Ann Thorac Surg 2006;81:1472-1479
© 2006 The Society of Thoracic Surgeons
New technology
Successful Endothelialization of Porcine Glutaraldehyde-Fixed Aortic Valves in a Heterotopic Sheep Model
Helmut Gulbins, MD
a
,
*
,
Anita Pritisanac, MD
a
,
Korbinian Pieper, MD
b
,
Angelika Goldemund, MD
a
,
Bruno M. Meiser, MD
a
,
Bruno Reichart, MD
a
,
Sabine Daebritz, MD
a
a Department of Cardiac Surgery, University Hospital Grosshadern, Munich, Germany
b Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität, Munich, Germany
Accepted for publication November 4, 2005.
* Address correspondence to Dr Gulbins, Department of Cardiac Surgery, University Hospital Ulm, Ulm, D-89070 Germany (Email: helmut.gulbins{at}medizin.uni-ulm.de).
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Abstract
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PURPOSE: The purpose of our study was to evalute the stability of an artificially seeded endothelial cell layer on porcine aortic prostheses under in vivo conditions in the arterial system.
DESCRIPTION: Ten female sheep were divided into two groups. Animals of the study group (n = 7) had dissection of their right external jugular vein for cell harvesting. Myofibroblasts and endothelial cells were labelled with PKH-26, seeded onto pretreated (10% citric acid) porcine glutaraldehyde-fixed aortic valves (Freestyle, Medtronic Inc, Duesseldorf, Germany), and the valves were implanted into the descending aorta. Controls (n = 3) received pretreated but unseeded valves. A shunt between the aortic arch and the left atrial appendage ensured systolic or diastolic leaflet motions, or both, that were documented by sonography. After 3 months the valves were explanted. Specimens for scanning electron microscopy and immunohistochemical staining were taken prior to implantation and after explantation.
EVALUATION: A neointimal proliferation was detected in the control group. No endothelial cells were found on the leaflets and the sinuses, but erythrocytes and thrombocytes were seen entrapped within the collagen fibers. Thrombus formation was documented macroscopically and histologically on the leaflets and the sinuses. In the study group a confluent endothelial cell layer was documented on the walls and leaflets. Neither neointimal proliferation nor any clots were seen. Some cells were still labelled positively indicating their origin from the initial cell seeding. No dilatation of any prosthesis was observed, but all valves showed slight thickening of the leaflets.
CONCLUSIONS: The artificially seeded endothelial cell layers remained stable under in vivo conditions in the arterial system. Biocompatibility of the prostheses seemed to be improved by reduction of thrombogenicity.
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Technology
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The degeneration of glutaraldehyde-fixed valves with subsequent calcification and tissue failure is the main disadvantage of these valvular prostheses. Different strategies have been developed to increase biocompatibility and durability [1–4]. Covering the surface of porcine glutaraldehyde-fixed valve prostheses with autologous endothelial cells of the recipient may delay valve degeneration and further reduce thromboembolic events thus improving biocompatibility. Recently we were able to publish the results of our in vitro studies with successful endothelialization of commercially available porcine glutaraldehyde-fixed aortic prostheses [5]. The purpose of the animal experiments was to evaluate the stability of an artificially developed endothelial cell layer under in vivo conditions in the arterial system and to investigate potential influences on the biocompatibility of these prostheses.
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Background
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Ten adult female sheep were enrolled in the study and divided into a study group (n = 7) and a control group (n = 3). All animals were handled according to the German guidelines for animal experiments (ie, the German law of protection of animals in its present 2003 form) and were approved by the government. Animals of the study group had explantation of the right external jugular vein under general anesthesia for cell harvesting. After cell culturing and seeding, the prostheses (Freestyle, Medtronic Inc, Duesseldorf, Germany) were implanted. Animals of the control group received unseeded prostheses. At implantation the animals were set under general anesthesia with controlled ventilation. The chest was opened using a left-sided lateral thoracotomy in the third intercostal space. The descending aorta was prepared after ligation of the hemiazygos vein. The aorta was then mobilized from the origin of the left subclavian artery down to the first two pairs of intercostal arteries. Heparin was administered with 200 IU/kg and partial clamping of the aorta was done distally to the left subclavian artery. An 8-mm shunt (W.L. Gore & Assoc, Flagstaff, AZ) was anastomosed to the aorta. The distal end of this shunt was then anastomosed to the descending aorta in height of the first pair of intercostal arteries. The aorta was cross clamped, and the valve prosthesis was interponated with two running sutures. After removal of air, the clamps were removed but flow was still reduced by partial aortic clamping. The shunt was ligated distally and its end was anastomosed to the left atrial appendage. Left atrial pressures were directly monitored through a left atrial catheter and pressures of less than 18 mm Hg were accepted. Otherwise the shunt was bandled to achieve a left atrial pressure of less than 18 mm Hg. During this time period, diastolic arterial pressure was also monitored carefully. After air removal, the shunt was opened and the motions of the leaflets were documented by direct sonography. The chest was closed, anesthesia was discontinued, and the animals were extubated.
After 3 months, the sheep were anesthetized again. The chest was reopened using the same access, and the descending aorta was prepared. After heparin administration (200 IE/kg), the aorta was cross-clamped proximally and distally to the implanted prosthesis, and the valves were explanted. The animals were then sacrificed by potassium infusion. After macroscopic examination of the valves, the valves were examined by fluoroscopy, and the thickness of the free prosthetic wall was measured. Specimens were taken for immunohistochemical staining and scanning electron microscopy.
Cell Culture
Endothelial cells and myofibroblasts were isolated from the external jugular vein of the animals of the study group as previously described [5–7]. The vein pieces were cannulated, rinsed with buffered medium, and incubated with 0.1% collagenase for 20 minutes at 37°C and 5% CO2. After centrifugation at 1,000 units per minute for 10 minutes, the cell pellet was resuspended in endothelial cell growth medium (Promocell, Heidelberg, Germany) and plated on culture dishes. For isolation of the myofibroblasts, the same vein pieces were again filled with 0.1% collagenase and incubated for 30 minutes. This procedure was repeated twice. After centrifugation, the pellet was resuspended in myofibroblast growth medium (Promocell, Heidelberg, Germany) and plated on culture dishes. For passaging, both cell types were trypsinized after reaching confluence and then centrifuged, resuspended in cell medium, and plated again. For cell counting, 30 µL of the resuspended cell suspension were incubated with trypan-blue and counted using a Neubauer chamber.
For intravital fluorescence labeling, PKH-26 dye (Sigma Chemical Company, St. Louis, excitation maximum at 551 nm) was used. The dye is deposited in the membrane of viable cells. The body of labeled cells fluoresced red, whereas the nucleus remained unstained. After cell death, the dye leaves the cell membrane. Therefore detected fluorescing cells were considered viable. For labeling, endothelial cells were incubated for 3 minutes with 10 mL trypsin at 37°C and 5% CO2. They were centrifuged twice for 10 minutes at 4°C and 1,000 U/min. After resuspension of the pellet in endothelial cell medium, the cell number was determined using the Neubauer cell count chamber. For labeling, 4 µL pKH-26 were added to 1 mL Diluent C (Sigma, St. Louis) and mixed with the resuspended cells for 3 minutes. The reaction was stopped using 1% bovine serum albumine for 1 minute. To wash out any remaining dye, the suspension was centrifuged for 10 minutes again after adding 4 mL endothelial cell medium. Thereafter, the number of viable cells was evaluated. After plating the cells for 2 more days, the success of the labeling procedure was confirmed by fluorescence microscopy of the cultured cells.
Cell Seeding on Commercially Available Porcine Aortic Valve Prostheses
Commercially available, unstented porcine aortic valve prostheses (Freestyle, Fa. Medtronic, diameters 21 and 23 mm) were pretreated as previously described [5]. These prostheses were glutaraldehyde-fixed with alpha amino oleic acid detoxification. After incubation with serum-supplemented M-199 for 24 hours at 4°C, the valves were incubated with citric acid (10% by weight) for 5 minutes at a pH of 3 to 3.5. This pretreatment increases hydrophilsm of the surface, thus improving cell adhesion and attachment [5]. The pretreated, but unseeded valves exhibited a cell-free surface of free collagen fibers prior to cell seeding (Fig 1). Thereafter, the prostheses were rinsed 3 times and buffered to a physiologic pH using PBSB buffer. After the final washing procedure, the valves were pre-seeded with myofibroblasts, followed by endothelial cell (EC) seeding. For myofibroblast seeding, the seeding device was filled with fibroblast growth medium and a cell suspension containing 2.4 x 107 myofibroblasts on average (range, 1.2 to 4.1 x 107 cells). The seeding procedure lasted for a period of 24 hours. After 30 minutes the device rotated for 150 seconds and stopped in a different position for the next resting period. This ensured that all parts of the aortic roots were exposed to cell seeding. After the seeding procedure the valves were kept under culture conditions for 7 days, followed by EC seeding. The seeding was done analogously to myofibroblast seeding with a mean of 0.9 x 107 ECs (range, 0.7 to 1.9 x 107 cells). Specimens of the free aortic wall were taken directly after the seeding procedure and 7 days thereafter.
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Fig 1. Scanning electron microscopy image of a pretreated, but unseeded free wall of a valve prosthesis (magnification, x500). No endothelial cell layer is seen, but the collagen fibers are lying free on the surface.
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Immunohistochemical Staining
Specimens of the free wall of the prostheses were obtained directly prior to implantation. After explantation, the free walls, the sinuses, and the leaflets were processed. Specimens were frozen and stored in liquid nitrogen. Sections of 8 µm were stained using monoclonal antibodies against factor VIII, CD90, 
-actin, CD31, CD34 (Dako, Hamburg, Germany), laminin Is, and collagen IV (Sigma, Deisnhofen, Germany). After incubation with the primary antibody, an anti-mouse IgG antibody (Dako, Hamburg, Germany) was added and stained with AEC (Dako, Hamburg, Germany). Negative controls were done analogously to the fluorescence staining.
Scanning Electron Microscopy
From each explanted valve, one complete leaflet and one sinus were taken out for scanning electron microscopy (SEM) examination. The specimens taken prior to implantation were cut in half and taken for immunohistochemical staining or SEM. Only cell layers with the typical cobblestone morphology were accepted as endothelial cell layers. For analysis, 10 visual fields of each specimen were evaluated under 1,000x magnification. Defects were marked and the precentage in relation to the visual field was calculated by the computer. Both fibrosal and ventricular surfaces of the leaflets were evaluated. The endothelial cell covering was assessed semi-quantitatively by two examiners and was classified as 100%, 75%, 50%, or 25%. A mean value of 95% and greater was believed to represent a confluent endothelial cell layer.
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Results
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Initial cell harvesting and culturing were successful in all 7 animals of the study group. After 21 days, the myofibroblasts had reached cell numbers above 107 cells. The cells were stained positively for CD90 and also particularly for -actin. Labeling of the endothelial cells with PKH-26 proved to be a reliable and reproducible method. Approximately 8% of the initially marked cells were lost due to the procedure. All specimens taken prior to implantation showed a confluent endothelial cell layer covering a layer of myofibroblasts on the luminal surface of the valves.
At surgery, implantation was tolerated well by all animals. Intraoperative sonography documented sufficient leaflet motion after opening of the shunt to the left atrium. In 3 animals, left atrial pressure rose above 20 mm Hg immediately. Therefore these shunts were bandled with a 5-0 Prolene suture. Thereafter the left atrial pressures were below 16 mm Hg continously by maintained leaflet motions on sonography (Fig 2A–C). Diastolic arterial pressure fell slightly from mean 54 mm Hg (range, 42 to 66 mm Hg) to 48 mm Hg (range, 40 to 59 mm Hg). One sheep of the control group died at valve implantation due to severe air embolization after opening of the aortic left atrial shunt. All other animals were extubated successfully and no neurologic complication occurred. One animal of the study group died on the first postoperative day because of a pulmonal complication. This sheep had its valve explanted and prepared for immunohistochemical and SEM examinations. All other animals reached the study end point at 3 months after implantation.
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Fig 2. Direct sonography after implantation of the prosthesis and opening of the shunt to the left atrium. (A) The images show the systolic and diastolic leaflet motions, beginning with a complete closure of the cusps. (B) During systole the leaflets opened widely. (C) When the pressure dropped at the beginning of the diastole, the leaflets closed again.
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On the valve of the animal that died on the first postoperative day, a confluent endothelial cell layer was documented on the free walls, the sinuses, and the leaflets (Fig 3A). The cells were positive labeled with PKH-26 (Fig 3B), proving them to be the originally seeded cells. No thrombotic material was found on this valve.
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Fig 3. (A) Immunohistochemical staining against CD31, leaflet of a seeded prostheses, first postoperative day (magnification, x20). Clearly positive reaction indicating viable endothelial cells. (B) Same section as Figure 3A; fluorescence microscopy (magnification, x20), 565 nm. This animal (study group) had died on the first postoperative day due to a pulmonary complication. On the specimen obtained from the implanted valve, positively labelled cells were found on both sides of the leaflet (see arrows). This gave strong evidence for the stability of the initially achieved endothelial cell layer on the porcine bioprostheses.
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At explantation after 3 months (Table 1), all shunts were found to be open. Shunt flow was documented by color Doppler imaging. Direct sonography revealed leaflet motions of all valves. However, the cusps seemed somewhat thickened and stiffer (Fig 4). At fluoroscopy, no evident calcifications were detected. The wall thickness of the free prosthetic wall had increased from mean 3.1 mm (range, 2.8 to 3.2 mm) to 3.8 mm (range, 3.4 to 4.3 mm). On light microscopy, an unspecific immunologic reaction with granulocytes infiltrating the free walls was seen on some sections. Immunohistochemical stainings against CD34 revealed an inflammatory response that seemed to be restricted to the outer surface of the free walls with CD34+ cells and neovascularization. Beneath the luminal endothelial cell layer, only rare CD34+ cells were found.
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Fig 4. Bioprosthesis of the study group at explantation. The leaflets, walls, and sinuses are free from thrombotical material and seem to be cell-covered (reflecting surface). The leaflets appear thickened, but motions were normal at direct sonography. The anastomoses showed no neointimal formation (see arrows).
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On the valves of the control group, a neointimal ingrowth was found originating from the anastomotic sites, covering approximately the first 5 mm on each end of the valve prostheses (Fig 5A). However, the remaining luminal surfaces of the valves were free of any cell covering. Macroscopically, clots were found within the sinuses of both valves of the control group. Microscopically, thrombotic material was also found on the sinuses and the leaflets (Fig 5B) on SEM and erythrocytes, and thrombocytes were found to be entrapped within the collagen fibers of the valves. At immunohistochemical evaluation, no CD31 or -actin-positive cells were found on the surface of the valves supporting the SEM results.
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Fig 5. (A) Scanning electron microscopy of free wall of a control group valve (magnification, x200). Neointimal ingrowth with a clear and sharp transition to the native (thick arrows), cell-free surface of the prosthesis. Between the collagen fibers, entrapped erythrocytes and thrombocytes were found (thin arrows). (B) Scanning electron microscopy of leaflet of a control group valve (magnification, x500). Thrombus formation (thin arrows) on the free collagen fibers of the leaflet of this control group valve.
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None of the valves of the study group showed a neointimal formation as it was seen in the control group. No clots were seen, neither macroscopically nor on SEM images. On the luminal surface of all valves of the study group, SEM revealed a still confluent endothelial cell layer (Fig 6), especially on the leaflets and the sinuses. The cells were still particularly labeled with PKH-26 (Fig 7), giving evidence for their origin from the initial seeded cells. Immunohistochemical staining for factor VIII and CD31 supported the SEM findings (Fig 8) and showed viable endothelial cells covering the luminal surface of the study group valves. The myofibroblasts and endothelial cells had synthetized extracellular matrix proteins, such as collagene IV (Fig 9) and laminin Is as a marker of physiologic function. These important components of the basement membrane were found as a confluent layer beneath the cell layers.
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Fig 6. Scanning electron microscopy of leaflet of a study group valve (magnification x500). Confluent endothelial cell layer with cells oriented to blood flow (arrows). No thrombocytes or erythrocytes are seen.
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Fig 7. Fluorescence microscopy at 565 nm, cross section of a leaflet of the study group after 3 months (magnification, x20) on both sides of the leaflets. Still positively labelled cells were found indicating them to originate from the initially seeded cells.
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Fig 8. Immunohistochemical staining against factor VIII of free wall of a study group valve (magnification, x10). Positive reaction on the luminal surface (arrows) of the valve indicating viable endothelial cells. These immunohistochemical stainings supported the results of the scanning electron microscopy that had shown a confluent endothelial cell layer on all valves of the study group at explantation.
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Fig 9. Immunohistochemical staining against collagene IV of a leaflet of a seeded prostheses. Both sides exhibit a clearly positive reaction indicating the synthesis of this important component of the basement membrane.
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Comment
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Seeding of human endothelial cells on biological valve prostheses was reported by several authors [2–4, 8–12]. Recently, we were able to publish the results of our in vitro experiments with successful endothelial cell seeding onto glutaraldehyde-fixed porcine aortic valves [5]. In these experiments it was shown that poor cell adhesion on glutaraldehyde-fixed porcine surfaces was also a result of a change in the physico-chemical properties caused by the cross-linking. Especially the reduced hydrophily prevented the cells to attach properly. This could be changed by introducing a strong hydrophilic substance through the way of a chemical salt formation on the surface. For this we successfully used citric acid or ascorbic acid, which are both strong organic acids [5]. Pre-seeding with autologous myofibroblasts resulted in further improvement of the endothelial cell seeding and shear-stress resistance. The transfer of the cell culturing techniques from human to sheep was successful. Sheep pocess myofibroblasts, they could not be distinguished from smooth muscle cells by staining against -actin alone. Because smooth muscle cells also synthetize extracellular matrix components, a low admixture of these cells to the preseeding culture should not affect the results negatively. Based on these results, the intention of the presented animal study was to evaluate the stability of such an artificially seeded cell layer under in vivo conditions.
The sheep is an accepted model for testing valve prostheses. It is known for its fast degeneration of foreign materials after implantation, especially in the growing sheep. However, in our experiments the evaluation of degeneration was not a primary study goal but the evaluation of the stability of an artificially seeded endothelial cell layer. Therefore we chose adult sheep versus juvenile sheep, because the adult sheep show a slower degeneration process. In addition, the valves were explanted after 3 months because the labelling technique that was used was only reliable for a 4-month period under in vivo conditions [13], and the proof of survival of initially seeded cells was one major goal of the study.
The coagulation system of the sheep is much more pro-coagulatoric compared with humans. Because one of the possible benefits of an endothelial cell layer was a reduction in thrombogenicity of the prostheses, this was one more advantage of this animal model. Because of the difficulties arising from extracorporal circulation in sheep, we decided to use a heterotopic model. To simulate systolic and diastolic leaflet motions, we installed a shunt from the distal aortic arch to the left atrial appendage, which has already been published [14]. The implanted valves and their cusps were exposed to arterial pressure, high flow, and leaflet motions.
The use of an intravital fluorescence dye was reported in the literature [13]. The mean half-time of this labeling was assumed to be 100 days, so it was sufficient for our study period of 3 months. The results showed that this was a reliable method. Because many animals, including sheep [15], are able to endothelialize different prostheses implanted, cell labeling was important to prove the cells to originate from the initial cell layer. The results of the animal that had died on the first postoperative day gave clear evidence for the stability of the cell layer, and the reliability of the labeling method in which a still confluent, labeled cell layer was found. It was expected that not all cells were still positive for PKH-26 after the 3-month period, as the dye was not transmitted sufficiently to daughter cells by cell division [13]. Because there is always a physiologic turnover of endothelial cells under in vivo conditions, some of the initially seeded cells were replaced by new ones. In addition, small endothelial cell defects of any reason are replaced by circulating endothelial progenitor cells, thus replacing labeled cells. The valves of the study group showed a confluent endothelial cell layer, whereas those of the control group did not. This showed that the underlying layer of myofibroblasts in combination with the endothelial cells had synthetized a physiologic extracellular matrix and basement membrane, thus enabling for the physiologic mechanisms of repair.
The neointimal formation starting at the anastomotic sites was a usual process when prostheses were implanted in the arterial system. The stop of this ingrowth after a few millimeters showed that the surface of the untreated glutaraldehyde-fixed prostheses was not suitable for cell covering. The free collagen fibers were thrombogenic, demonstrated by the presence of thrombotic material, especially in the sinuses and on the leaflets. On SEM, numerous erythrocytes and thrombocytes were found to be entrapped between the fibers, which could be a potential source for thrombosis. The sinuses had two features increasing thrombogenicity (ie, a turbulent flow and no anti-thrombotic properties on the surface). In contrast, the valves of the study group had a still confluent endothelial cell layer. The antithrombotic function of these cells had obviously prevented any thrombus formation because none was found, neither macroscopically nor histologically nor on SEM.
The degeneration found in all explanted valves was similar to previous animal experiments with these valves [16]. Although there was no evident calcification, and the motion of the leaflets was maintained, they were thickened and stiffer compared with implantation. Because the degeneration process in sheep is not only related to immunologic reasons, but even much more to calcium metabolism, it was not surprising that the endothelialization did not alter this process. Despite the luminal covering of the foreign material avoiding direct blood contact, the complete prosthesis was subject to a kind of foreign body reaction targeting the outer surface of the protheses. However, the pretreated grafts also maintained form and function, thus giving no signs for any structural weakening due to the citric acid pretreatment. The fact that we found CD34 positive cells mainly on the outer walls of the prostheses gives no evidence for an unaltered immunogenicity, but makes it unlikely.
Many scaffolds have been studied for tissue engineering of heart valves [17–21] and different cell types were also used [5, 10, 22]. Obtaining the cells from veins was an uncomplicated method that could be used even in clinical practice [23]. Resorbable materials possess the big advantage that after degradation no foreign material remains in the recipient. The stability, not only of the achieved cell layers, but also of the whole constructs, needs to be proven before any clinical use. Recently, implantation of such engineered grafts as conduit in patients with Fontan-circulation was published [24]. Their success stimulates tissue engineering, because they proved the feasibility of these techniques even in a clinical setting. Due to the implantation in a circulation with the lowest hemodynamic stress, the results can not be transferred to the arterial system. The implantation of a seeded graft into the pulmonary position in the Ross procedure, and our own report about implantation of an endothelialized aortic homograft also supported the feasibility of these techniques in principle. With the presented technique of tissue engineering onto commercially available biological aortic valve prostheses, a demand for further clinical research is evolving. These valves are already tested and in clinical use, and they are not expected to perform worse after cell seeding. In contrary, patients may benefit from the improved biocompatibility of these constructs, as it was already shown for patients receiving endothelialized vascular prostheses [25]. Due to the disability to grow of these prostheses, they cannot be used in pediatric patients, although this special group would profit most from tissue-engineered valves. However, for the researcher they can be valuable tools to gain more experience with the clinical application of tissue engineering. They should not be the end of heart valve tissue engineering, but one further step.
In conclusion, the artificially achieved cell layer proved to be stable under in vivo conditions in the arterial system. The endothelial cell covering seemed to reduce the thrombogenicity of the prostheses.
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Disclosures and Freedom of Investigation
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This experimental study was supported by a grant of US$ 30,000, sponsored by Medtronic Inc, Germany (Duesseldorf, Germany). All authors had full control of the design of the study, the methods used, the outcome measurements, the analysis of data, and the production of the written report.
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Footnotes
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Disclaimer The Society of Thoracic Surgeons, the Southern Thoracic Surgical Association, and The Annals of Thoracic Surgery neither endorse nor discourage use of the new technology described in this article.
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References
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