1Department of General, Visceral, Thoracic and Vascular Surgery, Medical Faculty of the Humboldt University, Charitè, Campus Mitte, Berlin and 2Cell-Lining GmbH, Gesellschaft für Zellkulturen, Berlin, Germany
Correspondence and offprint requests to: Julian Mall, MD, Department of General, Visceral, Thoracic and Vascular Surgery, Medical Faculty of the Humboldt University, Charitè, Campus Mitte, Schumannstrasse 20/21, D-10117 Berlin, Germany. Email: julianmall{at}hotmail.com
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Abstract |
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Methods. Pig endothelial cells were harvested from an external jugular vein. Following processing of the endothelial cells, seven ePTFE grafts were coated with an inner cell layer and were kept under pulsed perfusion. Each graft was then cannulated three times with a standard shunt needle. The endothelium was then left to regenerate for a maximum of 48 h. The grafts were stained with haematoxylin/eosin before histological study.
Results. All grafts were endothelialized over the puncture sites within 48 h. Histological analysis revealed a confluent endothelial cell lining at each puncture site. Cell morphology and cell pattern over puncture sites were not different from randomly picked locations over the graft lumen.
Conclusion. Our results underline the potential of endothelial tissue engineering in vascular shunt surgery. Vascular bio-hybrids that have the properties of pristine vascular endothelium may be a key step forward in maintaining angio-access in patients who require haemodialysis.
Keywords: angio-access; ePTFE graft failure; haemodialysis; tissue engineering
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Introduction |
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Although recent data indicate that complications and failure rates of heterotope AV grafts, such as expanded polytetrafluoroethylene (ePTFE) or Gore-Tex grafts, are often higher when compared with autologous AV fistulae [13], heterotope AV graft usage has increased over the last decade and has exceeded the rate of conventional AV fistula (Cimino fistula) implantation, especially in the USA [3,4]. Recent studies suggest that ePTFE grafts are a valuable second choice for vascular access in end-stage renal disease (ESRD) in patients in whom an autologous fistula is not suitable [5].
The factors limiting ePTFE graft survival are arterial thrombosis and anastomotic stenosis. Despite intensive research on surgical shunting techniques or adjuvant treatments, the occlusion rate of this shunt remains a challenging problem in vascular surgery [5].
While early occlusion may be due to haemodynamic problems, mismatch between the elastic or compliance properties of grafts and the adjacent native artery may be additional reasons for graft failure [6,7]. Many authors believe that failure to develop endothelial lining contributes to AV graft failure [8,9]. An important factor in the development of artificial graft failure seems to be the paucity of substances produced by endothelial cells that maintain a balance between thrombolytic and thrombotic activity in native vessels, thus leading to higher thrombogenesis in the heterotope small-diameter graft.
Recent publications indicate that cells that are seeded onto the graft under static conditions fail to remain adherent to the graft's surface under normal blood flow. We previously reported a new method to endothelialize ePTFE grafts under shear stress, where cells stay adherent in vitro under physiological conditions [10]. In a recent study, we examined the structural and histopathological changes of ePTFE grafts, that were endothelialized under shear stress, following implantation in pigs. We found that the confluent endothelial cell lining sustained a typical confluent endothelial morphology in vivo, and no thrombotic formations were found after 6 weeks [11].
With regard to the potential use of flow-processed grafts for haemodialysis access, we hypothesized that the endothelial cell lining of the grafts grows to confluence over the damaged puncture site and will seal the graft wall defect after puncture with a standard dialysis needle. Therefore, we performed an in vitro study aimed at evaluating re-endothelialization after repeated puncture of endothelialized ePTFE grafts that were constantly exposed to pulsed perfusion.
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Subjects and methods |
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Each vein segment was processed as follows. The vein was flushed repeatedly with Dulbecco's phosphate-buffered saline (PBS; Biochrom, Berlin, Germany) and filled with 1 U/ml Collagenase P® (Boehringer, Mannheim, Germany) diluted in PBS. The vein was then incubated at 37°C (gassed with 5% CO2 at 100% humidity) for 15 min. The endothelial cell solution was placed in centrifugation tubes and the collagenase activity was stopped by adding 10% fetal calf serum (FCS)-supplemented standard RPMI medium (Biochrom, Berlin, Germany). After centrifugation at 1000 r.p.m. for 5 min, the supernatant was discarded and the pellet resuspended in PBS. This procedure was repeated twice before the pellet was suspended in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, Steinheim, Germany) and endothelial cell growth medium (ECGM) (PromoCell GmbH, Germany), mixed 1:1. Cells were seeded in an incubator at 37°C (gassed with 5% CO2 at 100% humidity) and grown to subconfluence (80% confluent) before passaging. Harvesting was performed by incubating the cell monolayer for 5 min with PBS-diluted trypsin/EDTA solution (Biochrom, Germany).
Trypsin was inactivated by adding 10% FCS-supplemented standard RPMI medium. After centrifugation and washing in PBS, the cells were seeded again at a ratio of 1:21:4 until final harvest.
Cell number and viability were controlled with an electronic cell counter (Casy®, Schärfe System GmbH, Reutlingen, Germany). Cell identity was confirmed in situ with von Willebrand/factor VIII staining (Sigma-Aldrich, Steinheim, Germany) using the EnVisionTM AP detection system (Dako, Hamburg, Germany) and by observing the typical cobblestone morphology of endothelial cells in the confluent monolayer. Contamination by Mycoplasma and other bacteria was tested routinely by a fluorescence DNA staining kit (Venor GeM-Mycoplasmen Diagnostic Kit, Cat. No. WVGM-025, Biochrom AG, Berlin, Germany). To compare proliferative activity, the population doubling level (PDL) and population doubling time (PDT) were calculated as follows: primary cells multiply exponentially, since one primary cell divides into two second-generation cells. Given a determined number of primary cells (No), after n divisions there will be N cells overall or:
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Photo documentation of cell growth was performed with light microscopy (200 x magnification, Zeiss microscope, Jena, Germany) daily and before new passages of cells. Control cultures of endothelial cells were carried out for each harvest to check growth characteristics and cell viability. Data were tested for normal distribution by Spearman's correlation test. Categorical data are displayed as median and range.
ePTFE graft processing
The ePTFE graft (Model: T04030C30, Baxter, Vascular Systems Division, IL) was placed in an sterile perfusion chamber, TCS2c (Cell-Lining Gmbh, Berlin, Germany), which was configured with two separate circulations, inner and outer. The inner circulation was adjustable to different graft lengths. The outer perfusion circuit was run static (no pump connected, medium reservoir on the same level as the perfusion chamber and outer circulation). The medium reservoirs of the inner and outer perfusion circuits were fitted with sterile membrane filters, guaranteeing gas exchange between the medium and the controlled atmosphere within the incubator, in order to keep the pressure inside the chamber constant (for further chamber specifications, see Figure 1). The inner circulation's outlet was sealed and the graft filled air bubble-free with Beriplast P® fibrin glue (Aventis Behring, Liederbach, Germany). The inlet was then pressurized using a simple 20 ml syringe until small fibrinogen drops could be observed on the graft's exteriorindicating total sweating of the porous material, hence displacement of air. Surplus fibrinogen was removed by blowing air over the graft. The graft was then filled with diluted thrombin solution, air bubble-free, which was immediately blown out and removed. This procedure was repeated until the blown-out thrombin stayed liquid. The inner perfusion circuit was then filled with the prepared endothelial cell suspension and, immediately after a seeding period of 4 h, was connected to a peristaltic pump (Figure 2). The perfusion experiments were all conducted with DMEM containing 10% FCS. A constant flow was applied and slowly increased over the first 10 h with a maximum flow rate of 12.25 ml/s corresponding to a maximum shear stress rate of 15 dyn/ cm. After 10 h, the flow was pulsed with shear rate peaks of 15 dyn/cm2 to simulate human blood flow dynamics. Shear rate values were calculated with the HagenPoiseuille equation: = 4
x Q/(
x r3) [
= shear rate (dyn/ cm),
= dynamic viscosity (Pa x s), Q = flow (ml/min), r = radius (cm)]. Viscosity of DMEM was determined at 37°C with Low-Shear-Viscosimeter LS 40 (Fa. Mettler Toledo, Switzerland). The end point of endothelial cell confluence was determined via the established method based on the Bernouilli principle as follows.
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Shunt puncture
After 4 days of graft perfusion, the graft was cannulated with a standard haemodialysis needle for AV shunts [size 16 gauge x 25 mm (Kawasumi Laboratories, Japan)] under strictly sterile conditions. The ePTFE graft was partly taken out of the perfusion chamber by moving the adjustable lids at top and bottom. Graft perfusion in the inner circuit was not stopped at any time. Punctures were then made twice on each graft following standard clinical procedures, at a 45° angle opposite to the direction of flow. Puncture sites were placed opposite each other and offset by 1.52 cm, and the needles remained in situ for 15 min each. No flow was applied through the needles. Puncture sites were marked permanently with a waterproof felt-tip pen. Penetration sites were then covered with one layer of sterile gauze (ES-Mullkompressen, Paul Hartmann AG, Germany) moistened with fibrin glue Beriplast®. This method was developed by our research group in order to simulate surrounding adventitia and tissue which will occlude puncture defects of shunts in vivo. With this method, the transmural fluid current is reduced to an extent that allows endothelial cells to overgrow the defect. After moving the graft back in place, the perfusion chamber was sealed and the outer circulation refilled with the medium.
The medium loss from the outer to the inner perfusion system due to the graft damage following puncture was monitored closely, according to the confluence determination by Bernouilli [12]. Perfusion was continued for a maximum of 48 h after cannulation.
In addition, three grafts coated with fibrin glue without endothelialization served as controls.
Histological evaluation
The grafts were taken out of the perfusion chamber after 48 h and fixed in 4% formalin. For histological study, the grafts were cut in half longitudinally and stained with haematoxylin/eosin, following routine procedures. Histological evaluation was performed with light microscopy under 200 x magnification by an independent pathologist blinded to the procedure. Photographic documentation was made of each puncture site.
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Results |
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As determined according to the established method based on the Bernouilli effect, all grafts were covered with a monolayer of endothelial cells on the inner luminal surface after a median time of 36 h (range 2448 h) [12]. During the entire process, cell viability was 8095%. No signs of infection or bacterial contamination were seen throughout the repeated testing of the entire series.
Following puncture of the ePTFE grafts, no signs of bacterial contamination were observed. All perfusion systems worked according to protocol, and no leaks, other than those caused deliberately, were observed. Penetration sites were all covered with one layer of sterile gauze moistened with fibrin glue. Experiments conducted without artificial closure of the puncture site showed that fluid influx was strong and the puncture site did not close. We supposed that the medium flux through the defect was too strong to allow either endothelial cell migration or proliferation over the puncture site. Therefore, the method presented was developed by our research group in order to simulate surrounding adventitia and tissue that will occlude puncture defects of shunts in vivo and reduce the fluid current.
Perfusion chamber measurements were started immediately after both chamber lids, with the prosthesis still clamped between them, were fixed in the chamber's outer casing and the outer perfusion system was refilled with fluid. The inlet and outlet stopcocks for the outer perfusion system were closed and the scale chamber was calibrated to zero (tare). Measurements, taken up to 48 h after puncture, are depicted in Figure 3. In the first hour after puncture, chamber weight decreased (Figure 3). The fluid loss reached maximum levels during the first hour, decreasing during the next 47 h as endothelial cell regrowth occurred at the endoluminal site. The average time until fluid outflow decreased to a minimum was 36 h. The puncture defect was considered to be closed when the chamber's weight loss was at an equilibrium of 0.34 g on average. Histological analysis validated that the puncture site closure was due to endothelial cell regrowth (Figures 4 and 5). In all grafts, a confluent endo-thelial cell lining could be detected over the half-moon-shaped damage caused by the dialysis needle.
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Discussion |
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For many patients with ESRD, a native AV fistula cannot be created, and there is no choice but to use a prosthetic AV graft. Poor graft patency and the subsequent treatment of graft thrombosis, however, contribute to significant morbidity and mortality among ESRD patients [2,4,15]. The most common reasons for AV graft failure are stenosis due to neo-intimal hyperplasia (NIH) and thrombogenic occlusion of the graft's lumen. All underlying mechanisms, however, have not been understood so far. Altered mechanical stress has been shown to cause NIH. In addition to this, the absence of autologous endothelium may contribute to an imbalance of endocrine factors that promote and prevent NIH [8,9,1618]. The few effective methods developed to prevent NIH, e.g. using non-penetrating clips [19,20] or vein cuff interpositions, are not universally effective. In order to maintain graft patency in the face of thrombogenic graft properties in difficult cases, permanent anticoagulation may be necessary, carrying with it the additional risks of complications in patients with ESRD. New methods in tissue engineering have led to the development of a new generation of vascular bio-hybrids which can be seeded with autologous endothelial cells, with the ability to withstand high shear rates in vivo [12].
We recently were able to show in a study that ePTFE grafts endothelialized under shear stress sustained a confluent endothelial cell lining after being implanted in vivo for 6 weeks in the absence of anticoagulants, and without any detectable thrombogenic deposits [11]. The data from our study show that this new generation of vascular bio-hybrids not only can withstand high shear rates without losing endothelial integrity, but may regenerate to a confluent endothelial cell layer after being punctured by a haemodialysis needle under flow conditions in vivo.
It can be assumed that the endothelium will revert to its pristine condition in a shunt used in vivo and will prevent thrombus formation at the puncture site. Further studies will have to evaluate whether or not a graft endowed with primordial vascular features provides more reliable angio-access in the long term. Still, it was possible to demonstrate that endothelial cells seeded under dynamic conditions in vitro have the ability to overgrow a defect after repeated controlled damage, something which may be applicable to clinical situations in patients with ESRD. However, several problems remain to be solved. First, the need for endothelial cell harvesting will subject patients to two operations to receive an endothelialized graft. Especially in ESDR patients, many of whom have undergone several operations before, this might be difficult; and therefore additional research data are needed before starting a clinical evaluation. Secondly, processing the graft takes 23 weeks. During this time, patients will need conventional haemodialysis via Sheldon catheters, increasing the risk of thrombotic complications. Presently we are studying the patency rates of endothelialized ePTFE grafts used as haemodialysis shunts in vivo, but results are not yet available.
The potential applications of this bio-hybrid are not limited to haemodialysis. Patients with vascular diseases, e.g. coronary artery disease in need of bypass surgery, could benefit from small calibre, endothelialized under flow PTFE grafts, resulting in higher patency rates than the commonly used grafts. Another area of interest would be peripheral artery bypass surgery (pedal bypasses, femoro-popliteal below-knee bypass) which currently have high failure rates and high thrombotic occlusion rates within the first year.
Certainly, the elastic properties of prosthetic grafts remain an impediment, and the compliance mismatch persists. Nevertheless, the results of our study point to a first step in ameliorating the biological properties of the currently used graft material. Additional studies will be needed to improve the knowledge of tissue engineering of PTFE grafts. It is a promising approach towards improved angio-access in haemodialysis patients and, as such, deserves further research.
Conflict of interest statement. None declared.
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References |
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