| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Department of Cardiovascular Surgery, Ludwig-Maximilians-University, Munich, Germany
c Institute of Anatomy, Ludwig-Maximilians-University, Munich, Germany
b Department of Physiology, Charité University Medicine Berlin, Berlin, Germany
Accepted for publication November 19, 2009.
* Address correspondence to Dr Sodian, Department of Cardiac Surgery, Laboratory for Tissue Engineering and Cell Transplantation, Ludwig-Maximilians-University, Marchioninistr 15, Muenchen D-81377, Germany (Email: ralf.sodian{at}med.uni-muenchen.de).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Methods: After expansion and differentiation of CD133+ cells, phenotypes were analyzed by immunohistochemistry and cryopreserved. Heart valve scaffolds fabricated from a biodegradable polymer (n = 8) were seeded with blood-derived myofibroblasts and subsequently coated with blood-derived endothelial cells. Afterward, the heart valve constructs were grown in a pulse duplicator system. Analysis of all heart valves, including histology, immunohistochemistry, electron microscopy, fluorescence imaging, and biochemical and biomechanical examination, was performed.
Results: The tissue-engineered heart valves showed endothelialized layered tissue formation including connective tissue between the inside and the outside of the scaffold. The notion of an intact endothelial phenotype was substantiated by fluorescence imaging studies of cellular nitric oxide production and Ca2+ signaling. Electron microscopy showed that the cells had grown into the pores and formed a confluent tissue layer. Biochemical examination showed extracellular matrix formation (77% ± 9% collagen of human pulmonary leaflet tissue [HPLT], 85% ± 61% glycosaminoglycans of HPLT and 67% ± 17% elastin of HPLT).
Conclusions: Importantly, this study demonstrates in vitro generation of viable human heart valves based on CD133+ cells derived from umbilical cord blood. These findings constitute a significant step forward in the development of new clinical strategies for the treatment of congenital defects.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
A different concept in heart valve replacement is tissue engineering [5], introducing the prospect of an ideal heart valve substitute that lasts throughout the patient's lifetime and has the potential to grow with the recipient and to remodel. Since Shinoka's report in 1995, the ultimate goal has been to fabricate a functional and viable trileaflet heart valve substitute from autologous cell sources [6]. Cells of various origins have been used for tissue engineering of heart valves, including peripheral vascular cells [7, 8], bone marrow [9], progenitor cells from blood or amniotic fluid [10, 11], and umbilical cord vascular cells [12, 13]. Although numerous important results have been reported by several investigators, tissue engineering of human heart valves is still in an early stage of development. Optimal scaffold design and advanced cell cultures are required to create a tissue-engineered construct that will eventually become a clinically relevant valve prosthesis.
We have created a tissue-engineered heart valve utilizing cryopreserved human umbilical cord blood derived progenitor cells as a single cell source. In our concept, fetal echocardiography in high-risk pregnancies should be performed so that abnormal cardiac anatomy is identified before birth. Directly after birth, blood derived cells will be isolated, expanded, and differentiated in vitro into cardiovascular cell types. The cells will be cryopreserved and later recultivated at an ideal time point for surgery. Afterward, a biodegradable heart valve scaffold will be seeded with myofibroblastic—and subsequently with endothelial—cells. After maturation in a bioreactor system, the tissue-engineered heart valve can be implanted into the same patient as an autologous valve substitute. In this context, we investigated the feasibility of using cryopreserved differentiated umbilical cord blood derived cells as the single progenitor cell source for tissue-engineered heart valve constructs.
|
Material and Methods |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Cell Isolation and Differentiation
Directly after delivery, 15 mL fresh umbilical cord blood was treated with RosetteSep depletion agent (StemCell Technologies, Vancouver, BC) for 20 minutes and diluted to 35 mL with phosphate-buffered solution–ethylenediamine tetra-acetic acid (PBS-EDTA [Lonza, Basel, Switzerland]). Density gradient centrifugation was carried out on Biocoll (Biochrom AG, Berlin, Germany). Mononuclear fraction was removed and washed with PBS-EDTA. Cells were resuspended in 500 µL Iscove's modified medium (Biochrom) and 2% fetal calf serum (Promocell, Heidelberg, Germany). The CD133+ cells were sorted using an isolation kit according to the manufacturers instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Here, we were able to isolate approximately 1 x 105 cells to 2 x 105 cells from 15 mL fresh umbilical cord blood. After a few days, the cells had formed spheroidlike structures that were transferred to a collagen-coated 6-well plate cell culture dish (as many as 4 x 105 cells per well [TPP, Trasadingen, Switzerland]). Endothelial-like cells were cultured and differentiated in Iscove's medium (Biochrom) supplemented with 5% fetal calf serum (Promocell), 5 µg/mL Insulin (Sigma, St. Louis, MO), 5 ng/mL human epidermal growth factor (Peprotech Tebu-bio, Offenbach, Germany]), 2 ng/mL basic fibroblast growth factor (Peprotech Tebu-bio]), and 1% penicillin-streptomycin (Gibco; Invitrogen, Carlsbad, CA). Myofibroblast-like cells were cultured in standard fibroblast growth medium (Promocell) containing growth factors and supplements, for example, basic fibroblast growth factor (Tebu-bio), 10% fetal calf serum (Promocell), and penicillin-streptomycin (Gibco). After 4 to 6 weeks, sufficient cell numbers (30 x 106 myofibroblast-like cells and 5 to 8 x 106 endothelial-like cells) had been obtained for cryopreservation. In parallel, the endothelial-like and myofibroblast-like phenotypes were analyzed using immunocytochemistry.
Immunocytochemistry
Immunocytochemistry was performed as described before [15], using the following primary antibodies on differentiated myofibroblast cells: smooth muscle 
-actin (SMA [Dako, Glostrup, Denmark]), fibroblast-specific CD90/AS02 (Dianova, Hamburg, Germany) and desmin (Dako) and on differentiated endothelial cells CD31, vascular endothelial (VE)-cadherin, factor VIII, and von Willebrand factor (all Dako). Secondary antibody reaction and detection were performed with a LSAB2 Kit (Dako).
Cryopreservation of Cell Culture and Recultivation
For cryopreservation, cells were transferred to 10% DMSO (Sigma) medium. Subsequently, the cell suspension was transferred into cryotubes (Nunc; Thermo Fisher, Waltham, MA) and cooled to –80°C. The cryotubes were stored in liquid nitrogen for as long as 12 weeks.
After this cryopreservation period, the cryotubes were transferred to 37°C for 2 minutes. Cells were washed in PBS (Biochrom) and seeded in appropriate standard culture medium in 25-cm2 Falcon flasks (BD Biosciences, Erembodegem, Belgium). After cryopreservation and recultivation, 40% to 80% of the cells seemed to be viable, and nutrient media were changed every 3 days over a recultivation time of 3 to 4 weeks until sufficient numbers of cells were obtained for the 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; molecular weight 700 kD) with a porosity of approximately 80% (pore size 200 to 400 µm [Tepha, Cambridge, MA]). 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 at 2-mm slice distance and 0.1 x 0.1 mm pixel size within all slices. The data from computed tomography scans were processed using special software to reconstruct a three-dimensional model, as described previously. Based on this model, a heart valve scaffold including the sinus of Valsalva was fabricated (Fig 2A) [14].
|
Analysis of Tissue-Engineered Heart Valve Constructs
Histology
Sections of all dynamically conditioned tissue-engineered heart valves (leaflets and conduits) were fixed in Karnowsky for 1 hour at ambient temperature, followed by postfixation in 1% OsO4 solution (in 0.1 M phosphate buffer). Sections were rinsed and dehydrated in ascending alcohol series before embedding in Epon. Semithin sections of samples were stained 1 to 2 minutes in 1% Toluidine Blue (Merck, Darmstadt, Germany), rinsed several times in purified water, and examined under a light microscope (Axiophot 100; Zeiss, Jena, Germany).
Immunohistochemistry
The samples of all tissue-engineered heart valves were immersed in O.C.T. embedding medium (CellPath; Newtown, UK) and immediately frozen in liquid nitrogen. Then,10-µm cryosections were cut and methanol-fixed for 10 minutes. Sections of cultures were incubated with primary antibodies collagen I, elastin (both Novocastra Laboratories, Newcastle upon Tyne, UK), and integrin beta 1 (Dako, Glostrup, Denmark) [16], 1:30 in PBS. Secondary antibody was a goat anti-rabbit or goat anti-mouse immunoglobulin conjugated with FITC, GAM-FITC, or GAR-FITC (Dako). The sections were washed, air-dried and covered with Fluoromount mountant. Sections were examined under a light microscope (Axiophot 100, Zeiss, Jena, Germany).
Scanning electron microscopy
A sample of a dehydrated tissue-engineered heart valve construct was sputter-coated with gold at 5 x 10–2 mbar in an evacuated argon atmosphere. Examination of the specimen was performed with a Leo scanning electron microscope (Carl Zeiss SMT AG, Oberkochen, Germany) at 250x magnification [21].
Transmission electron microscopy
Samples of the dynamically conditioned tissue-engineered heart valve constructs were fixed in Karnowsky for 1 hour at ambient temperature followed by postfixation in 1% OsO4 solution (in 0.1 M phosphate buffer) [17]. Cultures were rinsed and dehydrated in ascending alcohol series before embedding in Epon. Sections were cut on a Reichert Ultracut and contrasted with 2% uranyl acetate/lead citrate. A transmission electron microscope (TEM 10; Zeiss, Jena, Germany) was used to examine the samples.
Fluorescence imaging
For fluorescence microscopic analyses, heart valve constructs were positioned under a custom-built upright intravital microscope on a vibration-free table, as previously described [18]. Fluorescence was excited by a near monochromatic beam from a Polychrome IV digitally controlled galvanometric scanner (T.I.L.L. Photonics; Agilent Technologies, Santa Clara, CA). Fluorescence emission was collected through the microscope (AxiotechVario 100 HD; Zeiss) equipped with an apochromat objective (UAPO 40x W2/340; Olympus, Hamburg, Germany) and appropriate dichromic and emission filters (FT 510 and LP 520; all Zeiss) by a CCD camera (Sensicam; PCO, Kelheim, Germany) and subjected to digital image analysis with TILLvisION 4.01 (T.I.L.L. Photonics).
For indirect immunofluorescence, construct samples of 5 x 5 mm2 size were incubated with primary goat anti-human CD31 polyclonal Ab or goat anti-human VE-cadherin polyclonal Ab (both 10 µg/mL [Santa Cruz Biotechnology, Heidelberg, Germany]) for 10 minutes at 37°C, 21% O2 and 5% CO2. Constructs were washed three times in PBS-Dulbecco (Biochrom AG, Berlin, Germany), reincubated with secondary donkey anti-goat FITC-labeled polyclonal Ab (20 µg/mL [Santa Cruz Biotechnology]) for 10 minutes, washed again, and viewed at an excitation wavelength of 480 nm.
For functional imaging, construct samples were loaded with Ca2+ and nitric oxide (NO)-sensitive fluorescent dyes, as previously described [19]. To measure NO production, constructs were incubated for 30 minutes with cell-permeant 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate (5 µM [Molecular Probes, Eugene, OR]), which intracellularly deesterifies to NO-sensitive DAF-FM. Intracellular DAF-FM is converted through an NO-dependent mechanism to an intensely fluorescent benzotriazole derivative with fluorescence intensity linearly reflecting NO concentration. The DAF-FM fluorescence was excited at 480 nm, and fluorescence intensity (F) recorded 5 minutes after stimulation with acetylcholine (0 to 1 mM [Sigma-Aldrich, Taufkirchen, Germany]) was expressed relative to its individual baseline (F0). For quantification of the intracellular calcium concentration ([Ca2+]i), constructs were incubated for 30 minutes with Fluo-3 AM (5 µM [Molecular Probes]), which is intracellularly converted to the Ca2+ sensitive fluorophore Fluo-3. Constructs were viewed at 480 nm at baseline and 5 minutes after stimulation with either histamine (10 µM [Sigma-Aldrich]) or thapsigargin (2 µM [Molecular Probes]), which releases Ca2+ from endosomal stores and stimulates store-operated Ca2+ influx.
Quantification of extracellular matrix elements
To quantify extracellular matrix constituents, Biocolor assays (Biocolor, Carrickfergus, UK) were performed in all tissue-engineered heart valves and in native human pulmonary heart valve leaflet tissue (excised during corrective procedures), according to the manufacturer's instructions. Unseeded polymer was used as control specimen. Type I to type V collagens were extracted with 0.5 M acetic acid (Sigma, St. Louis, MO), 1:50000 protease inhibitor cocktail at 4°C. Sulfated glycosaminoglycans were extracted with a 24-hour, 60°C papain (Sigma) treatment. Elastin was extracted with 100°C 0.25 M oxalic acid (Sigma). Samples and calibrators were treated with the respective dyes and measured in a Spectra Rainbow plate reader (Tecan, Maennedorf, Switzerland) after removal of unbound dye and release of bound dye.
Biomechanical analysis
Mechanical properties of heart valve tissue were analyzed at room temperature using a material testing device (Zwick Roell Group, Ulm, Germany). The specimen was stretched with appropriate clamps until complete tear in uniaxial tension. Passive tensile strength 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. As a control, samples from pulmonary arteries were taken from heart transplant recipients.
|
Results |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
-actin (Fig 1C), and desmin (Fig 1D). The cultured endothelial-like cells formed cobblestone like morphology and were grown in a confluent monolayer and stained positive for CD31 (platelet/endothelial cell adhesion molecule-1 [PECAM-1]; Fig 1E), von Willebrand factor (Fig 1F), VE-cadherin (Fig 1G), and factor VIII (Fig 1H). Sufficient cell numbers for the tissue engineering of heart valves were obtained from both cultures.
|
Histology and immunohistochemistry
Hematoxylin and eosin and Toluidine Blue-stained paraffin section showed compact tissue formation organized in a layered manner with a dense surface layer on both sides of the scaffold and ingrowth into the porous polymer scaffold with a lower cellularity in the inner part (Fig 3A). An endothelial cell lining was found in the CD31 and von Willebrand factor staining on the surface of the tissue-engineered constructs, confirmed by fluorescence imaging (Fig 3B).
|
SMA positive cells were detected throughout the whole construct. Collagen (Fig 3C) and integrin beta 1 (Fig 3D) were found throughout the leaflets and the vascular wall of the bioreactor conditioned constructs. The extracellular matrix proteins connected the inside and the outside of the scaffold and formed a compact tissue-engineered heart valve construct. In fluorescence immunostaining for elastin, a unilateral alignment as well as fiberlike expression regions within the constructs were detected (Fig 3E).
Electron microscopy
Transmission electron microscope of the construct showed dense tissue formation in several layers of cells on the surface with following characteristics: after attaching to the polymeric surface, the endothelial-like cells on the top (asterisk) and the myofibroblast-like cells on the bottom (arrowheads) proliferated at the surface, developing a tissuelike structure (Fig 4A). The endothelial-like cells on the top showed tight cell-cell contact and contained high numbers of vesicles and organelles. The myofibroblast-like cells on the bottom displayed are large, elongated, and contain large numbers of euchromatin in the nucleus, well-developed rough endoplasmic reticulum, many mitochondria in the cytoplasm, and are embedded in large amounts of extracellular matrix. Longitudinal cut of the scaffold showed that a network of extracellular matrix (arrows) fibrils expressed by these cells was observed consisting of small, singularly and irregularly running fibrils in the intercellular spaces that are closely attached to the plasma membrane. Cells contained a well-developed rough endoplasmic reticulum, a large Golgi apparatus, and vacuoles, the usual components of mitochondria and glycogen granules (Fig 4B).
|
Extracellular matrix analysis
Extracellular matrix analysis shows that the amount of collagen in tissue-engineered heart valve constructs reached values up to 77% ± 9% compared with native human pulmonary leaflet tissue. Moreover, 67% ± 17% of elastin compared with native tissue could be detected in the tissue-engineered heart valve constructs. Content of sulfated glycosaminoglycans was 85% ± 61% of native human leaflet tissue (Fig 5).
|
|
|
|
Comment |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Tissue engineering of heart valves aims to overcome the limitations of currently used prostheses by fabricating autologous tissue constructs with the ability to grow and to remodel. This strategy has worked well in animal experiments for patch, vascular, and heart valve tissues [21–23]. Human applications of this concept are rare. Shinoka and colleagues [24] reported the transplantation of a tissue-engineered pulmonary prosthesis fabricated from a polymeric scaffold and autologous venous cells. Seven months after implantation the patient was doing well, with no evidence of graft failure [24].
Previous investigators have 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 [25]. Moreover, they reported the use of amniotic fluid as single cell source enabling the prenatal fabrication of heart valves for potential repair of congenital defects shortly after birth [11]. In contrast to previously reported findings, cryopreservation of cells was used to have a cell source not only for the early weeks after birth (fresh umbilical cord cells or amniotic cells), but also one that lasts several months to years, to potentially fabricate multiple tissue-engineered heart valves and conduits for the ideal time point of surgery. We were able to isolate CD133+ cells from human umbilical cord blood and differentiated these cells into endothelial and myofibroblast cells, indicating similarity to human valvular cells. In this study, sufficient numbers of all phenotypes needed for the tissue engineering of heart valves were obtained. Our findings suggest that creation of tissue-engineered, autologous constructs from a single cell source is possible, even later in infancy or adulthood.
The observation drawn from previous experiments that a dynamic tissue environment stimulates extracellular matrix formation and accelerate the maturation of surgically feasible and implantable, human tissue-engineered heart valves is widely confirmed. Therefore, we used a pulsatile in vitro system that provides biomechanical stimulation combined with continuous pulsatile flow to grow viable, human tissue-engineered heart valves. Seven intact heart valve constructs were generated after 4 weeks of in vitro conditioning (3 weeks static and 1 week dynamic conditioning in a pulsatile flow system) that showed a pale yellow color and shiny surface, indicating connective tissue formation. The macroscopic hypothesis was confirmed by histologic and electron microscopic examination showing compact tissue formation organized in a layered manner and ingrowth into the porous polymer scaffold. Additionally, these examinations demonstrated the formation of extracellular matrix proteins known from native heart valve tissue. Formation of elastin as an important factor of cardiovascular extracellular matrix in our tissue-engineered heart valve constructs was proven. The extracellular matrix proteins connecting the inside and the outside of the scaffold formed a compact tissue-engineered heart valve construct.
In addition to previous experiments by ourselves and other investigators, in this study, we were able to fabricate a construct with an endothelial cell lining surface (positive for CD31, von Willebrand factor, and vascular endothelial cadherin). This finding was corroborated by fluorescence imaging studies of cellular NO production and Ca2+ signaling. Moreover, Fluo-3 imaging of surface cells revealed spontaneous [Ca2+]i oscillations that are characteristic of endothelial cells in a physiological microenvironment. These [Ca2+]i oscillations are a characteristic of endothelial cells in a physiological microenvironment [19], usually absent in cultured endothelia [26], and demonstrate endothelial viability of the construct with characteristic active signaling pathways preserved.
The quantitative matrix analysis showed reduced contents of collagen, elastin, and glycosaminoglycans in all tissue-engineered constructs compared with native pulmonary heart valve tissue. This finding shows that the tissue-engineered constructs are still immature and do not have identical characteristics to native heart valve tissue. Nevertheless, we found a higher matrix concentration of all constructs compared with those fabricated in previous studies using cryopreserved vascular umbilical cord cells. This again shows the positive effect of the viability and functionality of the differentiated progenitor cells in a dynamic cell culture environment.
That we found in vitro advances in tissue formation leads to an appropriate mechanical tissue strength comparable to samples of native tissue. Moreover, the elastic properties were different from those of native heart valve tissue. One potential explanation for this observation might be that the polymer was not degraded, and the mechanical properties of the scaffold are still dominant. Therefore, modification and further improvement of the scaffold material and preseeding treatment is currently under active investigation.
One limitation of our study was a microbiologic contamination of one construct. Although this experiment is one of the early studies that attempt to create human tissue-engineered heart valves from human umbilical cord progenitor cells, there is no question that a sterile cell harvesting technique in an unsterile environment (childbirth) and in vitro conditioning in a highly sterile bioreactor system are critical factors for potential human application of this tissue engineering concept.
In conclusion, this study demonstrates the possibility of the in vitro generation of human cardiovascular constructs based on cryopreserved human umbilical cord blood progenitor cells (CD133+ cells) as a single cell source for the tissue engineering of heart valves. The CD133+ cells showed mostly differentiated properties as well as cardiovascular tissue formation in vitro. Based on these findings, the novel single cell source is feasible for the tissue engineering of viable and functional heart valves. In terms of the potential clinical application of tissue-engineered heart valves, several important questions remain. They relate to a perfect cell source, the optimal scaffold material, advanced in vitro conditioning, and most importantly, full functionality in an in vivo environment. Despite these and other potential questions, our study represents a further improvement in the tissue engineering of heart valves. Therefore, our laboratory is currently focusing on animal experiments to further evaluate the concept and on the development of modified in vitro conditions for potential human application. Moreover, we are currently establishing a tissue engineering concept with undifferentiated umbilical cord blood cells from cord blood banks to more and more simplify the process of tissue engineering of heart valves from umbilical cord blood as a single source.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
