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Cardiovascular Research 2003 60(3):497-509; doi:10.1016/j.cardiores.2003.09.002
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Complete dynamic repopulation of decellularized heart valves by application of defined physical signals—an in vitro study

K. Schenke-Layland*,a, F. Opitza, M. Grossa, C. Döringa, K.J. Halbhuberb, F. Schirrmeisterc, Th. Wahlersa and U.A. Stocka

aDepartment of Cardiothoracic and Vascular Surgery, Friedrich-Schiller-University, Bachstrasse 18, 07743 Jena, Germany
bDepartment of Anatomy II, Friedrich-Schiller-University, Jena, Germany
cDepartment of Materials Engineering, University of Applied Sciences, Jena, Germany

*Corresponding author. Tel.: +49-3641-934850; fax: +49-3641-933441. Email address: katja.schenke{at}med.uni-jena.de

Received 14 May 2003; revised 14 August 2003; accepted 3 September 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Cardiovascular tissue engineering is a novel concept to develop ideal heart valve substitutes. The objective of this study was to use decellularized porcine pulmonary valves, ovine cells and dynamic tissue culture to obtain viable and biomechanically stable constructs, resembling native aortic heart valves. Methods: Endothelial cells and myofibroblasts were obtained from ovine carotid arteries. Porcine pulmonary valves were decellularized enzymatically, reseeded and cultured using a hydrodynamic bioreactor system over a time period of 9 or 16 days. Controls were grown over an equivalent time period under static conditions. Specimens of each valve were examined biochemically (cell proliferation, DNA, collagen, 4-hydroxyproline, elastin and glycosaminoglycans), histologically (hematoxylin–eosin, Movat-pentachrome and immunostains) and mechanically (radial and circumferential strength). Results: Histology and biochemical assays demonstrated the removal of almost all cells after decellularization with preservation of the extracellular matrix. Recellularization under pulsatile conditions was significantly improved after 9 and 16 days compared to static conditions. Biochemical and mechanical analysis revealed a continuous increase of cell mass, collagen and elastin contents and strength under pulsatile culture conditions compared to significantly lower values in the static controls. Conclusion: This study demonstrated the superiority of the hydrodynamic approach of cellular reseeding to replace decellularized porcine heart valves with ovine cells with almost complete preservation of extracellular matrix integrity.

KEYWORDS Cell culture/isolation; Extracellular matrix; Remodelling; Tissue engineering; Valve

This article is referred to in the Editorial by W.-H. Zimmermann and T. Eschenhagen (pages 460–462) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiovascular tissue engineering represents a new multidisciplinary approach which combines basic research such as matrix, developmental and cell biology, biochemistry, and materials science with clinical disciplines such as surgery and immunology to replace diseased natural tissues. A major focus is the creation of viable heart valves with the feature to grow, repair and remodel, without immunological response and the ability to be implanted into the systemic circulation. The basic approach uses scaffolds made out of acellular or decellularized bioresorbable materials, formed in the shape of the organ structures and cellular reseeding [1]. Biodegradable polymer scaffolds have been used to reconstruct heart valves. But limitations in cellular adhesion and tissue regeneration remain as unsolved problems [2]. Utilization of biological scaffold material for cardiovascular tissue engineering might be an alternative to overcome these limitations [3,4]. In a recently published study decellularized allogeneic matrix, cellular reseeding, and static tissue culture were used to tissue engineer pulmonary heart valves [5]. Attempts to apply the same principles to engineer aortic heart valves failed due to insufficient mechanical stability (Stock UA, unpublished data, 2000).

The present concept represents the successful dynamic in vitro repopulation of almost completely preserved decellularized porcine matrices using exposure of defined physical signals for tissue engineering of viable and biomechanically stable heart valves with distinct morphologic and mechanical characteristics of aortic heart valves.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
The general approach to cell isolation, culture and seeding, as well as the decellularization procedure were previously described in detail [5]. However, in this study several modifications have been applied. This study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.1. Cell and tissue culture medium
Standard cell and tissue culture medium was Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), 1% penicillin–streptomycin (PS) (10000 U/10 mg/ml, Invitrogen) and 1.5 µg/500 ml recombinant human Fibroblast Growth Factor, Basic (rhFGF, Basic) (Promega, Mannheim, Germany).

2.2. Cell isolation and culture
For cell isolation, segments of ovine carotid arteries ({approx}5 cm length) were harvested from 6- to 8-week-old lambs using a sterile surgical technique. Endothelial cell (EC) and myofibroblast (MF) isolation and culture were performed using previously described methods [5]. Approximately 26 (±4) days (passage 6–8) were needed to obtain 15 confluent T75 tissue culture flasks of MF (1 T75 flask equals approximately 3 x 106 cells). An adequate number of EC (approximately 1 x 106 cells) was achieved after 10 (±4) days.

2.3. Decellularization procedure
The decellularization of the pulmonary valve conduits was performed as previously described in detail [5]. For desinfection, the valves were washed prior to the trypsin/EDTA treatment for 30 min at room temperature (RT) in povidone–iodine-solution (Mundipharma, Limburg, Germany) and sterile PBS, and followed by an overnight incubation at 4 °C in an antibiotic solution (1.2 mg amikacin; 3 mg flucytosin; 1.2 mg vancomycin; 0.3 mg ciprofloxacin; 1.2 mg metronidazol in 1 ml aqua ad inject.). In addition, a washing step with PBS for another 24 h followed the enzymatic processing to remove residual cell detritus. The decellularized matrices were finally stored in Hanks' buffered saline solution (HBSS, Biochrom) at 4 °C prior to further processing and seeding.

2.4. Reseeding and dynamic culture procedure
Following cell expansion to a total quantity of approximately 3 x 107 cells, MF were trypsinized and re-suspended in culture medium. Decellularized pulmonary valves were placed in sterile glass bottles, followed by a dribble-seeding of the inner and outer surfaces. After the reseeding procedure the valves were placed for 2 days in an incubator to allow attachment of the MF. Cell medium was carefully changed on a daily base. Subsequently, the valves were placed in a pulsatile flow system (bioreactor) (specified in Fig. 1) and exposed to defined physical signals with a fixed frequency (1 Hz) and pressure conditions (3 l/min and 60/40 mm Hg) over a time period of either 9 or 16 days to achieve ingrowth of the MF into the scaffolds. Control valves were grown over an equivalent time period without mechanical stimulation (static culture) under the same conditions. A final coating of the inner and outer surfaces of the entire valves with EC (9 x 106 cells) for 2 days under static conditions completed the reseeding. This 2-day period allowed the sedimentation and attachment of the EC on the valve wall and the leaflets. The entire culture period took place in a standard incubator at 37 °C and 5% CO2. PS supplement (5 ml) was added every 4 days.


Figure 1
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  		Fig. 1 The tissue culture process was performed in a hydrodynamic pulse replicator system (A, C). The bioreactor (60 cm height, 15–20 cm diameter) consists of a compliance chamber, a culture medium reservoir and can be easily and repeatedly autoclaved. The system is pre-pressurized with a 5% CO2–gas mix. A pressure and vacuum driven and microprocessor controlled compliance chamber creates an additional pressure gradient resulting in a physiologic systolic and diastolic pressure profile and flow. The design and the arrangement of the system enables the simulation of a pressure and flow profile identical to the human aortic valve. Pressure can be individually adjusted up to 180 mm Hg systolic and 120 mm Hg diastolic pressure. The frequency can be varied up to 150 bpm. Picture (B) shows the synchronous opening and closing of the implanted leaflets as observed during all in vitro experiments.

 
2.5. Phenotypical cell identification by immunocytochemistry
Immunocytochemistry was conducted as previously described [6]. EC were identified by the presence of factor VIII-related antigen (von Willebrand factor [vWF]; 1:200; Dako Cytomation, Hamburg, Germany). MF were characterized by incubation with monoclonal mouse antibodies for {alpha}-smooth muscle actin ({alpha}-SMA, clone 1A4, 1:400; Sigma), desmin (clone DE-R-11, 1:50; Dako), vimentin (clone V9, 1:50; Dako) and fibronectin (1:250; Dako). For the vWF and fibronectin staining procedure a fluorescein isothiocyanate-conjugated (FITC) swine anti-rabbit antibody (1:20) (Dako) and, for the other primary antibodies, a FITC-conjugated rabbit anti-mouse antibody (1:20) (Dako) served as secondary antibodies. Negative controls with the absence of the primary antibodies were demonstrated. For counterstaining of DNA 4',6-diamidino-2-phenylindole (DAPI) (Sigma) was added to the final PBS washing.

2.6. Histology and immunohistochemistry
Specimens of each heart valve construct (each n = 4) were embedded in paraffin (Merck, Darmstadt, Germany). For general morphology serial sections were stained with hematoxylin–eosin (HE) stain (cellular components, nuclei). A modified Movat pentachrome stain [7] was carried out with the Russel–Movat–Pentachrom-stain kit (Mastertechs, Lodi, CA, USA) to demonstrate extracellular matrix components. Immunohistostaining was carried out using immunofluorescence techniques (FITC). Monoclonal primary antibodies were {alpha}-SMA (1:250), desmin (1:50), vimentin (1:50) and vWF (1:200). A FITC-conjugated rabbit anti-mouse antibody and a FITC-conjugated swine anti-rabbit antibody served as secondary antibodies (1:20). Negative controls with the absence of the primary antibodies were demonstrated. VECTASHIELD mounting medium with DAPI (Vector Laboratories) was used for preserving fluorescence sections and to visualize DNA. Sections were analyzed and documented by using routine bright field light microscopy (Axiovert S 100, Zeiss, Jena, Germany).

2.7. Scanning electron microscopy
Scanning electron microscopy (SEM) was demonstrated as previously described [8]. For analysis, 15 visual fields of each specimen (each n = 4) were evaluated using magnification 33X till 3200X. Both inflow (ventricularis) and outflow (arterialis) surfaces of the leaflets were scanned. Only cell layers with the typical cobblestone morphologic characteristics were accepted as EC layers.

2.8. Biochemical assays
Biochemical assays were performed for analysis of cellular and extracellular components of a representative number of native (porcine and ovine tissue), decellularized and tissue-engineered (TE) valve leaflets. Crude protein extracts were prepared by manual disruption of the entire leaflet using a pistil and mortar. Tissue sample extraction was prepared as previously described [9]. All samples were normalized according to equivalent dry weight. Total DNA was isolated and purified by sequential organic extractions with phenol (Carl Roth, Karlsruhe, Germany) and phenol/chloroform/isoamyl alcohol (Roth) and quantified by spectrophotometry (Helios β, Thermo Spectronic, Rochester, NY, USA). Tissue preparation and total 4-hydroxyproline (4-OHP) determination was performed as described before [9]. Total collagen, elastin and GAG contents were quantified using SIRCOL, FASTIN and BLYSCAN assays (Biocolor, Belfast, Northern Ireland). To detect metabolic cell activity, MF and EC were isolated by the incubation of the leaflets with 0.5% collagenase A and 0.1% elastase (Serva Electrophoresis, Heidelberg, Germany) in PBS for 90 min at 37 °C and 5% CO2 under rotating conditions [10]. Afterwards, the cells were assayed with the CellTiter® Non-Radioactive Cell Proliferation Assay (Promega) and measured with the 96 well plate Elisa reader "Sunrise" (Tecan Group, Maennedorf, Switzerland).

2.9. Biomechanical tests
Tensile testing was carried out at RT with a universal TIRAtest tensile tester (model 2420; TIRA, Schalkau, Germany) equipped with a 100 Newton (N) load cell at the University of Applied Sciences Jena, Department of Materials Engineering. Tissue specimens of native porcine, native ovine, decellularized and TE valves were attached at both ends to an atraumatic clamp and subjected to unaxial tensile loading to failure (radial and circumferential directions) [11]. During clamping, care was taken to mount the specimens under zero stress conditions. At the beginning of measurements, the initial crosshead speed was 2.0 mm/min. When a tensile force of 0.4 N was reached, the final crosshead speed increased up to 3.0 mm/min until complete rupture of the specimens.

2.10. Statistics
All values are shown as mean±standard deviation. The significance of differences between heart valve constructs was estimated by analysis of variance (non-parametric Mann–Whitney Test) using the commercially available software package SPSS for windows, version 10.0 (SPSS Software, München, Germany). P values less than 0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Isolated EC and MF
The confluent EC displayed a cobblestone morphology of flattened cells with large rounded nuclei and demonstrated strong granular perinuclear immunofluorescence for vWF. No signal was observed following antibody staining for {alpha}-SMA (Fig. 2A).


Figure 2
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  		Fig. 2 Images of isolated, fixed and DAPI counterstained EC (A) and MF (B). (a/b) Routine bright field light microscopy (a, b: bars equal 100 µm); (c/d) DAPI conterstaining; (e/f) {alpha}-SMA (FITC) and (g/h) vWF (FITC) immunostained cells (c–h: bars equal 50 µm). (A) A positive staining for vWF was detectable, which indicated the presence of EC, with no expression of {alpha}-SMA. (B) Expression of {alpha}-SMA and negative staining for vWF identified MF.

 
Immunocytochemistry of isolated and fixed carotid artery medial cells revealed a strong expression of {alpha}-SMA, vimentin and fibronectin, which proved the MF phenotype according to prior publications [12,13]. In contrast, no positive signal was detected for desmin and vWF among the isolated cell population (Fig. 2B).

3.2. Decellularized matrix
Histological analysis of the HE stained decellularized valve tissue sections showed that treatment with trypsin/EDTA for 24 h followed by a 24-h washing with PBS converted native porcine pulmonary valves into an almost cell free scaffold (Fig. 3). Furthermore, the typical valve structure consisting of arterialis, fibrosa, spongiosa and ventricularis [14] was optimally preserved without apparent disruptions of the leaflet histoarchitecture.


Figure 3
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  		Fig. 3 Demonstration of the gross morphology with HE stain (nuclear counterstaining) of native porcine and decellularized tissues (arterialis/fibrosa). Pictures A and C (higher magnification) show native porcine tissue with positive cell nuclei staining throughout the leaflet (black arrows). Images B and D (higher magnification) visualize heart valve leaflets after a successful 24-h decellularization. No nuclear counterstaining was detectable, identifying a total cell removal. Section images show representative parts of the leaflets (scale bars equal 50 µm).

 
DNA and proliferation assays showed a significant reduction of cells and cell debris (n = 10). Assays for quantification of extracellular matrix demonstrated an almost complete preservation of the major structural components (collagen, 4-OHP, elastin) (each n = 12). The amount of detectable collagen increased during the decellularization process due to an enhanced collagen extraction effect of the trypsin/EDTA. Total GAG contents decreased significantly after the 24-h trypsin/EDTA treatment (n = 12) (Table 1).


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  		Table 1 Assays for quantification of cell mass (DNA) and extracellular matrix components

 
3.3. Histology and immunohistochemistry of reseeded valves
In all TE valves synchronous opening and closing of the leaflets could be observed during the entire in vitro culture period in the hydrodynamic pulsatile flow system (Fig. 1B). Gross appearance showed intact and pliable constructs. HE and DAPI staining of the 16-day dynamic reseeded TE valves revealed a confluent cell coverage with a complete ingrowth of ovine MF into the matrix, comparable to the native tissue (Figs. 4–7)GoGoGo. A confluent endothelial lining of the surface of native ovine and 16-day dynamic reseeded valves could be demonstrated by positive staining for vWF and scanning electron microscopy (Figs. 4 and 5)Go. SEM images of the TE constructs showed a large number of microvilli-like structures, which increased the surface area (Fig. 5). However, immunohistochemistry visualized significant differences between the TE constructs and native specimens. In particular, a very strong positive staining for {alpha}-SMA throughout the entire TE leaflet was detectable, whereas native specimens showed mainly vimentin-rich cells. A thin layer of MF that expressed {alpha}-SMA was only found at the inflow side of native ovine aortic leaflets (Fig. 6).


Figure 4
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  		Fig. 4 Images of outflow (a) (A–D) and inflow (v) (E–H) sides of TE leaflets (bioreactor, 16 days) (blue (A, E): DAPI; red (B, F): vWF; green (C, G): {alpha}-SMA). Immunohistochemical processing of the sections showed vWF positive cells only on the valve surfaces, confirming viable EC (B, F). Cells positive for {alpha}-SMA were detectable throughout the entire leaflet, indicating MF (C, G). Figures (D) and (H) illustrate an overlay of images of the outflow (A–C) and inflow (E–G) sides. For an improved presentation of both cell types, a color transformation from green to red of the vWF stain was applied using a photo imaging program (Adobe® Photoshop 5.0, Adobe Systems, San Jose, CA, USA) (bars equal 40 µm).

 

Figure 5
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  		Fig. 5 SEM appearance (original magnification 1330 x) of representative parts of inflow (ventricularis) and outflow side (arterialis) of native ovine aortic and 16-day TE valve leaflets, demonstrating a comparable confluent endothelial cell layer. Cells on the surface of the TE constructs are characterized by a large number of cellular finger-like projections (microvillar structures), indicating high-grade metabolic active cells with an increased absorptive capacity. (A) Native arterialis; (B) TE arterialis; (C) native ventricularis; (D) TE ventricularis (bars equal 6 µm).

 

Figure 6
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  		Fig. 6 DAPI counterstaining and immunohistochemical staining of MF in native (A) and TE (B) (bioreactor, 16 days) leaflets, for {alpha}-SMA (microfilament; marker used for smooth muscle cell and activated MF detection) and vimentin (intermediate filament). Native tissues showed mainly vimentin-rich cells and only at the inflow side (ventricularis; v) a thin layer of MF that expressed vimentin and {alpha}-SMA. Cells from TE constructs showed a strong expression of vimentin and {alpha}-SMA throughout the entire leaflet. Bars equal 200 µm; a=outflow side.

 

Figure 7
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  		Fig. 7 Images of representative HE stained longitudinal sections of native ovine (A), static (2+9+2d) (B), dynamically reseeded TE heart valve leaflets (2+9+2d) (C) and (2+16+2d) (D). Valves grown under static culture conditions (B) revealed isolated islands of cell clusters without signs of migration into the scaffold (black arrow) on the outflow (arterialis=a) and inflow (ventricularis=v) side of the leaflets. Sections of the 9-day dynamically cultured grafts (C) revealed a beginning increase of cell proliferation and migration onto and into the scaffold material from the outside to the inside (black arrow) only on the outflow side. TE constructs after 16 days under dynamic conditions (D) were comparable to native valve leaflets (scale bars equal 50 µm).

 
Compared to the confluent populated native and 16-day bioreactor constructs (Fig. 7A,D), TE leaflets after 9 days of dynamic reseeding showed a compact cellular outflow side (arterialis), with ingrowth of MF and a sporadic endothelialization. The inflow side (ventricularis), however, consisted only of decellularized matrix, with isolated EC (Fig. 7C). Static controls revealed a loose, acellular tissue formation with isolated islands of cell clusters (EC) without signs of migration into the scaffold (Fig. 7B). Use of Russel–Movat pentachrome stain visualized the extracellular matrix components in the four well-defined tissue layers, as previously described [14] (Fig. 8).


Figure 8
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  		Fig. 8 Movat-pentachrome staining of native ovine (A), static (B; 2+9+2d), dynamic cultured TE valve tissues (C; 2+9+2d) and (D; 2+16+2d). Physical signals exposed heart valves (16 days) (D) demonstrated extracellular matrix components and a well-organized tissue formation, comparable to native ovine specimens (A). Arterialis (a) and fibrosa (f) were identifiable by a distinct staining for collagenous fibers (yellow) and some proteoglycans (blue/green). The spongiosa layer (s), as the non-fibrous supporting component of the ECM, showed typical properties of an extensively hydrated semisolid gel with a predominantly positive staining for proteoglycans. In the ventricularis (v), a slight fragmentation of elastin (blue/black) and collagen (yellow) was visible. In contrast, static controls (B) as well as 9-day dynamically cultivated valves (C) showed less ECM deposition and less organized tissue formation (scale bars equal 200 µm).

 
3.4. Biochemical assays
Quantitative tissue analysis data are summarized in Table 1. Phenol/chloroform extraction of the 16-day TE constructs revealed a significant increase in DNA content, compared with lower values of native porcine and native ovine samples (each n = 10). Analysis of the 9-day bioreactor, as well as the 16-day static control valves, showed almost comparable DNA rates (each n = 6). Static controls after 9 days showed significantly lower values (n = 6). Almost the same results were calculated for the cell number of viable, metabolic active cells in the 16-day dynamic reseeded valves (n = 10). Static reseeded controls after 16 days and the 9-day dynamic cultured valves (each n = 6) showed comparable lower values. Cell numbers of statically reseeded controls after 9 days were significantly lower compared to native porcine and ovine tissues (n = 6).

In addition, the synthesis of extracellular matrix proteins was significantly enhanced in valves which were subjected to shear and intermittent mechanical deformation signals. Compared to native porcine and ovine tissues (each n = 8), collagen and 4-OHP contents increased significantly after 16 days of dynamic culture (n = 6). In contrast, detection of elastin showed a minor loss during the 24-h decellularization process (n = 12). However, the 16-day bioreactor reseeded constructs and native porcine and ovine tissues revealed almost comparable amounts (each n = 8). Significant higher contents of total GAG were detected only in the 16-day bioreactor cultured constructs (n = 6).

3.5. Mechanical tests
In comparison to native porcine tissue, trypsin/EDTA-treated valves revealed a significant loss of strength in radial and circumferential direction. Biomechanical testing of the 16-day bioreactor cultivated TE constructs demonstrated a significant increase in stability, compared to decellularized valve leaflets. Mechanical properties were comparable to those of native ovine and porcine tissues (each n = 20). Interestingly, native porcine pulmonary leaflets proved to be stronger than native ovine aortic leaflets. Static controls (9 and 16 days; each n = 16) and decellularized scaffold materials (n = 20) showed weaker properties compared to the dynamic reseeded leaflets (9 and 16 days; each n = 16). Furthermore, tensile tests clearly indicated a reduced radial strength compared with an almost four times greater circumferential strength of the leaflets. This was due to the anisotropy of the leaflet tissue: the majority of the structural collagen fibers is arranged in the circumferential direction and carry most of the stress [15]. The radial and circumferential stress-strain behavior is shown in Fig. 9.


Figure 9
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  		Fig. 9 Radial and circumferential tensile strength testing demonstrated mechanical properties of the dynamic TE constructs (16 days) comparable to native porcine pulmonary and ovine aortic valves. Static controls, cultivated over an equivalent time, were mechanically weaker. Investigations of decellularized specimens revealed a dramatical loss of strength particularly with regard to radial direction. All presented results are means±S.D. (*P<0.05 versus native porcine tissue; {dagger}P<0.05 versus decellularized tissue).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The current heart valve replacement concept, with the use of mechanical or biological prostheses, has limitations because these structures have no growing, repairing and remodeling ability. This is particularly a problem for pediatric patients who require several operations prior to being suitable for the implantation of adult-size constructs. Accordingly, it is apparent that a more viable heart valve replacement remains an elusive goal for the cardiac surgeon, especially for these who deal with growing patients. Three important questions regarding the tissue engineering of heart valves that are suitable for implantation into the systemic circulation were addressed in this study: (I) Is a decellularized xenogeneic matrix a feasible scaffold material for tissue engineering of aortic valves? (II) Is it possible to precondition these decellularized valves in order to enhance mechanical stability by the use of applied physical signals and (III) if this is possible, how long does it take, to create such an optimal heart valve construct?

The present concept demonstrated a successful in vitro application of cardiovascular tissue engineering to create viable and biomechanically stable tissue-engineered heart valves on the basis of decellularized porcine scaffolds, repopulation with ovine cells and applied physical signals utilizing a hydrodynamic pulsatile flow system. A complete biochemical analysis of all types of the major ECM molecules including collagen, elastin and glycosaminoglycans, characterization of the distinct cellular phenotypes, precise histology and histochemistry as well as extensive biomechanical testing was performed.

Several decellularization protocols were previously used to obtain acellular matrices [16–19]. In our concept, a trypsin/EDTA method [16] was applied for cell elimination of porcine tissue. This technique preserved the well-organized valve structure, without apparent matrix disruptions or functional loss. More importantly, the major extracellular matrix components, which play a fundamental role in cell function and adhesion [20], were mostly preserved. Previous reports on tissue engineering have focused on biodegradable polymer constructs [2,21]; however, the use of synthetic polymer material is concomitantly with a variety of limitations regarding regulation of cell attachment and tissue remodeling. For instance, the important extracellular matrix proteins such as collagen and elastin, well known as the critical elements for the structural integrity and biomechanical profile of heart valves, are completely missing [16]. This was a major factor in deciding to use a biological tissue as starter matrix for our study. The second reason to choose the decellularized material was the knowledge of most favorable hydrodynamic qualities [22] and an excellent cellular adhesive property of the surface. However, the discussion whether a reseeding pre-implantation is necessary or not remains controversial. With respect to cell seeding, we believe that it is wise to distinguish between tissue engineering as a concept of implantation of in vitro seeded matrices and guided tissue regeneration, and the approach of acellular matrices which are repopulated after implantation by the host [23,24]. The reseeding of large surface areas such as vascular prosthesis (DacronTM) from the adjacent native tissue is a phenomenon seen regularly in animals, but seems to occur only to a limited extent in humans [25]. It remains highly speculative if the observed repopulation of acellular and unseeded matrices, as observed in recent studies [26] can be transferred to humans. Furthermore, first clinical results from decellularized, non-reseeded heart valves implanted in the pulmonary circulation proofed to fail with fatal outcomes (P. Simon, M.D. Early failure of the tissue-engineered porcine heart valve synergraft in pediatric patients, Oral Presentation at the 16th Annual Meeting of the European Association for Cardiothoracic Surgery, Monte Carlo, Monaco, 22–25 September 2002).

We believe that reseeding of decellularized or acellular matrices prior implantation is crucial. However, the ideal cell source for tissue engineering of heart valves is still not well defined. Accordingly, a broad variety of cells were used for the reseeding in prior investigations [27–29]. Recently published results [30] showed that a pure endothelial cell seeding is not adequate for complete repopulation of heart valves. The decision to use MF and EC, isolated from ovine carotid arteries, based on the knowledge, that the phenotypical characteristics of arterial wall medial cells were similar to those of interstitial cells of valve leaflets [31], and the crucial thromboresistant function of endothelial cells [32]. With regard to a potential clinical application of our experimental work, we believe that a less invasive source of autologous cells is required. New research with mesenchymal stem cells [33], circulating bone marrow-derived endothelial cells [34] and peripheral blood cells [35] will represent an alternative cell source for tissue engineering.

In order to achieve constructs, with mechanical characteristics of native aortic valves, this study revealed that pure static culture conditions are not sufficient. The decision to use a bioreactor culture system was based on previous reports indicating that the dynamic cell-seeding results in mechanically more stable tissue constructs [36–38]. We demonstrated that in vitro exposure of TE heart valves to hydrodynamic pulsatile flow for 16 days resulted in significantly enhanced tissue formation and mechanical properties, resembling native heart valves. Furthermore, the dynamic culture conditions improved cellular adhesion in contrast to the static control valves. Histology and immunohistochemical examinations showed a well-organized, fibrous tissue with confluent coverage of viable and secretionally active cells and a repopulation throughout the entire TE leaflets. With unequivocal images we visualized an ingrowth direction of the MF from the outside to the inside of the scaffold. However, immunohistochemistry revealed significant differences between the TE constructs and native specimens. The high level of detected {alpha}-SMA positive MF represents a potential indicator for intense remodeling. If this will have a beneficial or unfavorable impact on the currently conducted in vivo studies requires further in depths studies.

Cells on the surface of the TE valves stained positively for vWF, confirming the presence of EC. The complete repopulation was additionally proved by scanning electron microscopic images of outflow and inflow sides of the TE constructs. SEM also revealed numerous microvillis, particularly on the surface of the TE leaflet cells. Microvillis represents precursor structures for macropinocytosis and, at this stage, they can be interpreted as an indicator for the activated status of the lumenal plasmalemm of the TE heart valve cells. However, more explicit ultrastructural investigations are necessary to explain this phenomenon in detail.

The extracellular matrix analysis of dynamically cultured TE constructs (16 days) revealed a dynamic process of growth and remodeling, with values comparable to those of native tissues. One question is, if the higher amount of collagen after 16 days of bioreactor cultivation will turn out to be a limitation with negative influence on ongoing in vivo studies. In accordance with the more pronounced matrix formation, mechanical testing demonstrated superior stability of the bioreactor cultured constructs (16 days), compared to significantly weaker properties of the equivalent dynamic 9-day samples and static controls.

Taken together, the time span from vessel or cell harvest, cell isolation, culture, matrix seeding and tissue culture requires an over all in vitro culture period of 48 days prior implantation. With respect to a potential clinical situation, this appears to be a rather long time period. In fact, for patients with acute aortic valve failure due to endocarditis, this approach is not feasible. However, for the majority of patients requiring aortic valve surgery, this procedure is elective and a preoperative waiting time of 48 days is tolerable.

In the present study, we could demonstrate for the first time the feasibility of a complete in vitro cellular replacement, on the basis of a preserved decellularized porcine matrix and ovine cell reseeding under application of defined in vitro shear and intermittent mechanical deformation signals. The recellularization was proved by a cell migration from the outside to the inside of the scaffold. Biomechanical tests, carried out in radial and circumferential direction, revealed an increase of strength caused by the dynamic reseeding and culture procedure. The investigated TE constructs showed morphological features and mechanical properties resembling native aortic heart valves.

Based on these promising results we are currently conducting an in vivo study using the engineered heart valves in the systemic circulation in juvenile lambs.


   Acknowledgements
 
The authors would like to thank Ingemarie Herrmann (Institute of Ultrastructural Research) for excellent assistance with the SEM and Karl-Heinz Holzfuss (Department of Materials Engineering, University of Applied Sciences) for the help with the TiraTest device. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Sto359/2-1+2, S.U.A.) and the Bundesministerium für Bildung und Forschung (IZKF-Jena, S.U.A.).


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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