Tissue Engineering of Autologous Human Heart Valves Using Cryopreserved Vascular Umbilical Cord Cells

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1 Tissue Engineering of Autologous Human Heart Valves Using Cryopreserved Vascular Umbilical Cord Cells Ralf Sodian, MD, Cora Lueders, PhD, Liv Kraemer, MD, Wolfgang Kuebler, MD, Mehdi Shakibaei, MD, Bruno Reichart, MD, Sabine Daebritz, MD, and Roland Hetzer, MD Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum Berlin, Departments of Physiology and Cell-Biology and Neurobiology, Charité University Medicine Berlin, and Department of Cardiovascular Surgery, Ludwig-Maximilian- University, München, Germany Background. Tissue engineering of autologous heart valves with the potential to grow and to remodel represents a promising concept in pediatric cardiovascular surgery. Currently we are exploring the impact of cryopreserved human umbilical cord cells (CHUCCs) for the fabrication of tissue-engineered heart valves for patients diagnosed prenatally with congenital heart lesions, potentially enabling heart valve replacement in the early years of life. Methods. Human umbilical cord cells were isolated from vascular segments of umbilical cords and cryopreserved in a cell bank. After 12 weeks the cryopreserved cells were again expanded in culture and characterized by histology, immunohistochemistry, and proliferation assays. Trileaflet heart valve scaffolds were fabricated from a porous polymer (P4HB, Tepha Inc, Cambridge, MA) and sequentially seeded with CHUCCs (n 10). Five of the heart valve constructs were grown for 7 days in a pulse duplicator and, as a control, five constructs were grown under static cell culture conditions for 7 days. Analysis of all tissue-engineered heart valves included histology, immunohistochemistry, electron microscopy, functional analysis, and biomechanical and biochemical examination. Results. We found that CHUCCs remained viable after 12 weeks of cryopreservation and showed a myofibroblast-like morphology that stained positive for -actin and fibroblast specific marker. Histology of the tissueengineered heart valves showed layered tissue formation, including connective tissue between the inside and the outside of the porous scaffold. Immunohistochemistry was positive for collagen (types I, III, and IV), desmin, laminin, and -actin. Electron microscopy showed that the cells had grown into the pores and formed a confluent tissue layer during maturation in the pulsatile flow system. Biochemical examination showed an increase of extracellular matrix formation in constructs after pulsatile flow exposure compared with the static control group. Functional analysis demonstrated a physiological increase of the intracellular Ca 2 concentration of the recultivated cells and the conditioned constructs after stimulation with histamine. Conclusions. This study demonstrates in vitro generation of viable and functional human heart valves based on CHUCCs and biomimetic flow culture systems. The CHUCCs demonstrated excellent growth potential and abilities of in vitro tissue formation. These findings suggest the potential benefit of establishing autologous human cell banks for pediatric patients diagnosed intrauterinely with congenital defects that will potentially require heart valve replacement in the early years of life. (Ann Thorac Surg 2006;81: ) 2006 by The Society of Thoracic Surgeons Accepted for publication Dec 20, Address correspondence to Dr Sodian, Department of Cardiac Surgery, Laboratory for Tissue Engineering and Cell Transplantation, Ludwig- Maximilians-University, Marchioninistr. 15, München, Germany; ralf.sodian@med.uni-muenchen.de. Numerous congenital cardiac defects (eg, tetralogy of Fallot, truncus arteriosus) most often necessitate cardiac valve replacement, using mechanical or tissue valve substitutes [1]. Many heart valve replacements with currently used prostheses increase the survival of patients, especially pediatric patients with congenital defects [2]. Nevertheless, all devices are nonliving and consist of foreign body materials which limit the longterm function. Moreover, beside the typical valve-related problems such as thromboembolic complications, structural or nonstructural dysfunction, and prosthetic valve endocarditis, all heart valve substitutes lack the potential for growth and remodeling [3]. Therefore, many patients who undergo these types of corrections require reinterventions related to these valve-associated problems [4]. Thus, the search for the ideal heart valve substitute that lasts throughout the patient s lifetime and has the potential to grow with the recipient and to remodel is still ongoing. One approach to potentially reaching that goal is tissue engineering [5]. Several investigators, including our own 2006 by The Society of Thoracic Surgeons /06/$32.00 Published by Elsevier Inc doi: /j.athoracsur

2 2208 SODIAN ET AL Ann Thorac Surg CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2006;81: group, are using the techniques of tissue engineering to fabricate a living heart valve substitute. The basic idea is to transplant autologous cells onto a biodegradable scaffold in the shape of a heart valve and thus generate a cell polymer construct in vitro as a living tissue-engineered heart valve [6]. Using this technique, favorable results were reported in vitro and in vivo in several experimental animal models (eg, sheep) [7, 8]. After these studies, researchers started focusing on the tissue engineering of human cardiovascular structures [9, 10]. Regarding tissue engineering of human heart valves, Hoerstrup and colleagues [11] reported the fabrication of functional trileaflet heart valves from human marrow stromal cells. Other researchers described the feasibility of using umbilical cord vascular cells for tissue engineering of cardiovascular patches [12]. In this experiment, polymeric patches were seeded with fresh human umbilical cord cells and investigated after exposure to continuous laminar flow for 14 days. Although numerous important results have been reported by several investigators, tissue engineering of human heart valves is still in a very early stage of development. Currently, tissue engineering of human valve substitutes is limited by the fact that an optimal heart valve scaffold design is required and advanced cell culture conditions are important to create a tissueengineered construct that, it is hoped, will eventually be used to replace human heart valves. In our study we investigated a new approach to fabricating a custom-made tissue-engineered heart valve using cryopreserved human umbilical cord cells. In our concept, congenital cardiac defects are diagnosed intrauterinely by echocardiography. After the birth, the umbilical cord is harvested and vascular cells can be isolated. In order to provide a cell source not only for the early weeks after birth, we cryopreserved vascular umbilical cord cells and investigated the feasibility of using these cells to fabricate tissue-engineered heart valve constructs. Additionally, we used a dynamic cell culture environment, which showed appropriate tissue formation in previously reported experiments [13]. Material and Methods Principal Concept Early fetal echocardiography in high risk pregnancies should be performed in order to diagnose abnormal cardiac anatomy that may necessitate heart valve replacement in the early years of life. Vascular-wall cells of the umbilical cord of such children will be isolated, expanded in vitro, and cryopreserved (cryopreserved human umbilical cord cells, CHUCCs). At the ideal time point for surgery CHUCCs will be recultivated and seeded on a biodegradable heart valve scaffold. The cell polymer construct will be transferred into a dynamic cell culture system ( bioreactor ) and grown in vitro. After maturation in the bioreactor-system the tissueengineered heart valve will be implanted as an autologous valve substitute into the same patient (Fig 1). Cell Isolation and Cultivation Umbilical cords (n 10) were collected and processed within one hour after delivery following the strict guidelines of the Ethical Review Board at the University Hospital Charité, Humboldt University of Berlin, Germany. The artery of human umbilical cord sections, 15 cm in length, was washed with Dulbecco s phosphatebuffered saline (PBS; Gibco BRL, Rockville, MD), minced into 1-mm pieces, and placed in petri dishes (Sarstedt, Germany). The arterial wall sections were cultured in Dulbecco s modified Eagle s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Sigma) and 1% antibiotic solution (Gibco). The cell culture was placed in a humidified incubator at 37 C and 5% carbon dioxide (CO 2 ). The cells were expanded and the medium was changed every 4 days. After 3 to 4 weeks sufficient cell numbers had been obtained and cryopreservation was initiated. Cryopreservation of Cell Culture and Recultivation For cryopreservation, cells were transferred to the previously described cell culture medium supplemented with 10% dimethyl sulfoxide (DMSO; Sigma, St Louis, MO). Subsequently, the myofibroblast cell suspension was transferred into cryotubes (Nunc, Roskilde, DK) and cooled to 50 C with the Icecube 1810 computer freezer (HCI Cryogenics, The Netherlands). Afterward the temperature was reduced to 120 C at 1 C/minute. The cryotubes were stored in liquid nitrogen for up to 12 weeks. After this cryopreservation period the cryotubes were transferred to a water bath at 37 C for 2 minutes. Cells were washed in PBS and seeded in appropriate standard culture medium in 25-cm 2 flasks (Falcon Plastics, Inc, Brookings, SD) as described previously. The recultivated cells reached confluency after 4 to 7 days. The nutrient media were changed every 3 days and over a recultivation time of 3 to 4 weeks a sufficient number of cells were obtained for tissue engineering of heart valves. Polymer Scaffold and Heart Valve Fabrication The polymer used for heart valve scaffold fabrication is a porous and biodegradable poly-4-hydroxybutyrate (P4HB; MW 700,000 by gel permeation chromatography) with a porosity of approximately 80% (pore size 200 to 400 m). The P4HB is a biopolyester produced through a proprietary fermentation process (Tepha Inc, Cambridge, MA) [14]. The polymer is a semicrystalline, thermoplastic elastomer with a melting point of 56 C and a glass transition temperature of 51 C. For heart valve scaffold fabrication a human aortic homograft was scanned with computed tomography (CT) at a 2-mm slice distance and a mm pixel size within all slices. The data from CT scans were processed using special software to reconstruct a three-dimensional stereolithographic model as described previously [15]. Based on this stereolithographic model (Fig 2A) a heart valve scaffold, including the sinus of Valsalva and resembling the anatomic design

3 Ann Thorac Surg SODIAN ET AL 2006;81: CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2209 Fig 1. Tissue engineering technique for fabrication of autologous human heart valves. Fetal echocardiography will be performed in order to diagnose abnormal cardiac anatomy (eg, pulmonary valve malformation). Vascular-wall cells of the umbilical cord will be isolated, expanded in vitro, and cryopreserved. At the ideal time point of surgery the cryopreserved human umbilical cord cells will be recultivated and seeded on a biodegradable heart valve scaffold. The cell polymer construct will be transferred into a dynamic cell culture system ( bioreactor ) and grown in vitro. After maturation in the bioreactor system the tissue-engineered heart valve will be implanted as an autologous heart valve into the same patient. of a native heart valve for potential reconstruction of the right ventricular outflow tract, could be fabricated (Fig 2B). Fig 2. (A) Three-dimensional reconstructed stereolithographic model from the inside of an aortic homograft. (B) Trileaflet heart valve scaffold from porous poly-4-hydroxybutyrate including sinus of Valsalva (seen from the aortic side) fabricated from the stereolithographic model. Cell Seeding and Dynamic In Vitro Environment After recultivation of a sufficient number of the cryopreserved cells, the myofibroblasts were seeded onto polymeric heart valve scaffolds using a conventional dripping technique [16]. In a first step, third passage CHUCCs ( cells/valve) were seeded onto the scaffold and cultured under static conditions for one hour at room temperature. This procedure was repeated twice before the constructs were cultivated under static conditions in a humidified incubator at 37 C and 5% CO 2 for an additional 7 days. The media was changed every 3 days. After incubation, the heart valve constructs (n 5) were transferred into a bioreactor, exposing them to gradually increasing pulsatile flow (300 to 500 ml/minute) and pressure (5 to 15 mm Hg) for 7 days [17]. In parallel, controls (n 5) were seeded with exactly the same cryopreserved and recultivated vascular cell suspension and cultured under static conditions using standard cell culture medium for 14 days. All heart valves (dynamic and static conditioned heart valves) were first conditioned for 7 days, before they were conditioned for an additional 7 days under dynamic or static cell culture conditions. Analysis of Cryopreserved Human Umbilical Cord Cells (Indirect Immunofluorescence Microscopy) Indirect immunofluorescence microscopy was performed with recultivated CHUCCs grown on coverslips in 6-cm cell culture dishes (Sarstedt, Inc, Newton, NC). These cell

4 2210 SODIAN ET AL Ann Thorac Surg CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2006;81: cultures on the coverslips were washed with PBS and fixed for 15 minutes with 4% paraformaldehyde. Cells were incubated for 45 minutes with the following cell specific antibodies: mouse antibodies directed against CD-31 (PECAM-1; Sigma-Aldrich, Münich, Germany), smooth muscle actin (Calbiochem, San Diego, CA), collagen I, III, and IV (Sigma-Aldrich), fibronectin (BD, PharMingen, Heidelberg, Germany), and the fibroblast specific antibody ASO2 (Dianova, Hamburg, Germany) against CD-90 and visualized with goat antimouse immunoglobuline G coupled to fluorescein isothiocyanate (Dianova). Micrographs were taken with an Olympus microscope (BX 61, Hamburg, Germany), SIS ColorView II camera set and the SIS analysis documentation software (Soft Imaging Systems, Stuttgart, Germany). Analysis of the Tissue-Engineered Heart Valves HISTOLOGY. Sections of the static and dynamic conditioned tissue-engineered heart valves were fixed in 4% phosphate-buffered formalin and embedded in paraffin. Afterward, paraffin sections were cut at 2 m and stained with hematoxylin and eosin. IMMUNOHISTOCHEMISTRY. Immunohistochemistry was performed by incubation with monoclonal mouse antibodies for collagen I, III, and IV (Novocastra Lab. Ltd, Newcastle upon Tyne, UK), fibronectin (Novocastra), and -smooth muscle actin (DAKO Inc, Carpinteria, CA), as described previously. TRANSMISSION ELECTRON MICROSCOPY. Sections of the statically and dynamically conditioned tissue-engineered heart valve constructs were fixed in Karnofsky s solution and processed for electron microscopy as described previously [18]. The samples were than embedded in Epon, cut on a Reichert Ultracut (Reichert, Vienna, Austria), and finally contrasted with 2% uranyl acetate-lead acetate. Afterward samples were examined using a transmission electron microscope (TEM 10, Zeiss, Oberkochen, Germany). Functional Analysis of Tissue-Engineered Heart Valves (Imaging of Intracellular Ca 2 Concentration) Cell permeant fura-2 AM (50 g) was diluted in pluronic DMSO solution (20%) and centrifugated at 8,000 rpm. Samples were incubated with 10- M fura-2 solution for 30 minutes in a humidified incubator at 37 C with 5% CO 2. After subsequent washing with PBS, Ca 2 concentration of cells was examined as described previously [19]. Fluorescence images were obtained at 5-second intervals with an exposure time of 50 ms. The CHUCC [Ca 2 ] I was determined from the computer-generated 340 nm:380 nm ratio. Histamine (10 M) was added to stimulate the cells and Ca 2 concentration was measured as described above. Analysis of the Extracellular Matrix Biochemical assays for total collagen (Blyscan assay; Biocolor, Galway, Ireland) was performed as described previously [20]. Biomechanical Analysis Representative sections of statically and dynamically conditioned tissue-engineered heart valves were examined for mechanical properties using an Instron (Series IX Automated Materials Testing System ; Instron, High Wycombe, UK). Biomechanical uniaxial tension testing was carried out at room temperature (22 C). A rectangular-shaped specimen was cut from the tissueengineered heart valve (15 5 mm) using a scalpel, and attached at opposite ends to the test apparatus. One arm of the Instron progressively stretched the specimen (at 2.0 mm per minute) until failure (complete tear). The passive tensile strength of each specimen was continuously recorded during the displacement. Maximal stress (index of maximal tensile strength) was determined by the peak of the curve, whereas tissue resistance to stretch was determined by the slope of the curve. Statistics Quantitative data from the biochemical analysis were shown as mean standard error of the mean. The SPSS software (SPSS Inc, Chicago, IL) was used for statistical analysis. The Mann-Whitney test was performed, considering a p value less than 0.05 as statistically significant. Results As a general result of our evaluation of cryopreserved human umbilical cord cells we were able to obtain an appropriate number of cells for tissue engineering of heart valves. In fact, we obtained from one umbilical cord more than three times the number of cells needed to fabricate one tissue-engineered construct, which theoretically offers the opportunity to generate another construct at a different point in time. The in vitro conditioning process was uneventful and after 7 days no leakage or contamination of the pulse duplicator system was detected. Analysis of CHUCCs The fixed and recultivated CHUCCs showed myoblastlike and elongated fibroblast-like morphology. Staining of the cells revealed expression of anti-aso2, fibronectin, anti-alpha-actin, and collagen (Figs 3A to 3D). The cryopreserved and recultivated cell culture did not stain positive for the endothelial cell marker CD-31 or von Willebrandt factor. Structure of the Tissue-Engineered Heart Valves MACROSCOPY. After maturation in the bioreactor system, explanted heart valves opened and closed correctly and seemed to be intact. All conditioned heart valves showed a shiny yellow surface on the inside and the outside of the heart valve scaffold (Fig 4). Macroscopically there were no differences between the static controls and the dynamically conditioned constructs. HISTOLOGY. Hematoxylin and eosin staining of representative sections of the tissue-engineered heart valves dem-

5 Ann Thorac Surg SODIAN ET AL 2006;81: CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2211 Fig 3. The fixed cryopreserved human umbilical cord cells showed myofibroblast-like morphology. Immunofluorescence staining revealed intracellular expression of anti-aso2 (A), fibronectin (B), anti-alpha actin (C), and collagen (D). onstrated layered tissue formation and cellular ingrowth in the dynamically conditioned constructs (Fig 5A). The controls showed hardly any cellular adherence and tissue formation with only a small number of cells attached to the scaffold and almost no cellular ingrowth (Fig 5B). IMMUNOHISTOCHEMISTRY. Immunohistochemistry revealed positive expression for collagen, fibronectin, and actin in the dynamically conditioned heart valves. Static controls showed less organized extracellular matrix formation (Figs 6A to 6F). ELECTRON MICROSCOPY. Electron microscopy of the dynamically conditioned heart valves showed dense tissue formation and a confluent surface. In contrast, static controls showed only few cells, no cell ingrowth, and a nonconfluent surface (Figs 7A,7B). Functional Analysis of Tissue-Engineered Heart Valves (Imaging of Intracellular Ca 2 Concentration) The functional analysis showed an intact intracellular Ca 2 concentration after stimulation with histamine in the recultivated and cryopreserved cells as well as in the dynamically conditioned constructs. These findings suggest that the cells are still viable and functional after cell culture, cryopreservation, recultivation, and in vitro conditioning (Figs 8A to 8D). Quantitative Analysis Quantitative analysis of the tissue-engineered constructs showed a higher concentration of collagen in the dynamically conditioned heart valves compared with static controls. The quantitative analysis of collagen showed no significant differences between the groups (Table 1). Fig 4. Tissue-engineered heart valve after 7 days of static conditioning and an additional 7 days of dynamic conditioning in a bioreactor system. Mechanical Testing The mechanical properties of the conditioned constructs showed an increase of the tensile strength in longitudinal direction in dynamically conditioned heart valves compared with static controls (Fig 9). The extensibility (strain

6 2212 SODIAN ET AL Ann Thorac Surg CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2006;81: Fig 5. Hematoxylin and eosin staining of the tissue-engineered heart valves shows (A) layered tissue formation and cellular ingrowth in the dynamically conditioned constructs (black arrows). The white arrows show the degrading polymeric scaffold. (B) The controls showed hardly any cellular adherence and tissue formation with only a small number of cells attached to the scaffold and almost no cellular ingrowth (black arrows). The white arrow shows the degrading polymeric scaffold. at maximal load) was decreased, as described in previous experiments, compared with that of native aortic tissue [21]. Comment The inability of heart valves to grow or to remodel is an important source of morbidity and mortality after repair or reconstruction of cardiovascular structures in children and young adults [22]. Over the past 40 years, the prostheses for heart valve replacement during infancy were limited to allografts, autograft pulmonary valves, bioprosthetic valves, and synthetic valves. Each type has limitations, which include their inability to grow, repair, and remodel, and this leads to repeated valve replacement and an increased risk of thromboembolic events and extensive pannus formation [23]. To create a new autologous and viable heart valve, our laboratory focused on the tissue engineering of human heart valves. One approach in tissue engineering is to use autologous cells, seed those cells onto a biodegradable scaffold, and form viable tissue while the matrix degrades. The process of in vitro formation of cardiovascular tissues can be directed through dynamic cell culture conditions. This strategy has worked well in animal experiments for patch, vascular, and heart valve tissues [24, 25]. Human applications of this tissue engineering concept are rare. Shin oka and colleagues [26] reported the transplantation of a tissue-engineered pulmonary artery in a patient with single right ventricle and pulmonary atresia, corrected with pulmonary artery angioplasty and the Fontan procedure, which revealed total occlusion of the right pulmonary artery 7 months after primary correction. For the second operation autologous, venous cells were isolated, expanded in vitro, and seeded on polymeric scaffolds. Ten days after seeding the construct was implanted. Seven months after implantation the patient was doing well, with no evidence of graft failure. Hoerstrup and colleagues [21] reported the use of human umbilical cord cells to fabricate tissue-engineered conduits. In their in vitro experiment fresh human umbilical cord cells were used to generate a vascular construct with morphologic and mechanical properties approximating those of human pulmonary arteries. In our current experiment we introduced the use of cryopreserved human umbilical cord cells to fabricate autologous human tissue-engineered heart valves. This experiment was designed to evaluate CHUCCs as a new cell source for tissue engineering of autologous human heart valves. Our concept presupposes the performance of fetal echocardiography in high-risk pregnancies in order to diagnose abnormal cardiac anatomy that will potentially necessitate heart valve replacement. In these patients vascular-wall cells of the umbilical cord should be isolated, expanded in vitro, and cryopreserved. After 12 weeks of cryopreservation CHUCCs were recultivated and seeded onto polymeric heart valve scaffolds. The cell polymer constructs were transferred into a pulse duplicator system (bioreactor) and grown in vitro. After maturation in the bioreactor system the tissue-engineered constructs were explanted and characterized with histology, immunohistochemistry, electron microscopy, functional analysis, and biomechanical and biochemical examination. In our concept these constructs would be implanted as an autologous valve substitute into the patient from whom the cells were taken. In contrast to previously reported findings we used cryopreserved cells in order to have a cell source not only for the early weeks after birth (fresh umbilical cord cells) but one that lasts several months to years, to potentially fabricate multiple tissue-engineered constructs for the

7 Ann Thorac Surg SODIAN ET AL 2006;81: CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2213 Fig 6. Immunohistochemistry of the heart valves revealed positive expression for collagen (A, black arrows), fibronectin (B, black arrows), and actin (C, black arrows) in the dynamically conditioned heart valves. Static controls showed less organized extracellular matrix formation such as collagen (D, black arrows), fibronectin (E, black arrows), and actin (F, black arrows). The white arrows show the degrading polymeric scaffold. Fig 7. Electron microscopy of the dynamically conditioned heart valves (A) showed layered tissue formation and a confluent surface. In contrast, static controls showed only few cells and no cell ingrowth (B).

8 2214 SODIAN ET AL Ann Thorac Surg CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2006;81: Fig 8. Functional analysis showed an intact intracellular Ca 2 concentration after stimulation with histamine in the recultivated and cryopreserved cells (A and B) as well as in the dynamically conditioned constructs (C and D). ideal time point of surgery [12]. Although we do not know exactly how long we are able to cryopreserve our cells, in the current experiment it was possible to recultivate three times the number of cells potentially needed for the tissue engineering of one heart valve. This suggests that the creation of multiple tissue-engineered, autologous constructs from one single cell source is theoretically possible. In the current study we present a pulsatile in vitro system that provides biomechanical stimulation combined with continuous pulsatile flow to grow viable, human tissue-engineered heart valves. In order to fabricate such constructs in vitro it has been widely confirmed that a dynamic tissue environment stimulates extracellular matrix formation using vascular ovine cells. We hypothesized that dynamic in vitro conditions might be beneficial for developing human cardiovascular structures from CHUCCs and might accelerate the maturation of surgically feasible and implantable, human tissueengineered heart valves. In our experimental setting we worked in a highly sterile environment and found no leakage or microbiological contamination of the system. This is an important finding in the tissue engineering of potentially implantable human constructs. The modification and further improvement of the whole system for Table 1. Quantitative Analysis of the Tissue-Engineered Constructs Showed Higher Concentration of Collagen in the Dynamically Conditioned Heart Valves Compared With Static Controls. The Concentration of Collagen was not Significantly Different Between the Groups (p 0.05) Collagen in % of human aortic wall Static Conditions Dynamic Conditions 30.27% 5.51% 43.87% 12.38% eventual human application is currently under active investigation. Furthermore, we found that CHUCCs can be cryopreserved and recultivated without losing any growth potential physiological characteristics. The cells showed myofibroblast-like morphology, and immunohistochemistry revealed intracellular expression of a fibroblast specific marker (anti-aso2), fibronectin, collagen, and anti-alpha-actin. A limitation of this experiment was that no endothelial cells were seeded and the staining of CD-31 and von Willebrandt factor was, as expected, negative. There is no question that a viable endothelial cell layer would be beneficial for the long-term durability of tissue-engineered human heart valves as well as the reduction of thromboembolic events. However, in this study we focused on the feasibility of using a new cell source (CHUCCs) for the fabrication of functional human heart valve constructs including adequate matrix formation and a high viability of the cells and constructs. Histologic and electron microscopic examination showed that the cells attached to the porous, polymeric scaffold and formed viable, layered tissue in the dynamically conditioned constructs. In contrast, the controls showed only loose and unorganized tissue formation with poor cellular ingrowth. These findings confirm our previous results with vascular human and animal cells. Additionally, they demonstrate the formation of extracellular matrix proteins known from native heart valve tissue. These included expression of collagen types I, III, and IV, fibronectin, and alpha-actin. The quantitative matrix analysis showed reduced contents of extracellular matrix proteins in all tissueengineered constructs compared with native tissue. This finding shows that the tissue-engineered constructs are all immature and do not have identical characteristics to native heart valve tissue. Nevertheless, we found a

9 Ann Thorac Surg SODIAN ET AL 2006;81: CHUCCS FOR TISSUE-ENGINEERED HEART VALVES 2215 Fig 9. Mechanical properties of the conditioned constructs showed an increase of the tensile strength in longitudinal direction in dynamically conditioned heart valves compared with static controls. higher matrix concentration in dynamically conditioned heart valves compared with static controls, which demonstrates the positive effect of pulsatile flow for advanced tissue formation. Modification of the in vitro environment and the polymeric heart valve scaffold may be an important factor for in vitro formation of tissue resembling native tissue and is currently under investigation in cooperation with our research partners. Biomechanical analysis of the constructs demonstrated appropriate tissue strength for potential implantation. The dynamically conditioned heart valve seemed to have slightly better mechanical profiles but the differences did not reach statistical significance. One reason for this finding might be that the mechanical characteristics of the polymer are still dominant after two weeks in an in vitro environment (7 days of seeding and incubation and 7 days of conditioning) because the scaffold is mainly not degraded and still provides the three-dimensional structure for the tissue-engineered constructs. This would also be a potential explanation of why the elastic properties were different from those of native tissue. These findings are in accordance with the quantitative analysis of the matrix formation and the suggestion that tissue formation was still ongoing and the scaffold was not degraded after a relatively short in vitro conditioning time. Furthermore, functional analysis of recultivated CHUCCs and tissue-engineered heart valves showed a physiological increase of intracellular Ca 2 concentration after stimulation with histamine. These findings suggest that the cells not only attached to the scaffold and formed layered tissue but that they were still functional and showing physiological characteristics after cell culture, cryopreservation, recultivation, and in vitro conditioning. This is an important finding of our experiment and shows that it is possible to generate viable and functional human heart valve constructs using CHUCCs and biomimetic flow and pressure conditions. One limitation of our study was that we did not seed any endothelial cells onto our constructs. Although this experiment is a study that attempts to create human tissue-engineered heart valves from cryopreserved vascular human umbilical cells, there is no question that a functional endothelial coverage would be beneficial in a tissue-engineered heart valve. Therefore, to develop a feasible seeding and in vitro conditioning is one of our ultimate future goals. In conclusion, this study demonstrates the possibility of the in vitro generation of human heart valve constructs based on cryopreserved human umbilical cord cells and dynamic flow culture systems. The CHUCCs showed excellent growth properties and thus represent a new feasible cell source for tissue engineering of heart valves and other cardiovascular structures. These findings suggest the potential benefit of a cell source that is not only available for the early weeks after birth (like fresh umbilical cord cells) and does not necessitate additional surgical harvest of an intact autologous vascular structure but lasts several years with the potential to fabricate multiple tissue-engineered constructs for surgery at different time points. Based on these findings it may be desirable to establish an autologous human cell bank for pediatric patients diagnosed intrauterinely with abnormal cardiac anatomy, who are likely to require heart valve or vascular replacement in the early years of life. Although our early in vitro results appear promising and we have fabricated a viable and functional tissueengineered heart valve construct based on CHUCCs, the results are still preliminary and multiple issues remain to be addressed before this concept reaches clinical application. Therefore, our laboratory is currently focusing on animal experiments to further evaluate this concept and on the development of modified in vitro conditions for potential human application. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (462/1-1). We thank David P. Martin, PhD (Tepha, Inc, Cambridge, MA), for his generous support of P4HB and his advice concerning the polymeric scaffold, and Professor Dr Monica Bauer for mechanical testing (Fraunhofer Institut, Teltow). Furthermore, we would like to thank Anne Gale, ELS, of the Deutsches Herzzentrum Berlin for editorial assistance and Annette Gaussmann and Carla Weber for their graphic assistance in the preparation of the manuscript. References 1. Hanley FL, Heinemann MK, Jonas RA, et al. Repair of truncus arteriosus in the neonate. J Thorac Cardiovasc Surg 1993;105: Erez E, Kanter KR, Isom E, Williams WH, Tam VK. Mitral valve replacement in children. J Heart Valve Dis 2003;12: 25 9; discussion Hammermeister K, Sethi GK, Henderson WG, Grover FL, Oprian C, Rahimtoola SH. Outcomes 15 years after valve replacement with a mechanical versus a bioprosthetic valve: final report of the Veterans Affairs randomized trial. 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