Human Fibrin as a Cell Carrier for Heart Valve Tissue Engineering

Transcription

1 Human Fibrin as a Cell Carrier for Heart Valve Tissue Engineering R.W.E. Pikaart January 2008 BMTE Committee: prof.dr.ir. Frank Baaijens dr. Anita Mol dr. Rene van Donkelaar dr. ir. Maria Stekelenburg Eindhoven University of Technology Department of Biomedical Engineering Division Soft Tissue Biomechanics and Engineering

2 Abstract Background In heart valve tissue engineering, the optimization of tissue properties has to be combined with the realization of a completely autologous tissue engineering approach to obtain a functional and living heart valve replacement. Autologous conditions already have been improved by using human cell sources. However, culture conditions and scaffolds still have to be optimized. This study is based on the successful use of the scaffold system of PGA/P4HB combined with bovine fibrin as a cell carrier. However, to create a fully autologous approach the use of animal-derived fibrin has to be avoided and transferred to human fibrin. Methods and Results Human fibrin characteristics which are important for tissue engineering applications such as gelation time and cell distribution, were optimized by varying thrombin and fibrinogen concentration in a fibrin disk model system. Thrombin concentration was shown to influence gelation time, and thrombin and fibrinogen concentration both influenced cell density and distribution. The optimized human fibrin gels were further tested by analyzing their suitability as a cell carrier in a well-known tissue engineering system. Human fibrin seeded constructs showed similar quantitative and qualitative tissue formation and mechanical properties as bovine fibrin seeded constructs. Finally, human fibrin was successfully used as a cell carrier to tissue engineer a trileaflet heart valve. Conclusion This study demonstrated the feasibility of human fibrin as a cell carrier in combination with a PGA/P4HB scaffold for heart valve tissue engineering, bringing a completely autologous tissue engineering approach one step closer to reality. 2

3 Contents Abstract 2 1 Introduction 4 2 Material and Methods Fibrin disk model system Preparation fibrin disks Gelation time measurement Cell distribution Preparation and culture conditions constructs and heart valve leaflets Constructs Trileaflet heart valve Analysis of constructs and heart valve leaflets Histology Biochemical Assays Evaluation of Mechanical Properties Functionality Test Statistics Results Optimization fibrin characteristics Gelation time Cell distribution Tissue engineered constructs Histology Quantitative evaluation of tissue formation Evaluation of mechanical properties Tissue engineered human heart valve leaflets Macroscopic appearance Functionality test Histology Discussion 18 Bibliography 21 3

4 Chapter 1 Introduction Valvular heart disease is a significant cause of morbidity and mortality world-wide being responsible for nearly 20,000 deaths each year in de United States and is a contributing factor in about 42,000 deaths annually. The majority of these cases involve disorders of the aortic valve (63 %) or the mitral valve (14%) [1]. Surgical replacement of diseased heart valves by mechanical and bioprosthetic valve substitutes is the most common treatment for end-stage valvular heart diseases, with approximately 85,000 valve replacements performed each year in the US and 275,000 worldwide [2]. Although survival and quality of life are enhanced for many patients, prosthesis-associated complications and failure are frequent and have considerable impact on patient outcome [3]. With currently available valves, approximately 60% of substitute valve recipients develop an important prosthesis-related complication within 10 years postoperatively [4]. Major disadvantages are long-term anticoagulation therapy to control thromboembolism, infection risk, and degeneration. In addition, a major shortcoming in pediatric applications of these valve replacements is the lack of growth, repair and regeneration once implanted in the body, which frequently results in several re-operations during the patient s lifetime [5]. To address the above-mentioned problems, heart valve tissue engineering is a new research area, which aims to overcome the limitations of currently used prostheses by seeding autologous cells onto a synthetic polymer or biological material that serves as a scaffold. Subsequently, the cells are stimulated by biological stimuli via the culture medium, and/or mechanical stimulation in a bioreactor system, which will induce tissue formation in vitro. As a result, a completely autologous, living, and functional heart valve replacement develops, which can be implanted in the body if the tissue properties are sufficient to withstand the in vivo conditions [2]. During the past 10 years, several research groups have demonstrated the feasibility of the autologous tissue engineering concept for heart valve applications [6, 7, 8]. The first successful replacement of a single pulmonary valve leaflet with a tissue-engineered equivalent, based on a synthetic biodegradable scaffold, was demonstrated in lambs in 1995 [9, 10]. However, the leaflets in the referred studies did lack the mechanical integrity for systemic pressure application, such as the replacement of the aortic valve. Therefore, several studies today concentrate on improving the mechanical properties and tissue formation of the engineered heart valve leaflets to be able to withstand the pressures and flows in the aorta [11, 12, 13, 14]. For future clinical applications, the optimization of tissue properties has to be combined with the realization of a complete autologous tissue engineering approach. Taken together, this 4

5 Chapter 1 Introduction should finally result in a completely autologous, functional and living heart valve replacement with tissue properties comparable with a native heart valve. Progress has already been made to improve the autologous conditions by transferring the in vitro methodology from animal to human, by the use of human cell sources, such as bone marrow-derived mesenchymal stem cells, umbilical cord myofibroblasts and saphenous vein myofibroblasts [15]. However, culture conditions still have to be improved by using human-derived serum instead of animal-derived serum and an appropriate scaffold, either synthetic or biological, has to be chosen. Nowadays, materials used in tissue engineering are either fiber-based synthetic polymers, such as polylactic acid (PLA), polyglycolic acid (PGA) and poly(caprolactone) (PCL) or biological gels, such as collagen, chitosan and glycosaminoglycans (GAGs) [16]. A biological gel, fibrin, combines a number of important properties of an ideal scaffold and is, therefore extensively used in cardiovascular tissue engineering applications [17, 18, 19]. Fibrin gel allows for fast cell encapsulation, providing a spatial uniform cell distribution, which is associated with high rates of extracellular matrix (ECM) production [20, 18, 21]. The advantage of fibrin gel over the other gels is that it can be easily obtained autologous from the blood of the patient. Moreover, cells entrapped in fibrin gels were reported to produce more collagen [22, 23] and elastin [20] compared to cells entrapped in collagen gel. Limitation of fibrin as a scaffold are its inferior mechanical properties [17, 18]. Therefore, combining the advantages of fibrin as a cell carrier, providing a homogeneous cell distribution and less loss of freshly formed ECM in the surrounding medium, with the advantages of a fiber-based synthetic scaffold, providing structural integrity to the developing tissue, offers a favorable approach within tissue engineering applications. Combining bovine fibrin with a composite material of PGA and poly-4-hydroxybutyrate (P4HB) has been demonstrated to result in optimized tissue formation and organization [19]. However, for future clinical application, the use of animal-derived fibrin has to be avoided and human fibrin should be used to provide a completely autologous approach. In addition, optimization of fibrin parameters is still necessary, in terms of tissue engineering requirements, such as homogeneous cell encapsulation, handling convenience and cell proliferation. Much work has already been done to investigate the effect of various modifications to fibrin parameters. The most investigated parameters are thrombin and fibrinogen concentration [24, 25, 26, 27]. The aim of this study was to investigate the feasibility of human fibrin as a cell carrier for heart valve tissue engineering. First, a fibrin disk model system was developed to optimize fibrin characteristics, such as handling convenience (e.g. gelation time and mechanical stability) and cell distribution by variation of thrombin and fibrinogen concentration (resulting in different fibrin formulations). The properties of the human fibrin clots were compared to their bovine analogues used in the study of Mol et al. [19]. Second, the optimized fibrin gels were further analyzed in a tissue engineering pilot study. In this study, human vena saphena cells (HVSCs) were seeded onto rectangular PGA/P4HB scaffold constructs using human fibrin as a cell carrier and subsequently cultured under static constraint for three weeks. The constructs were examined for tissue formation and mechanical properties. The human fibrin constructs were compared to their bovine analogues. Finally, the optimized fibrin formulation, determined from the optimization study and tissue engineering pilot study, was used to tissue engineer a human heart valve. HVSCs were seeded into a trileaflet shaped PGA/P4HB scaffold and subsequently cultured under dynamic conditions for four weeks. The feasibility of the tissue engineered heart valve to withstand physiological aortic flow conditions was tested in the valve exerciser [28]. In addition, tissue formation was examined by histology. 5

6 Chapter 2 Material and Methods 2.1 Fibrin disk model system Preparation fibrin disks The bottom and capped ends of eppendorf tubes were cut off and discarded and the remaining tubes were placed in 100 x 20 mm tissue culture plates. Thrombin and fibrinogen were both reconstituted in tissue engineering (TE) medium as described in appendix A. TE Medium consisted of DMEM Advanced (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Greiner Bio One, USA), 1% Glutamax (Gibco, USA), 1% penstrep (10000 U penicillin/ml and µg streptomycin/ml; Cambrex, Belgium) and L-ascorbic acid 2-phosphate (0.25 mg/ml; Sigma). The thrombin solution was then delivered inside each tube on the tissue culture dish. Subsequently, fibrinogen solution was added to the tubes and mixed with the thrombin solution in a 1:1 (vol/vol) ratio. Fibrin clots were allowed to polymerize, and after 2 hours the tubes were removed, resulting in dime shaped fibrin clots (150 µl) (see appendix B). To create an optimal human fibrin scaffold for tissue engineering application several fibrin formulations were prepared by varying thrombin and fibrinogen concentrations. Thrombin concentrations of 1, 5, 10 and 15 IU/ml and fibrinogen concentrations of 2, 5, 10 and 15 mg/ml were chosen. In the remainder of the study formulations will be abbreviated. For example, a fibrin formulation with a thrombin concentration of 10 IU/ml and a fibrinogen concentration of 5 mg/ml will be referred to as Bovine fibrin will be referred to as reference. Human fibrin disks were prepared from human thrombin and fibrinogen (Kordia, Leiden) and from human thrombin and fibrinogen components from Tisseel Kit (Baxter, Austria). Specifications of Baxter fibrin are shown in appendix A. Bovine fibrin disks were prepared from 10 IU/ml bovine thrombin and 10 mg/ml bovine fibrinogen (Sigma, USA), which has been successfully used in the study of Mol et al. [19] and is used as a reference Gelation time measurement Gelation time of the fibrin formulations was determined by measuring the time required to observe gel-like behavior. Thrombin solution was mixed twice with fibrinogen solution and subsequently injected into a culture dish. Every five seconds the solution was checked for gelation by touching the solution with a pipet-tip. Fibrin solutions were defined as a gel, when solid fibrin solution was sticking to the pipet-tip. Fibrin formulations were appropriate for tissue engineering applications when gelation time was between seconds. Fibrin 6

7 Chapter 2 Material and methods gel was defined as mechanically stable when fibrin disk maintained its shape over time Cell distribution HVSCs were harvested from the vena saphena magna and expanded using regular cell culture methods [29]. The medium used for cell culture consisted of DMEM Advanced supplemented with 10% FBS, 1% Glutamax, and 0.1% gentamycin (Lonza, Belgium). Optimized fibrin formulations 5.5, 5.10, 5.15, 10.5, 10.10, 10.15, 15.5, and and reference as determined by the gelation time measurement, were prepared by mixing HVSCs with thrombin solution (10 X 10 6 cells/ml) and subsequently mixing with fibrinogen solution. After polymerization, fibrin disks were embedded in Tissue-Tek (Sakura, Netherlands) and snap-frozen. 10-µm sections were cut in the thickness direction of the fibrin disks and studied by DAPI (4-6-diamidino-2-phenylindole) staining, which binds strongly to DNA, for analysis of cell distribution and H&E staining for general fibrin structure. Fluorescent DAPI images were collected on a Zeiss Axiovert 200 MOT fluorescence microscope with Axiocam. H&E sections were analyzed on a Zeiss Axio Observer inverted microscope with Axiocam. 2.2 Preparation and culture conditions constructs and heart valve leaflets Constructs Scaffold constructs (5 mm x 35 mm; n=5) were cut out of non-woven PGA (thickness 1.0 mm; specific gravity 70 mg/cm 3 ; Cellon, Luxembourg) and coated with 1% P4HB (MW: 1 X 10 6 ; TEPHA Inc., USA) in tetrahydrofuran (THF; Fluka, Germany). The constructs were glued at the ends with a polyurethane (PU) glue (20% w/v; DSM, Netherlands) to a stainless steel ring (see figure 2.1b). This was performed in such a way that a piece of 5 mm x 25 mm was left over for seeding. The rings were placed in 6-well plates and dried overnight. Thereafter, the scaffolds were sterilized using 70% ethanol. The ethanol was allowed to evaporate overnight, after which the scaffold was washed with phosphate-buffered saline (PBS; Sigma, USA). TE medium was added to the scaffolds overnight before seeding to facilitate cell attachment by deposition of proteins. The seeding was performed using the optimized human fibrin formulations 10.5, and 15.10, determined from the cell distribution study, as a cell carrier. Bovine fibrin was used as a reference. The scaffolds were seeded with human vena saphena cells (passage 7) at a cell density of 2.0 X 10 6 cells per cm 2. The cell-seeded constructs were allowed to polymerize for 20 minutes in an incubator (37 o C with 5% CO 2 ) before adding TE medium. Constructs were statically cultured on a shaking table for three weeks and two third of the medium of all constructs was replaced every 3-4 days. A detailed protocol for tissue engineering constructs is provided in appendix C Trileaflet heart valve Heart valve leaflets and aorta wall were cut out of PGA sheets and attached to a radially selfexpandable nitinol stent (length=38 mm, diameter=25 mm when fully expanded at 37 o C; pfm AG, Germany) by stitches (Prolene 4-0, ETHICON Inc., USA). Thereafter, the scaffold was coated with a thin layer of P4HB in THF. Before evaporation of the solvent, the leaflets were positioned in the shape of a trileaflet heart valve (figure 2.1b). The scaffold was sterilized 7

8 Chapter 2 Material and methods and washed as described for the constructs above. Seeding was performed at a cell density of 2.0 X 10 6 cells (passage 7) per cm 2. Optimized human fibrin formulation 10.5, determined from the tissue engineered construct pilot study, was used as a cell carrier. After seeding, the scaffold was allowed to polymerize for 20 minutes in an incubator (37 o C with 5% CO 2 ) before it was placed into the Diastolic Pulse Duplicator (DPD), where the leaflets were exposed to dynamic strains at 1 Hz and continuous medium circulation, as previously described by Mol et al. [30]. Tissue engineered heart valve leaflets were cultured for four weeks in TE medium, of which two third was replaced every 3-4 days. A detailed protocol for tissue engineering of a human trileaflet heart valve is provided in appendix C. (a) (b) Figure 2.1: a) Rectangular PGA/P4HB scaffold constructs attached to stainless steel rings by PU glue; b) Top view of PGA/P4HB trileaflet heart valve scaffold attached to stent by stitches. 2.3 Analysis of constructs and heart valve leaflets Histology Tissue composition of both constructs (after three weeks of culture) and heart valve leaflets (after four weeks of culture) was studied by histology. Samples of TE constructs were fixed in phosphate-buffered formalin (Fluka, USA) and embedded in paraffin. Sections were cut at 10-µm and studied by Hematoxylin and Eosin (H&E) staining for general tissue morphology and Masson trichrome staining (commercially available kit; Sigma, USA) and Aniline blue with a Hematoxylin counterstaining for collagen formation. The sections were analyzed on a Zeiss Axio Observer inverted microscope with Axiocam. Sections were additionally stained by a picrosirius red staining according to Junqueira et al. [31] and analyzed by polarization microscopy. Sirius Red binds to collagen in a parallel fashion and enhances the normal birefringence of collagen fibers (the degree to which they retard polarized light), which can be detected using polarization microscopy [31, 32]. In this way also very thin fibrils, undetectable in normal light microscopy, become visible. 8

9 Chapter 2 Material and methods Biochemical Assays Tissue formation of the constructs was quantitatively determined from the amount of DNA, as an indicator of cell number, GAGs, and hydroxyproline, as an indicator of collagen content, per mg dry weight of tissue. For all analyses, lyophilized tissue samples were digested in papain solution (100 mm phosphate buffer, 5 mm L-cysteine, 5 mm ethylenediaminetetraacetic acid [EDTA], and 125 to 140 µg papain/ml) at 60 o C for 16 hours. The DNA content was determined using the Hoechst dye method and a standard curve from calf thymus DNA (Sigma, USA). The GAG content was determined using a modification of the assay described by Farndale et al. [33] and a standard curve from chondroitin sulfate from shark cartilage (Sigma, USA). The hydroxyproline content was determined using a modification of the assay described by Huszar et al. [34] and a standard curve from trans-4-hydroxyproline (Sigma, USA). By normalizing the collagen and GAG content for the amount of DNA, a measure for the amount of these matrix components produced per cell was obtained Evaluation of Mechanical Properties The mechanical properties of the constructs were measured using a uniaxial tensile tester (Kammrath & Weiss GmbH, Germany; load cell of 20N) with a constant strain rate of 1.7% per second. The dimensions of the tissue samples were 9 X 3 to 5 mm. The forces acting on the tissues as a response to elongation were represented in stress-strain curves. The ultimate tensile strength (UTS), indicative for tissue strength, and elongation at break, indicative for tissue extensibility, were obtained from the stress-strain curves. The modulus, indicative of tissue stiffness, was calculated as the slope of the linear part of the stress-strain curve Functionality Test The opening and closing behavior of the heart valve leaflets by exposure to aortic flow conditions was visualized in a custom designed valve exerciser [28] up to 15 minutes. The aortic flow was generated via a computer controlled pump. Images were obtained with a high-speed video camera (Phantom v9.0; Vision Research Inc, New Jersey). The flow, aortic pressure and left ventricular pressure were monitored using flow (Transonic, USA) and pressure sensors (Becton Dickinson, Belgium). Data acquisition was performed using LabView software (National Instruments, USA) Statistics Quantitative data was averaged per construct, subsequently averaged per group, and represented as average±standard deviation. Comparisons between groups were performed by one-way ANOVA using Tukey post-hoc tests to determine significant differences (P <0.05). 9

10 Chapter 3 Results 3.1 Optimization fibrin characteristics Gelation time Fibrin disks could be easily formed by mixing solutions of fibrinogen and thrombin. After gelation at room temperature, the transparant solution lost its fluidity and was transformed into an opaque hydrogel. The various fibrin formulations were analyzed by measuring gelation time and checking the mechanical stability. The results of the gelation time measurements of the various fibrin formulations are summarized in table 3.1. Table 3.1: Measured gelation times from fibrin formulations prepared from human and bovine thrombin and fibrinogen. Formulations with appropriate gelation times for tissue engineering are highlighted. Formulation Thrombin (IU/ml) Fibrinogen (mg/ml) Gelation times (s) Human fibrin (Baxter) Human fibrin (Kordia) Bovine fibrin (Sigma) Reference too weak too weak too weak

11 Chapter 3 Results The human fibrin formulations prepared from Kordia thrombin and fibrinogen demonstrated a gelation time which was too long for tissue engineering purposes. Human fibrin from Baxter showed appropriate gelation times for the formulations with a thrombin concentration between 5 and 15 IU/ml, which was comparable with the gelation time for bovine fibrin. However, the Baxter fibrin formulations with 2 mg/ml fibrinogen concentration showed weak mechanical stability and could not be used for tissue engineering. This resulted in 10 formulations useful for tissue engineering purposes (5.5, 5.10, 5.15, 10.5, 10.10, 10.15, 15.5, and and the reference), which are highlighted in table Cell distribution The 10 fibrin formulations were further analyzed by adding cells during the preparation of the fibrin disks. H&E stained sections showed a lot of cutting artefacts in fibrin formulations 5.5, 5.10, 15.5 and 15.15, which indicated lower mechanical stability in these fibrin gels (data not shown). This indirectly influenced cell distribution results of these formulations, which made them unreliable for further analysis. DAPI staining (figure 3.1) showed homogeneous cell distribution in most of the constructs, indicating appropriate mixing of cells, thrombin and fibrinogen. However, variation in cell density was observed between formulations and some formulations demonstrated cell accumulation at the surface of the constructs ((figure 3.1b,d,h). In comparison with the bovine fibrin (figure 3.1j), the best cell distribution and highest cell density were obtained for human fibrin formulations 10.5, and (see figure 3.1d,e,h). H&E staining confirmed these results, by showing most dense fibrin structure for above mentioned formulations compared to the reference (data not shown). 3.2 Tissue engineered constructs Optimized human fibrin formulations were further analyzed for their suitability as a cell carrier in tissue engineering. The constructs using human fibrin (10.5, and 15.10) as a cell carrier were sacrificed after three weeks and compared to similar constructs using bovine fibrin (reference) Histology Tissue morphology was shown by H&E staining (figure 3.2a,d,g,j) and Aniline blue staining (figure 3.2b,e,h,k). After three weeks of culturing, cells and tissue formation were present throughout the full thickness of the constructs and collagen was mainly deposited at the surface. Compared to bovine fibrin seeded constructs, constructs seeded with human fibrin showed similar tissue development. Picrosirius red staining, a measure for collagen maturity, showed hardly any birefringent fibers in all the groups. Only 10.5 demonstrated some birefringent fibers at the surface of the construct(figure 3.2f) Quantitative evaluation of tissue formation An overview of the amount of DNA, GAGs and hydroxyproline in the four groups after three weeks of culturing is shown in figure 3.3a. The amount of DNA, GAGs and hydroxyproline in the constructs did not show any significant differences among groups. To detect any changes in extracellular matrix synthesis activity of the cells, the amount of GAGs and hydroxyproline 11

12 Chapter 3 Results (a) 5.5 (b) 5.10 (c) 5.15 (d) 10.5 (e) (f) (g) 15.5 (h) (i) (j) Reference Figure 3.1: HVSCs, stained by DAPI, observed in the fibrin disks prepared from different thrombin and fibrinogen concentrations directly after seeding. The human thrombin and fibrinogen concentration of the formulations are shown below every figure with the thrombin concentration [IU/ml] being the first value and the fibrinogen concentration [mg/ml] being the second value (a-i). Bovine fibrin is referred to as reference (j). The most homogeneous cell distribution and most densely packed constructs are observed for 10.5 (d), (e) and (h) and the reference (j),. Bars represent 100 µm. 12

13 Chapter 3 Results (a) Reference (b) Reference (c) Reference (d) 10.5 (e) 10.5 (f) 10.5 (g) (h) (i) (j) (k) (l) Figure 3.2: Tissue morphology of the tissue engineered constructs after three weeks of culturing. H&E staining (a,d,g,h) showed general tissue formation, which was visible throughout the full thickness of the constructs. Aniline blue (b,e,h,k) stained collagen blue and demonstrated collagen deposition mainly at the surfaces of the constructs. Picrosirius red staining showed hardly any birefringent fibers in all groups. Only 10.5 demonstrated some birefringence at the surface of the construct (arrows) (f). Polymer remnants were clearly visible in Picrosirius red staining. Holes in the tissue (asterisks) represent cutting artefacts, probably due to polymer remnants. Bars represent 100 µm. 13

14 Chapter 3 Results produced per µg DNA were determined for all groups after three weeks of culturing as shown in figure 3.3b. These results showed similar collagen and GAG synthesis activity for all groups. DNA GAG Hyp GAG per DNA Hyp per DNA [ g/mg] ,9 10,6 19,2 12,0 19,8 18,7 11,3 11,3 [ g/ g DNA] ,3 6,2 6,2 6,2 3,8 3,4 3,3 3, ,9 3,1 3,2 3,0 Reference Reference (a) (b) Figure 3.3: a) The amount of DNA, GAGs, and hydroxyproline (Hyp) in the tissue engineered constructs (n=4) after three weeks of culturing; b) The amount of hydroxyproline (Hyp) and GAGs produced per µg DNA in the four groups (n=4) after three weeks of culturing. No significant differences were observed between human and bovine seeded constructs Evaluation of mechanical properties The mechanical properties of the constructs, characterized by UTS, modulus and strain at break, are depicted in figure 3.4. Human fibrin seeded constructs with a thrombin concentration of 10 IU/ml (10.5 & 10.10) demonstrated no significant differences in UTS, modulus and strain at break (data not shown) compared to their bovine analogues. Figure 3.5 shows representative stress-strain curves for all groups after three weeks of culturing, demonstrating typical non-linear tissue-like behavior in all groups. UTS Modulus 5,0 4,0 MPa 3,0 3,4 3,5 3,7 3,1 2,0 1,0 0,5 0,6 0,5 0,4 0,0 Reference Figure 3.4: The UTS and modulus for all four groups after three weeks of culturing. There were no significant differences observed between human and bovine seeded constructs. 14

15 Chapter 3 Results Cauchy Stress [MPa] Reference Strain [%] Figure 3.5: Representative stress-strain curves for all four groups after three weeks of culturing. The construct tissues of all groups show typical non-linear mechanical behavior, representative for tissue-like behavior. 3.3 Tissue engineered human heart valve leaflets Results from the tissue engineering study of constructs showed no significant differences in qualitative and quantitative tissue formation, and mechanical properties between bovine and human fibrin seeded constructs, indicating that human fibrin formulations 10.5, and all have potential as a cell carrier for heart valve tissue engineering. The only difference that was observed between human fibrin formulation, was that 10.5 showed some birefringent fibers by Picrosirius red staining indicating mature collagen formation. Based on this result, 10.5 has been chosen for the feasibility study to tissue engineer human heart valve leaflets Macroscopic appearance All leaflet tissues were intact after four weeks of culturing and showed dense tissue formation, illustrated in figure 3.6. The individual leaflets were grown together from the commissures towards the center of the valve. After cutting the free edges, the leaflets retracted because of pre-strain in the tissue figure 3.6c Functionality test For the functionality test of the human heart valve leaflets, the heart valve was placed into the valve exerciser and exposed to aortic flows. Images taken during one opening and closing cycle are shown in figure 3.7. The leaflets showed proper opening behavior and were able to sustain the aortic flow environment for the 15 minutes tested. However, because of the retraction of the leaflets and the resulting large opening at the center of the heart valve, regurgitation occurs after closure of the leaflets. As a consequence, the aortic and left ventricle pressure did not reach physiological values and flow drops below zero (data not shown). 15

16 Chapter 3 Results (a) (b) (c) Figure 3.6: Appearance of the tissue engineered human heart valve leaflets: top view (a), bottom view (b) and after cutting the free leaflet edges (c). Dense tissue formation was observed. (a) (b) (c) (d) (e) (f) (g) (h) Figure 3.7: Images obtained during testing of human heart valve leaflets at physiological flow conditions during one opening and closing cycle Histology Tissue formation of one representative heart valve leaflet is shown by H&E, Masson trichrome and Picrosirius red staining in figure 3.2. After four weeks of culturing, homogeneous tissue formation and abundant amounts of collagen were present throughout the full thickness of 16

17 Chapter 3 Results the constructs (figure 3.2a,b). Picrosirius red staining showed birefringent fibers, mainly at the surfaces of the construct, which suggest the presence of mature collagen. (a) (b) (c) Figure 3.8: Tissue morphology of one representative tissue engineered heart valve leaflet after 4 weeks of culturing. H&E staining (a) and Masson trichrome staining (b) showed homogeneous tissue formation and collagen formation respectively throughout the whole thickness of the constructs. Birefringent fibers could be identified by picrosirius red staining (c), indicating the presence of mature collagen. Bars represent 200 µm. 17

18 Chapter 4 Discussion Ten years of research in tissue engineering of heart valves has resulted in many improvements in mechanical properties and tissue formation [11, 12, 13, 14]. However, for future clinical applications, the optimization of tissue properties has to be combined with the realization of a completely autologous tissue engineering approach. Autologous conditions have already been improved by using human cell sources instead of animal cell sources. However, culture conditions and scaffolds still have to be improved. This study is based on the successful use of the scaffold system of PGA/P4HB combined with bovine fibrin as a cell carrier. This kind of hybrid scaffold system has shown to combine the advantages of fibrin as a cell carrier, providing a homogeneous cell distribution and less loss of freshly formed ECM into the surrounding medium, with the mechanical properties of the synthetic scaffold. Seeding scaffolds using fibrin as a cell carrier have demonstrated to result in optimized tissue formation and organization compared to conventional seeding [19]. However, to create a complete autologous approach, the use of animal-derived fibrin has to be avoided and transferred to human fibrin. The ultimate goal of this study was to test the feasibility of human fibrin as a cell carrier combined with the PGA/P4HB scaffold for heart valve tissue engineering. This has been performed by optimization of human fibrin characteristics and subsequently testing human fibrin as a cell carrier for tissue engineering applications. Before human fibrin could be utilized for heart valve tissue engineering, human fibrin characteristics, which are important for tissue engineering application, were first optimized by preparation of human fibrin gels with varying concentrations of thrombin and fibrinogen. Preparation was performed with commercially available human fibrin ordered from Kordia and Baxter. Human fibrin ordered from Baxter has successfully been used to achieve hemostases and tissue sealing in various surgical procedures [35]. To obtain proper cell encapsulation during seeding of tissue engineered constructs, sufficient handling time is needed to inject the fibrin solution into the synthetic scaffold before it polymerizes. On the other hand, gelation time should not be too long, otherwise cell seeding may be negatively influenced by gravity, resulting in an inhomogeneous cell distribution. According to Yamada et al. [36] gelation time is correlated with thrombin concentration. They have shown that a decrease in thrombin concentration resulted in a longer gelation time. Based on their results, it was decided in this study to investigate thrombin concentrations in the range of 1 and 15 IU/ml, which was expected to correspond to gelation times between 30 and 100 seconds. Fibrinogen concentration choice was based on the correlation between cell proliferation and fibrinogen concentration. Several studies [25, 26] have demonstrated that lower fibrinogen concentrations were found 18

19 Chapter 4 Discussion to be optimal for cell proliferation. Therefore, fibrinogen concentrations between 2 and 15 mg/ml were tested, resulting in 16 human fibrin formulations to be studied. Concerning cell proliferation, structural properties may also be an important parameter to consider. In future studies, it would be interesting to visualize structural properties of fibrin by scanning electron microscopy (SEM), and to investigate if they influence cell proliferation and tissue formation. Gelation time measurements confirmed the findings by Yamada et al., showing a correlation between gelation time and thrombin concentration. All human fibrin formulations prepared from Kordia thrombin and fibrinogen showed a gelation time which was too long for tissue engineering application. Baxter fibrin formulations with thrombin concentrations of 5, 10 and 15 IU/ml fulfilled the requirement to have a gelation time between 30 and 100 seconds. Furthermore, their mechanical stability was comparable to bovine fibrin. However, Baxter fibrin formulations with a fibrinogen concentration of 2 mg/ml demonstrated weak mechanical stability and could not be used for further experiments. This observation is in agreement with literature, which shows that a decrease in fibrinogen concentration results in a decrease in mechanical strength [24, 26]. Differences in gelation time between Baxter and Kordia fibrin may be caused by additional proteins present in thrombin and fibrinogen, which result in different thrombin and fibrinogen compositions. Optimized fibrin formulations 5.5, 5.10, 5.15, 10.5, 10.10, 10.15, 15.5, and and the reference, as determined by gelation time measurements, were further investigated by analyzing cell distribution and fibrin structure directly after seeding in fibrin disks. H&E staining showed a lot of cutting artefacts in fibrin formulations 5.5, 5.10, 15.5 and 15.15, which indicated lower mechanical stability in these fibrin gels. This indirectly influenced visualization of cell distribution in the fibrin, which made DAPI staining results of these fibrin formulations unreliable. DAPI staining showed homogeneous cell distribution in most of the formulations, which indicates proper mixing of thrombin, fibrinogen solutions and cells. However, differences in cell density were observed among formulations, with cell accumulation at the surface of fibrin formulations 5.10, 10.5 and An explanation for this behavior could be that slow polymerization of the fibrin caused the cells to accumulate at the bottom of the construct under influence of gravity. Based on the gelation time measurements, this behavior was expected in fibrin formulation with a low thrombin concentration. However, cell accumulation was shown at various thrombin concentrations, indicating other reasons for this behavior. Ultimately, the choice of fibrin formulations which were further used for tissue engineering experiments has been based on cell density and dense fibrin appearance, resulting in fibrin formulations 10.5, and and the reference. Optimized human fibrin formulations, by gelation time and cell distribution studies, were further analyzed for application as a cell carrier in tissue engineering. Human fibrin seeded constructs demonstrated no significant differences in qualitative and quantitative tissue formation and mechanical properties when compared to their bovine analogues. Therefore, all tested human fibrin formulations have the potential to be a cell carrier for heart valve tissue engineering. Histology sections showed homogeneous tissue formation and collagen deposition mainly at the surfaces of all constructs. This phenomenon might be due to a lack of diffusion of nutrients into the inner parts of the constructs. DNA, GAG and hydroxyproline amounts were comparable to values determined in a study by Mol et al. [37], where similar constructs using bovine fibrin were statically cultured for four weeks. Mechanical properties from the same study demonstrated a similar modulus, but showed a twice as large UTS compared to results determined in this study. This difference can be caused by the duration of the culture period. In summary, small changes in human fibrinogen and thrombin concentration 19

20 Chapter 4 Discussion did not result in significant differences in tissue and mechanical properties, which made fibrin formulations 10.5, and all suitable for heart valve tissue engineering. The only difference that was observed was the presence of mature collagen formation at the surface of the construct for fibrin formulation In future experiments, it would be interesting to investigate if this difference is also shown quantitatively by determining the amount of crosslinks in the constructs. For that reason, 10.5 was chosen to be used for tissue engineering heart valve leaflets. This study has successfully implemented human fibrin as a cell carrier for heart valve tissue engineering. The tissue engineered trileaflet heart valve demonstrated dense homogeneous tissue formation and abundant amounts of mature collagen throughout the construct. Overall qualitative tissue formation showed similarities to tissue formation demonstrated by tissue engineered heart valve leaflets prepared from bovine fibrin seeded constructs [38], which were cultured under similar conditions. Comparison by quantitative tissue formation still have to be performed. No three layered structure was observed as is shown in native aortic heart valves [39]. The heart valve was tested for functionality in the valve exerciser under physiological aortic flow conditions. Heart valve leaflets sustained the physiological flows for the 15 minutes tested and demonstrated proper opening behavior. However, because of retraction of the leaflet tissue after cutting the leaflet edges, a large opening at the center of the heart valve developed, resulting in suboptimal closure dynamics and subsequent leakage. For further experiments, leaflet design has to be improved by enlarging the leaflet scaffolds to include the effect of compaction in order to end up with sufficient coaptation of the leaflets. Before actually changing leaflet geometry, it is recommended to determine an optimized geometry, with respect to tissue compaction and coaptation, by computational modeling. In addition, it would be interesting to investigate mechanical properties of the heart valve leaflets and to compare them with the anisotropic properties of native heart valve leaflets. In conclusion, the human fibrin characteristics, gelation time and cell distribution, were optimized by varying of thrombin and fibrinogen concentrations in fibrin disks. Thrombin concentration was shown to influence gelation time, and thrombin and fibrinogen concentration both influenced cell density and distribution. The optimized human fibrin gels were further tested by analyzing their suitability as a cell carrier in a well-known tissue engineering model system. Human fibrin seeded constructs showed similar quantitative and qualitative tissue formation and mechanical properties as their bovine analogues. Finally, human fibrin was demonstrated to be feasible as a cell carrier in combination with a PGA/P4HB scaffold for heart valve tissue engineering, bringing a completely autologous tissue engineering approach one step closer to reality. 20

21 Bibliography [1] American heart association. [2] E. Rabkin and F.J. Schoen. Cardiovascular tissue engineering. Cardiovasc Pathol, 11: , [3] F.J. Schoen. Pathology of heart valve substitution with mechanical and tissue prostheses. In M.D. Silver, A.I. Gotlieb, and F.J. Schoen, editors, Cardiovascular Pathology, pages New York: Churchill Livingstone, [4] K. Hammermeister, G.K. Sethi, and W.G. Henderson. Outcomes 15 years after valve replacement with a mechanical versus a bioprosthetic valve: final report of the veterans affairs randomized trial. J Am Coll Cardiol, 36:1152 8, [5] J.E. Mayer. Uses of homograft conduits for right ventricle to pulmonary artery connections in the neonatal period. Semin Thorac Cardiovasc Surg, 7: , [6] S. P. Hoerstrup, R. Sodian, S. Daebritz, J. Wang, E. A. Bacha, D. P. Martin, A. M. Moran, J. Guleresian, J. S. Sperling, S. Kaushal, J. P. Vacanti, F. J. Schoen, and J. E. Mayer. Functional living trileaflet heart valves grown in vitro. Circulation, 102:III 44 III49, suppl III. [7] R. Sodian, S.P. Hoerstrup, J.S. Sperling, S. Daebritz, D.P. Martin, A.M. Moran, B.S. Kim, F.J. Schoen, J.P. Vacanti, and J.E. Mayer. Early in vivo experience with tissueengineered trileaflet heart valves. Circulation, 102:III 22 III 29, suppl III. [8] F.W.H. Sutherland, T.E. Perry, Y. Yu, M.C. Sherwood, E. Rabkin, Y. Masuda, A. Garcia, D.L. McLellan, G.C. Engelmayr, M.S. Sacks, F.J. Schoen, and J.E. Mayer Jr. From stem cells to viable autologous semilunar heart valve. Circulation, 111: , [9] T. Shinoka, C.K. Breuer, R.E. Tanel, G. Zund, T. Miura, P.X. Ma, R. Langer, J.P. Vacanti, and J.E. Mayer. Tissue engineering heart valves: valve leaflet replacement study in a lamb model. Ann Thorac Surg, 60:s13 6, [10] T. Shinoka, P.X. Ma, D. Shum-Tim, C.K. Breuer, R.A. Cusick, G. Zund, R. Langer, J.P. Vacanti, and J.E. Mayer. Tissue-engineered heart valves: autologous valve leaflet replacement study in a lamb model. Circulation, 94:II 164 II 168, suppl II. [11] S. Jockenhoevel, G. Zund, S. P. Hoerstrup, A. Schnell, and M. Turina. Cardiovascular tissue engineering: a new laminar flow chamber for in vitro improvement of mechanical tissue properties. ASAIO J, 48:8 11,

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23 Bibliography [26] W. Ho, B. Tawil, J.C.Y. Dunn, and B.M. Wu. The behavior of human mesenchymal stem cells in 3D fibrin clots: dependence on fibrinogen concentration and clot structure. Tissue Eng, 12: , [27] S.L. Rowe, S.Y. Lee, and J.P. Stegemann. Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels. Acta Biomaterialia, 3:59 67, [28] M.C.M. Rutten, M.W. Wijlaars, A. Mol, E.A. v. Dam, G.J. Strijkers, K. Nicolay, and F.P.T. Baaijens. The valve exerciser: a novel bioreactor for physiological loading of tissue-engineered aortic valves. J Biomech, [29] A.M. Schnell, S.P. Hoerstrup, G. Zund, S. Kolb, R. Sodian, J.F. Visjager, J. Grunenfelder, A. Suter, and M. Turina. Optimal cell source for tissue engineering. venous vs. aortic human myofibroblasts. Thorac Cardiovasc Surg, 49:221 5, [30] A. Mol, N.J.B. Driessen, M.C.M. Rutten, S.P. Hoerstrup, C.V.C. Bouten, and F.P.T. Baaijens. Tissue engineering of human heart valve leaflets: a novel bioreactor for a strain-based conditioning approach. Ann Biomed Eng, 33: , [31] L.C. Junqueira, G. Bignolas, and R.R. Brentani. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J, 11: , [32] J.G. Pickering and D.R. Boughner. Quantitative assessment of the age of fibrotic lesions using polarized light microscopy and digital image analysis. Am J Pathol, 138: , [33] R.W. Farndale, D.J. Buttle, and A.J. Barrett. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta, 883:173 7, [34] G. Huszar, J. Maiocco, and F. Naftolin. Monitoring of collagen fragments in chromatography of protein mixtures. Anal Biochem, 105: , [35] M.R. Jackson. Fibrin sealants in surgical practice: An overview. Am J Surg, 182:1S 7S, [36] Y. Yamada, J.S. Boo, R. Ozawa, T. Nagasaka, Y. Okazaki, K. Hata, and M. Ueda. Bone regeneration following injection of mesenchymal stem cells and fibrin glue with a biodegradable scaffold. Craniomaxillofac Surg, 31:27 33, [37] A. Mol. Functional tissue engineering of human heart valve leaflets. PhD thesis, Technical University Eindhoven, [38] A. Mol, M.C.M. Rutten, N.J.B. Driessen, C.V.C. Bouten, G. Zund, F.P.T. Baaijens, and S.P. Hoerstrup. Autologous human tissue-engineered heart valves: prospects for systemic application. Circulation, 114:I152 I158, [39] A. Stevens. Human Histology. Mosby, London, 2 nd edition,

24 Appendix A Preparation of fibrin Bovine fibrin was prepared from 10 IU/ml bovine thrombin and 10 mg/ml bovine fibrinogen as described in the study of Mol et al. [19]. Human fibrin was prepared with the same protocol at various concentrations. During preparation of bovine fibrin there were two points of attention. First, bovine thrombin solution needs to be cold before filter sterilizing, because otherwise the solution will not be able to go through the filter. Therefore, a cold syringe and a cold filter are needed. Second, bovine fibrinogen is difficult to dissolve in TE medium. In case of the preparation of human fibrin, these problems did not occur. Thrombin solution can be filtered at room temperature and fibrinogen can be easily dissolved in TE medium. Requirements TE medium Fibrinogen Thrombin 2.5 ml syringes 0.2 µm filters Centrifuge tubes Ice Procedures Preparation thrombin solution 1. Put a centrifuge tube, a syringe and a sterile filter on ice. 2. Calculate the amount of thrombin needed to obtain the desired thrombin concentration: weight a certain amount of thrombin and transfer to a centrifuge tube; check which thrombin activity the used thrombin has at the bottle. Calculate the amount of medium to be added with the following formula: TE medium to be added [ml] = amount of thrombin [mg] X thrombin activity [IU/mg] desired concentration [IU/ml] i

25 Appendix A Preparation of fibrin 3. Mix the solution until thrombin is dissolved (shake gently) and put on ice for about 10 minutes. 4. Sterile filter the solution with the syringe and the sterile filter and store the sterile solution on ice until use. Preparation fibrinogen solution 1. Calculate the amount of fibrinogen needed to obtain the desired fibrinogen concentration: weight a certain amount of fibrinogen and transfer to a centrifuge tube; check on the fibrinogen pot what the actual protein content is. Calculate the amount of medium to be added with the following formula: TE medium to be added [ml] = amount of fibrinogen [mg] X amount of actual protein [%/100] desired concentration [mg/ml] 2. Mix the solution until fibrinogen is dissolved (shake gently). 3. Sterile filter the solution with the syringe and the sterile filter and store the sterile solution on ice until use. ii

26 Appendix A Preparation of fibrin Figure 1: Specifications of Baxter Tisseel kit containing human thrombin and fibrinogen. iii

27 Appendix B Preparation fibrin disks a) Tubes are prepared and added to culture dishes b) Thrombin (either or not including cells) is mixed with fibrinogen c) Fibrin clots polymerize for two hours d) Tubes are removed e) Dime shaped fibrin clots Figure 2: Protocol for preparation of fibrin disks. Derived from Cox et al. [25] iv