätsrisiko dar.

Als Alternative zu den genannten Prothesen könnte ein Klappenersatz mit den Methoden des Tissue Engineerings (TE) hergestellt werden, die nicht thrombogen, weniger infektanfällig, und nicht

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bekannt und könnte ein wichtiger Faktor für die langfristige Funktion der Gewebetechnikkonstruktion sein. Hämodynamische Kräfte wirken auf das Endothelium in Form von Schubspannung, welche endotheliale Zellen-(EG)-Strukturen und -Funktionen modulieren kann. Hier präsentieren wir vorklinische In-vivo-Tests von Detergenz Dezellularizierten allogener Pulmonalklappenconduits (PV) re-endothelialized unter simulierten physiologischen Bedingungen in einem Klappen-Bioreaktorsystem im Vergleich mit nur Dezellularizierten, nicht rebesiedelte Pulmonalklappenconduits.

Alle Tierversuche und chirurgischen Eingriffe wurden in Übereinstimmung mit dem Leitfaden für die Pflege und Nutzung von Labortieren (National Institutes of Health Publication No. 85-23, revidiert 1996) und genehmigt von lokalen Tierpflegeausschüssen.

Klappenconduits, einschließlich kurz unterhalb der Herzmuskelmanschette, Klappenanulus, Klappensegeln und Pulmonalarterienwand (3 cm Länge) wurden von jugendlichen Schafen (Körpergewicht 15 bis 20 kg) unter sterilen Bedingungen entnommen. Dezellularizierung wurde durch Behandlung mit einer Lösung von 0,5 % Natrium-Deoxycholate und 0,5 % Natrium-Dodecylsulfate für 24 Stunden geführt, gefolgt von sechs Waschzyklen (jeder 12 Stunden) in PBS zusätzlich mit 100 IE/ml Penicillin-Streptomycin zur Entfernung etwaiger verbleibender Reinigungsmittel und Zelltrümmer.

Nach dezellularizierung der Pulmonalklappenconduits, gefolgt von DNase I-Verdauung, zeigten die DPV Proben 5 % zum restlichen DNA-Vergleich mit nativen Gewebeproben. Histologie, Immunhistochemie und SEM ergaben ein effizient erhaltenes, dreidimensionales Netzwert des

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Gerüstes, einschließlich Kollagene, elastischen Fasern, und Glykosaminogykanen mit vollständiger Erhaltung der Fundamentmembrane entlang der inneren Oberfläche der Pulmonalarterienwand und beider Seiten des Blättchens. Für die Zellisolation wurden Jugularvenen von jugendlichen Lämmern unter sterilen Bedingungen entnommen. Die endothelialen Zellen (EC) wurden aufgelöst von der Gefäßwand mit 2 % Kollagengase A in M199, resuspendiert in Kulturmedium, bestehend aus Endothelzellkultur Medium-2, ergänzt mit SingleQuot Kit, 10 % fötalem calfserum, Penicillin- Streptomycin (100 µg/ml), und schließlich Samen in Kulturflaschen.

Die Rebesiedelung wurde mit Hilfe eines speziell entwickelten Bioreaktorsystems für dynamische Kulturentwicklung durchgeführt. Das verwendete System war zur Nachahmung der physiologischen Bedingungen eines pulmonalen Kreislaufs in der Lage. Volumenstrom und Pulsation wurden kontinuierlich gemessen und entsprechend angepasst. Druck und Temperatur wurden ständig gewartet und überwacht. Der Gasaustausch in Bioreaktor fand durch eine ständige Oberflächenbelüftung innerhalb der Sauerstoffversorgung/Compliance-Kammer statt. Frisches Gas (durchschnittlich 94 % Luft, 6 % CO2) wurde in den Bereich mittels einer Roller-Pumpe transportiert.

Der PH-Wert in den Zirkulierten CM wurde angepasst durch die Höhe der CO2-Versorgung. Während der dynamischen Rebesiedelung, wurden Lactat-, Glucose-, pO2-, pCO2- und pH-Werte wiederholt kontrolliert. Die sterile Pulmonalklappencondutis wurden in die Bioreaktoren nach der Vorinkubationsmethode in CM für 24 Stunden hinein genäht. In drei Zyklen, insgesamt 1,2 x 107 ECs (zweiter oder dritter Zellpassage), wurde genau in das Klappenlumen durch eine speziell entwickelte Zellbesiedelungbucht injiziert. Auf jeden Besäungsschritt folgte eine 12-Stunden-Periode langsamer Rotation des Bioreaktors (0,1 Rotation/min), wodurch es gelang, für die gesamte Klappenoberfläche optimale Anlagebedingungen zu erreichen. Nach der Einsaat, wurden die Bioreaktoren an eine pulsatile Pumpe angeschlossen, mit einem initial pulsatilen Kreislauf von 0,1 l/min. Der Flussstrom wurde mit 0,1 l/min zweimal täglich erhöht bis ein maximaler Fluss von 2.0 l/min und eine Pulsationsrate von 20 Schlägen/min erreicht wurden. Das mittlere Systemdruck wurde bei 25 +/- 4 mm Hg während der gesamten dynamischen Besiedelung beibehalten.

Nach der Re-Endothelialisierung wurde die Oberfläche des Lumens des Pulmonalklappenconduits mit einer konfluenten Zellschicht bedeckt. Wieder gesetzte Zellen des Willebrand-Faktors und endotheliale

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transvalvulären Druck-Gradienten beobachtet, im Vergleich zu Messungen unmittelbar nach der Implantation. Drei Monate nach der Implantation zeigten alle DPV moderate thrombotische Strukturen auf den Blättchen auf, und im Klappensinus. Im Gegensatz dazu, zeigten alle EPV eine glatte Oberfläche der ausgezeichnet erhaltenen Conduit-Wand und feine sowie durchsichtige Blättchen ohne Vegetationen, Fensterung, Verkalkungen, und waren vergleichbar mit nativem Gewebe. Explantierte EPV zeigten nach einem oder drei Monaten eine homogene konfluente Zellenschicht mit typischer kopfsteinpflasterartiger EC-Morphologie, darüber liegend der BM der beiden Blättchenseiten und der Pulmonalarterienwand. Diese Zellen exprimierene eNOS und vWF, entsprechend einen endothelialen Charakter an. Im Gegensatz wurden einen Monat nach Implantation keine ECs auf der Oberfläche von DPV festgestellt. Einen Monat nach der Implantation wurden interstitielle Zellen in der Wand von beiden DPV und EPV gefunden, dahingegen waren die Segeln größtenteils frei von interstitiellen Zellen. Zellen im Klappeninterstitium drückten Aktin aus, anzeigend für eine myofibroblastale Identität des DPV. Die positive Prokollagen-I-Immunfärbung schlug eine neu eingedrungene interstitielle Zelle synthetisch neu in der Matrix-Komponente vor. In DPV neotintima Hyperplasie war signifikant höher als in EPV und mehr ausgeprägt im Sinus-Bereich. Es wurden keine Zeichen einer Kalzifikation im Inneren des Klappenfundaments in DPV oder EPV gefunden.

Hiermit haben wir gezeigt, dass das Dezellularisieren von Pulmonalklappencondutis mit Detergenzien eine adäquate Erhaltung der wichtigsten ECM-Strukturen einschließlich der Kollagene, Elastine und Glykosaminoglykanen erlaubt. Die Aufrechterhaltung von ECM-Komponenten direkt involviert in der Zell-Matrix-Interaktion ist ein entscheidender Aspekt für eine wirksame Re-Endothelialisierung nach der Dezellularisierung von Pulmonalklappenconduits. Es scheint, dass das Fehlen eines Endotheliums

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von decellularisierten Matrizen die ungeschützte Matrix-Oberfläche zu Thrombose und Intima- Hyperplasie mit späterem Transplantatausfall prädisponieren darf. In diesem Zusammenhang ist für die Aufrechterhaltung der EC-Tätigkeit eine Schubspannungsanwendung in vitro erforderlich, speziell für die Zell-Adhäsion. Trotz der höheren Inzidenz von thrombotischer Bildung und Neointima-Bildung in DPV drei Monate nach der Implantation, ergaben beide Studiengruppen (EPV und DPV) ausgezeichnete Klappenfunktionen mit einem geringen transvalvulären Gradienten und nur einem leichten Anstieg des wirksamen Öffnungsbereichs, ohne Anzeichen für eine signifikante Klappenregurgitation oder Stenose.

Diese Beobachtungen deuten daraufhin, dass unsere Dezellularisierungsmethode eine effiziente interstitielle Zellwiederbevölkerung erlaubt und das Wiedereinsäen hat keinen Einfluss auf die interstitielle Zellwiederbevölkerung der Klappen. Die Segeln als ein Ergebnis der anatomisch größeren Distanz der externen Klappenschichten wurden neu besiedelt, vorherrschend im proximalen Segment und waren daher nach drei Monaten immer noch nicht abgeschlossen. Wir glauben, nachhaltig zu den langfristigen Beobachtungen von anderen Gruppen, dass dezellularisierte Klappenblättchen eventuell gleichmäßig nach einer längeren Zeitspanne neu besiedelt werden.

Zusammenfassend präsentieren wir hier ein Protokoll für die Tissue engineering von endothelialisierenden Herzklappenprothesen, die eine exzellente Klappenfunktion im lebenden Organismus mit einem geringen transvalvulären Gradienten zeigen, mit nur einem leichten Anstieg des wirksamen Öffnungsbereichs, ohne Anzeichen für eine signifikante Klappenregurgitation oder Stenose, und ohne thrombotische Strukturen oder Hyperplasie.

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4. Lebenslauf

Persönliche Information

Name: Igor Tudorache Familienstand: verheiratet Staatsangehörigkeit: Rumänisch Geburtstag: 26 Juli 1974

Geburtsort: Kishinev, Moldawien Eltern: Vasile Tudorache Lidia Tudorache

Ausbildung 1981 – 1990 Schule Nr. 23, Kishinev, Moldawien

1990 – 1991 Rumänisch-Deutsches Gymnasium „M. Kogalniceanu“, Kishinev, Moldawien

1991 – 1997 Staatliche Medizinische und Pharmazeutische Universität

“N. Testemitanu“ Kishinev, Moldawien

1998 – 2002 Facharztausbildung in Allgemeinchirurgie, Klinik für Abdominalchirurgie und Lebertransplantation, Fundeni Institut, Medizinische Universität „Carol Davila“, Bukarest, Rumänien.

July 2001 Microsurgery Course, The University of Texas, MD Anderson Cancer Center, Department of Plastic Surgery, Texas, USA

2002 - 2003 Stipendiat in der Abteilung Herz-, Thorax-, Transplantation und Gefäßchirurgie der Medizinischen Hochschule Hannover und in den Leibniz Forschungslaboratorien für Biotechnologie und Bioartifizielle Organe (LEBAO).

2003 – 2005 Wissenschaftlicher Mitarbeiter in der Abteilung Thorax-, Herz- und Gefäßchirurgie der Medizinischen Hochschule Hannover und in den Leibniz Forschungslaboratorien für Biotechnologie und Bioartifizielle Organe (LEBAO).

Berufserfahrung

1998 – 2002 Assistenzarzt in der Abteilung für Abdominalchirurgie und Lebertransplantation, Fundeni Institut, Bukarest, Rumänien.

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Biological scaffolds for heart valve tissue engineering.Methods Mol Med. 2007;140:309-17.

Tudorache I, Cebotari S, Sturz G, Kirsch L, Hurschler C, Hilfiker A, Haverich A, Lichtenberg A. Tissue engineering of heart valves:

biomechanical and morphological properties of decellularized heart valves. J Heart Valve Dis. 2007 Sep;16(5):567-73; discussion 574.

Lichtenberg A, Tudorache I, Cebotari S, Suprunov M, Tudorache G, Goerler H, Park JK, Hilfiker-Kleiner D, Ringes-Lichtenberg S, Karck M, Brandes G, Hilfiker A, Haverich A. Preclinical testing of tissueengineered heart valves re-endothelialized under simulated physiological conditions. Circulation. 2006 Jul 4;114(1 Suppl):I559-65.

Cebotari S, Lichtenberg A, Tudorache I, Hilfiker A, Mertsching H, Leyh R, Breymann T, Kallenbach K, Maniuc L, Batrinac A, Repin O, Maliga O, Ciubotaru A, Haverich A. Clinical application of tissue engineered human heart valves using autologous progenitor cells. Circulation. 2006 Jul 4;114(1 Suppl):I132-7.

Lichtenberg A, Tudorache I, Cebotari S, Ringes-Lichtenberg S, Sturz G, Hoeffler K, Hurscheler C, Brandes G, Hilfiker A, Haverich A. In vitro re-

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endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions. Biomaterials. 2006 Aug;27(23):4221-9. Epub 2006 Apr 18.

Lichtenberg A, Cebotari S, Tudorache I, Sturz G, Winterhalter M, Hilfiker A, Haverich A. Flow-dependent re-endothelialization of tissueengineered heart valves. J Heart Valve Dis. 2006 Mar;15(2):287-93;

discussion 293-4.

Schultheiss D, Gabouev AI, Cebotari S, Tudorache I, Walles T, Schlote N, Wefer J, Kaufmann PM, Haverich A, Jonas U, Stief CG, Mertsching H.

Biological vascularized matrix for bladder tissue engineering: matrix preparation, reseeding technique and short-term implantation in a porcine model. J Urol. 2005 Jan;173(1):276-80.

Jagodzinski M, Cebotari S, Tudorache I, Zeichen J, Hankermeier S, Krettek C, van Griensven M, Mertisching H. Tissue engineering of long bones with a vascular matrix in a bioreactor. Orthopade. 2004 Dec;33(12):1394-1400.

Abstracts:

S.Cebotari, I.Cucu, I. Tudorache, et.al. “Diagnostic and surgical treatment of perforated peptic ulcer.” 3rd International Student Congress in Surgery “Recent News in Diagnostic and Treatment of Medical-Surgical Emergencies”1994, Kischinew, Moldawien

I.Tudorache, S. Cebotari, et al. "Our experience in surgical

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D. Schultheiss, A. Gabouev, A Pilatz, S. Cebotari, I. Tudorache, H.

Mertsching, N. Schlote, N. Wefer, A. Haverich, CG Stief, U Jonas.

"Neue Alternetiven für das tissue engineering der Harnblase:

Vascularisierte biologische Matrix und mesenchymale Stammzellen des Knochenmarks." 45. Tagung der Vereinigung Norddeutscher Urologen e.V. zusamen mit der 9 Tagung der Berliner Urologischen Gesellschaft e.V. vom 19 bis zum 21 Juni 2003 in der Handelskammer Hamburg – Posterpreis 2003

Cebotari S, Tudorache I, Lichtenberg A, Ciubotaru A, Haverich A. "

Tissue engineered intestinal vascularized patch for surgical reconstruction of myocardium." Anale Stiintifice ale Universitatii de Stat de Medicina si Farmacie “N.Testemitanu”. Probleme Clinico- Chirurgicale Vol. III, 2004, Kischinew, Moldawien

Lichtenberg A, Tudorache I, Cebotari S, Ringes-Lichtenberg S, Haverich A "Tissue Engineering of Heart Valves on Biological Matrix Using Controlled Physiological Dynamic Environment." 2004 Scientific Sessions of the American Heart Association 7-10 Nov. 2004, New Orleans Luisiana USA

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I. Tudorache, S. Cebotari, A.Lichtenberg and A. Haverich "Heart valves tissue engineering using continuous monitoring of biotechnological processes." Leibniz Symposium on Transplantation and Regeneration of Thoracic Organs, 3-4 Dec. 2004, Hannover, Germany. Posterpreis.

Cebotari S, Lichtenberg A, Tudorache I, Leyh R, Mertsching H, Teebken O, Ciubotaru A, Haverich A "Clinical Application of Tissue Engineered Human Heart Valves" Leibniz Symposium on Transplantation and Regeneration of Thoracic Organs, 3-4 Dec. 2004, Hannover, Germany

Lichtenberg, A.; Cebotari, S.; Tudorache, I.; Höffler, K.; Haverich, A.

"Behavior of endothelial cells on the tissue engineered pulmonary valves in high-flow pulsatile bioreactor circulation."

The Thoracic and Cardiovascular Surgeon; 2005,S 1, 53 Main Session – Experimental Cardiac Surgery/Basic Science II)

Igor Tudorache, Artur Lichtenberg, Serghei Cebotari, Gerrit Sturz, Andres Hilfiker, Ludger Kirsch, Christof Hurschler, Axel Haverich

"Heart Valves Tissue Engineering: Biomechanical And Morphological Properties Of Decellularized Porcine Scaffolds."

Third Biennial Meeting of The Society for Heart Valve Disease June 17- 20, 2005, Vancouver BC Canada

Artur Lichtenberg, Serghei Cebotari, Igor Tudorache, Andres Hilfiker, Gerrit Sturz, Michael Winterhalter, Axel Haverich "Cellular Instability Of Tissue Engineered Constructs In high Flow Dynamic Circulation." Third Biennial Meeting of The Society for Heart Valve Disease,June 17-20, 2005, Vancouver BC Canada

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"Detergent Decellularization Of Human Pulmonary Valve Conduits For Tissue Engineering Of Heart Valves." Third Biennial Meeting of The Society for Heart Valve Disease June 17-20, 2005, Vancouver BC Canada

Tudorache I., Cebotari S., Hilfiker A., Ternes W., Lichtenberg A., Haverich A. “Heart Valves Tissue Engineering: Assement Of Detergent Elimination And Susceptibility Of Resulted Matrix To Reseeding With Human Endothelial Cells." Interactive CardioVascular and Thoracic SurgerySuppl. 1 to Vol. 4 (May 2005)

Lichtenberg A, Tudorache I, Cebotari S, Sturz G, Goerler H, Ringes- Lichtenberg S, Karck M, Brandes G, Hilfiker A, Haverich A. "Preclinical testing of tissue engineered heart valves re-endothelialized under simulated physiological conditions." 2005 Scientific Sessions of the American Heart Association 14-16 Nov. 2005, Dallas, Texas, USA

Cebotari S, Mertsching H, Leyh R, Lichtenberg A, Tudorache I, Hilfiker A, Maniuc L, Batrinac A, Repin O, Ciubotaru A, and Haverich "A Clinical Application of Tissue Engineered Human Heart Valves

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Using Autologous Progenitor Cells." 2005 Scientific Sessions of the American Heart Association 14-16 Nov. 2005, Dallas, Texas, USA

A. Ciubotaru, O. Maliga, E. Cheptanaru, S. Barnaciuc, V. Corcea, L.

Maniuc, O. Repin, S. Manolache, A. Batrinac, V. Moscalu, A. Ciornii, S.

Cebotari, I. Tudorache, T. Breymann, G. Tudorache, A. Hilfiker, A.

Lichtenberg, A. Haverich. “Appllication of Tissue Engineered Pulmonary Valves in Surgical Correction of Congenital Heart Diseases.” “Late Breaking News” Session, 35th Annual Meeting of German Society for Thoracic and Cardiovascular Surgery. 19-22 Februar 2006, Hamburg, Germany

I. Tudorache, S. Cebotari, A. Lichtenberg, S. Barnaciuc, E. Cheptanaru, A. Batrinac, O. Repin, A. Ciornii, A. Ciubotaru, A. Haverich. “An Efficient Method for Decellularization of Human Aortic Valve Conduits.” 35^th Annual Meeting of German Society for Thoracic and Cardiovascular Surgery. 19-22 Februar 2006, Hamburg, Germany

Lichtenberg A, Tudorache I, Cebotari S, Suprunov M, Goerler H, Brandes G, Hilfiker A, Haverich A. ”Tissue engineered heart valves re-endothelialized under simulated physiological conditions:

Preclinical testing.” The Thoracic and Cardiovascular Surgeon; S 1, 2006 (Special Session – Basic Science I)

S Cebotari, I Tudorache, A Lichtenberg, A Hilfiker, A Haverich

“Detergent decellularization of heart valves for tissue engineering: Toxic effects of residual detergents on human endothelial cells.” Thorac cardiovasc Surg 2006; 54 S1

K Kallenbach, J Heine, A Schmiedl, A Lichtenberg, I Tudorache, S Cebotari, H Mertsching, A Haverich “Functional and morphological

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Tudorache, E. Cheptanaru, S. Barnaciuc, A. Ciornii, A. Ciubotaru, A.

Haverich. „Decellularization of Homografts and Xenografts for Pulmonary Heart Valve Tissue Engineering: Hannover Method.” 10th Annual Hilton Head Workshop and 2nd Biennial Heart Valve Meeting, March 1-5, 2006, SC, USA.

I. Tudorache, A. Lichtenberg, S. Cebotari, M. Suprunov, G.

Tudorache, S. Ringes-Lichtenberg, A. Hilfiker, A. Haverich

“Controlled Physiological Dynamic Environment in Heart Valves Tissue Engineering.“ 10th Annual Hilton Head Workshop and 2nd Biennial Heart Valve Meeting, March 1-5, 2006, SC, USA.

S. Cebotari, K. Kallenbach, S. Sorrentino, A. Lichtenberg, I.

Tudorache, A. Hilfiker and A. Haverich. „Decellularized versus pre-seeded valvular grafts: in-vivo testing using rat model.” 10th Annual Hilton Head Workshop and 2nd Biennial Heart Valve Meeting, March 1-5, 2006, SC, USA.

A. Hilfiker, A. Lichtenberg, S. Cebotari, I. Tudorache, W. Ternes, and A. Haverich “Assessment of Detergent Removal and

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Receptiveness Of Decellularized Valvular Scaffolds To Human Cells Seeding In Tissue Engineering.” 10th Annual Hilton Head Workshop and 2nd Biennial Heart Valve Meeting, March 1-5, 2006, SC, USA

S. Cebotari, I. Tudorache, A. Lichtenberg, E. Cheptanaru, S.

Barnaciuc, V. Sirbu. O. Maliga, L. Maniuc, O. Repin, V. Corcea, V.

Moscalu, A. Batrinac, A. Ciornii, T. Breymann, A. Hilfiker, A.

Haverich and A. Ciubotaru. “Tissue Engineered Heart Valves in Clinical Practice: First Results." Leibniz Symposium on Transplantation and Regeneration of Thoracic Organs, May 19th and 20th 2006, Hannover, Germany

A. Lichtenberg, I. Tudorache, M. Suprunov, S. Cebotari, G.

Tudorache, J-K. Park, H. Goerler, G. Brandes, T. Breymann, A.

Haverich, A. Hilfiker. „In-vivo repopulation of decellularized bovine jugular valved vein grafts with autologous cells occurs in sheep when implanted in pulmonary valve position.” 36. Jahrestagung der Deutschen Gesselschaft für Thorax-, Herz- und Gefäßchirurgie, 11-14 Februar 2007, Hamburg, Germany

A. Lichtenberg, M. Suprunov, I. Tudorache, A. Cebotari, G.

Tudorache, E. Bagaev, S. Ringes-Lichtenberg, J-K. Park, G.

Brandes, A. Hilfiker, A. Haverich. “Integrative capacity and functional competence of detergent-decellularized xenogenic pulmonary valves.” 36. Jahrestagung der Deutschen Gesselschaft für Thorax-, Herz- und Gefäßchirurgie, 11-14 Februar 2007, Hamburg, Germany

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Vasile Corcea, Liviu Maniuc, Serghei Cebotari; Igor Tudorache;

Thomas Breymann ; Artur Lichtenberg and Axel Haverich „Pulmonary Heart Valve Tissue Engineering: Detergent Decellularization of Human Allografts“

Historic Inaugural Meeting of the World Society for Pediatric and Congenital Heart Surgery May 3-4, 2007 Washington, D.C., United States

Serghei Cebotari; Igor Tudorache; Artur Lichtenberg; Andres Hilfiker;

Eduard Cheptanaru; Sergiu Barnaciuc; Anatol Ciubotaru; Axel Haverich

“fresh” Cell-free Valvular Scaffolds For Surgical Correction Of Congenital Heart Diseases In Pediatric Patients” Fourth Biennial Meeting of The Society for Heart Valve Disease, June 15-18, 2007,New

York NY United States

Serghei Cebotari, Igor Tudorache, Artur Lichtenberg, Andres Hilfiker, Anatol Ciubotaru and Axel Haverich “Heart Valve Tissue Engineering - Experimental and Clinical Results” Invited Lecture at Postgraduated Course “Basic Science”, 21^st Annual Meeting of the European Association of Cardio-Thoracic Surgery, 15-19 September, Geneva, Switzerland

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I. Tudorache, S. Cebotari, A. Hilfiker, G.Tudorache, E. Cheptanaru, S, Barnaciuc, A. Calistru, A. Ciubotaru and A. Haverich ^“Heart Valve Tissue Engineering: Results and Persperctives.”

SRCCV National Conference 2007, Sibiu, Romania

A. Calistru, I. Tudorache, S.Cebotari, A. Hilfiker, A.Batrinac, O. Repin, G.Tudorache, A.Ciubotaruand A.Haverich “Creation of Valve Grafts Using Detergent Decellularization of Human Pulmonary Conduits.”

A. Ciubotaru, E. Cheptanaru; S. Barnaciuc, L. Maniuc, O. Repin, V.

Corcea, O. Maliga, I. Tudorache and S.Cebotari “Cell-free Human Pulmonary Valves For Surgical Correction Of Congenital Heart Diseases.”

Weitere

Berufstätigkeiten

1994 – 1997 Krankenpfleger in der Abteilung Plastischenchirurgie Kischinew, Moldawien

Sprachkenntnisse Rumänisch (Muttersprache), Deutsch, Englisch, Franzosisch, Russisch.

Besondere Interessen

Transplantationchirurgie, Tissue Engineering

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5. Danksagungen

An dieser Stelle möchte ich besonderes meinem Doktorvater, Herrn Prof. Dr. med. Axel Haverich für die Bereitstellung des Themas sowie für die Möglichkeit der Promotion in seiner Klinik danken.

Darüber hinaus danke ich im für die Anleitung zum strukturierten wissenschaftlichen Arbeiten.

Weiterhin danke ich der Europäischen Gesellschaft für Herz- und Thorax- und Gefäßchirurgie und ins besonders Prof. Dr. med. Hans G. Borst, die mir als Osteuropäer die Möglichkeit eines Clinical and Research Fellowship in Herzchirurgie in der Medizinischen Hochschule Hannover ermöglichten.

Für die Unterstützung und Betreuung während meiner wissenschaftlichen Tätigkeit in Deutschland und für die allzeit freundschaftliche Zusammenarbeit bei diesem und anderen Projekten möchte ich ganz herzlich den Herren Dr. med. Serghei Cebotari, PD. Dr. Artur Lichtenberg und Dr. Andres Hilfiker danken.

Für die Hilfestellung und Betreuung im Labor danke ich den Mitarbeitern der Abteilung ‚Experimentelle Chirurgie’ Karin Peschel, Rosie Katt, Astried Diers-Ketterkatt und Petra Ziehme, die zum Erfolg der Arbeit beigetragen haben.

Besonderer Dank gilt Herrn Klaus Höffler für die exzellente Beratung und Unterstützung bei der operativen Durchführung der Versuche.

Und nicht zuletzt möchte ich meiner Familie für die stetige Unterstützung und Ermutigung nicht nur während meines Studiums danken.

Die Laborarbeiten fanden im Leibniz Forschungslaboratorium für künstliche Organe (LEBAO), der Klinik für Herz-, Thorax-, Transplantation und Gefäßchirurgie der Medizinischen Hochschule Hannover statt.

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keine anderen als die dort aufgeführten Hilfsmittel benutzt habe.

Ich habe diese Dissertation bisher an keiner in- oder ausländischen Hochschule zur Promotion eingereicht. Weiterhin versichere ich, dass ich den beantragten Titel bisher noch nicht erworben habe.

Ergebnisse der Dissertation wurden veröffentlicht:

• Lichtenberg A, Tudorache I, Cebotari S, Ringes-Lichtenberg S, Haverich A "Tissue Engineering of Heart Valves on Biological Matrix Using Controlled Physiological Dynamic Environment." 2004 Scientific Sessions of the American Heart Association 7-10 Nov. 2004, New Orleans Luisiana USA

• Tudorache, S. Cebotari, A.Lichtenberg and A. Haverich "Heart valves tissue engineering using continuous monitoring of biotechnological processes." Leibniz Symposium on Transplantation and Regeneration of Thoracic Organs, 3-4 Dec. 2004, Hannover, Germany.

Posterpreis.

Hannover, den

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(Igor Tudorache)