ePTFE Vascular Grafts Research Articles

Susceptibility of ePTFE vascular grafts and bioengineered human acellular vessels to infection

BackgroundSynthetic expanded polytetrafluorethylene (ePTFE) grafts are routinely used for vascular repair and reconstruction but prone to sustained bacterial infections. Investigational bioengineered human acellular vessels (HAVs) have shown clinical success and may confer lower susceptibility to infection. Here we directly compared the susceptibility of ePTFE grafts and HAV to bacterial contamination in a preclinical model of infection. Materials and methodsSections (1 cm2) of ePTFE (n = 42) or HAV (n = 42) were inserted within bilateral subcutaneous pockets on the dorsum of rats and inoculated with Staphylococcus aureus (107 CFU/0.25 mL) or Escherichia coli (108 CFU/0.25 mL) before wound closure. Two weeks later, the implant sites were scored for abscess formation and explanted materials were halved for quantification of microbial recovery and histological analyses. ResultsThe ePTFE implants had significantly higher abscess formation scores for both S. aureus and E. coli inoculations compared to that of HAV. In addition, significantly more bacteria were recovered from explanted ePTFE compared to HAV. Gram staining of explanted tissue sections revealed interstitial bacterial contamination within ePTFE, whereas no bacteria were identified in HAV tissue sections. Numerous CD45+ leukocytes, predominantly neutrophils, were found surrounding the ePTFE implants but minimal intact neutrophils were observed within the ePTFE matrix. The host cells surrounding and infiltrating the HAV explants were primarily nonleukocytes (CD45−). ConclusionsIn an established animal model of infection, HAV was significantly less susceptible to bacterial colonization and abscess formation than ePTFE. The preclinical findings presented in this manuscript, combined with previously published clinical observations, suggest that bioengineered HAV may exhibit low rates of infection.

Assessing the Safety of Expanded Polytetrafluoroethylene Synthetic Grafts in Living Donor Liver Transplantation: Graft Migration Into Hollow Viscous Organs - Diagnosis and Treatment Options.

BackgroundOur recent studies have highlighted the importance and safety of backtable venoplasty for middle hepatic vein (MHV) and inferior right hepatic veins (IRHV) reconstruction using expanded polytetrafluoroethylene (ePTFE) vascular grafts. In this study, we aim to analyze the complications associated with ePTFE graft use and discuss the management of the rare, but, potentially life threatening complications directly related to ePTFE conduits.Material/MethodsFrom January 2012 to October 2015 a total of 397 patients underwent living donor liver transplantation (LDLT). The ePTFE vascular grafts were used during the backtable venoplasty for outflow reconstruction in 262 of the liver allografts. Recipients who developed ePTFE-related complications were analyzed.ResultsePTFE-related complications developed in 1.52% (4/262) of the patients. One patient (0.38%) developed complete thrombosis with sepsis at 24 months post-transplantation and died due to multiorgan failure. Three patients (1.1%) developed graft migration into the second portion of the duodenum, without overt peritonitis. Surgical exploration and ePTFE graft removal was done in all the patients. One patient died due to overwhelming sepsis.ConclusionsePTFE graft migration into the duodenum causing perforation is a new set of complications that has been recently described in LDLT and can be treated effectively by surgical removal of the infected vascular graft and duodenal perforation closure. Despite of such complications, in our experience, ePTFE use in LDLT continues to have wide safety margin, with a complication rate of only 1.52%.

Surface Coating of Polytetrafluoroethylene with Extracellular Matrix and Anti-CD34 Antibodies Facilitates Endothelialization and Inhibits Platelet Adhesion Under Sheer Stress.

Expanded polytetrafluoroethylene (ePTFE) polymers do not support endothelialization because of nonconductive characteristics towards cellular attachment. Inner surface modification of the grafts can improve endothelialization and increase the long-term patency rate of the ePTFE vascular grafts. Here we reported a method of inner-surface modification of ePTFE vascular graft with extracellular matrix (ECM) and CD34 monoclonal antibodies (CD34 mAb) to stimulate the adhesion and proliferation of circulating endothelial progenitor cells on ePTFE graft to enhance graft endothelialization. The inner surface of ECM-coated ePTFE grafts were linked with CD34 mAb in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution and the physicochemical properties, surface morphology, biocompatibility, and hemocompatibility of the grafts were studied. The hydrophilicity of CD34 mAb-coated graft inner surface was significantly improved. Fourier transform infrared spectroscopy analysis confirmed ECM and CD34 mAb cross-linking in the ePTFE vascular grafts with our method. Scanning electron microscopy analysis showed protein layer covering uniformly on the inner surface of the modified grafts. The cell-counting kit-8 (CCK-8) assay confirmed that the modified graft has no obvious cytotoxicity. The modified graft showed a low hemolytic rate (0.9%) in the direct contact hemolysis test, suggesting the modification improved hemocompatibility of biopolymers. The modification also decreased adhesion of platelets, while significantly increased the adhesion of endothelial cells on the grafts. We conclude that our method enables ePTFE polymers modification with ECM and CD34 mAb, facilitates endothelialization, and inhibits platelet adhesion on the grafts, thus may increase the long-term patency rate of the prosthetic bypass grafts.

Biomimetic Heparan Sulfate-Like Coated ePTFE Grafts Reduce In-graft Neointimal Hyperplasia in Ovine Carotids

Thrombogenicity and neointimal hyperplasia are major causes for synthetic vascular graft failure. Bioactive coatings like heparin have improved patency by reducing thrombogenicity, but neointimal hyperplasia still remains an unsolved problem. Surface coatings with heparan sulfate (HS), the major component of the glycocalyx of endothelial cells, have shown reduced platelet and cell adhesion invitro. The aim of the study was to evaluate the invivo surface properties of expanded ePTFE vascular grafts with a semisynthetic HS-like coating (SSHS). ePTFE vascular grafts (n=16, diameter 3.5mm) covalently coated with SSHS were compared with uncoated grafts (n=16) of the same diameter in a carotid interposition model in 16 sheep. The grafts were harvested at 20wk for histological and morphometric analysis. SSHS-coated grafts showed less neointima formation than uncoated grafts (P<0.001). There was no evidence for cell or protein adhesion to SSHS-coated grafts, whereas the surface of uncoated ePTFE grafts was covered with a confluent circular layer of neointima. No difference was found concerning reactions at the anastomotic site of the genuine carotid vessel, both groups displayed neointimal hyperplasia. ePTFE grafts covalently coated with a semisynthetic SSHS-glycosaminoglycan successfully mimicked the endothelial glycocalyx. They displayed excellent antiadhesive properties preventing neointimal formation on the graft surface. The results indicate that a biomimetic SSHS coating may be a useful component of bioengineered grafts and an alternative to synthetic surfaces and endothelial seeding.

Reconstruction of Isolated Inferior Right Hepatic Vein(s) in Right Lobe Living Donor Liver Transplantation Using Polytetrafluoroethylene Grafts: A New Feasible Concept, Technique of 'Bridging Conduit Venoplasty' and Outcomes.

BACKGROUND Right lobe living donor liver transplantation (LDLT) remains the most common form of liver transplantation in Asia. However, reconstruction of the venous outflow in a right liver allograft may pose technical difficulties if hepatic venous variations are present. Recently, much emphasis has been given to the reconstruction of large and multiple inferior right hepatic veins (IRHVs). The method of reconstructive technique, type of vascular grafts, and the outcome after the procedure have been a point of debate. In this report we discuss the IRHV reconstruction techniques using expanded polytetrafluoroethylene (ePTFE) vascular grafts and the outcomes after such reconstruction. MATERIAL AND METHODS Out of 262 right liver allografts that underwent venous reconstruction using ePTFE vascular grafts, IRHVs required either venoplasty or second inferior vena cava (IVC) anastomosis in 99 recipients. Depending upon type of IRHV reconstruction, the recipients were divided in 2 groups: Group A (n=52): IRHV venoplasty using ePTFE graft, and group B (n=47): Direct IRHV-to-IVC anastomosis. The outcome after LDLT was compared for these 2 groups. RESULTS The ePTFE venoplasty group had significantly shorter warm ischemia time as compared to the direct to IVC anastomosis group (p<0.01, 95% confidence interval -10.96 to -2.92). There were no thrombotic complications in either group of recipients; 4.2% of the recipients from group B developed hepatic venous stenosis but with no clinical deterioration; and 1 patient from group A developed ePTFE graft migration in the second portion of the duodenum that required surgical exploration. CONCLUSIONS The IRHVs drain a considerable portion of the posterior sector of right liver allografts and thus must be reconstructed. Use of ePTFE vascular grafts for IRHV venoplasty is a safe and feasible concept that facilitates the outflow reconstruction of liver allografts.

E-WE Thrombin (ProCase) Inhibits Thrombin Mediated TAFI Activation and Accelerates TPA-Induced Thrombolysis

Thrombin-activatable fibrinolysis inhibitor (TAFI) activation during thrombolysis may limit the effectiveness of tissue plasminogen activator (tPA) treatment. Our data suggest that an antithrombotic human thrombin analog, E-WE thrombin (ProCase), which is a selective protein C activator under development for treating acute thrombotic emergencies, also promotes tPA-induced fibrinolysis. We found that E-WE thrombin is a poor activator of TAFI compared to wild-type (WT) thrombin. In the presence of thrombomodulin (TM), WT thrombin induced TAFI activation within 5 min. In contrast, detectable amounts of activated TAFI were not generated by E-WE thrombin in the presence of TM until 60 min. Further studies using a quantitative TAFI activity assay showed that E-WE thrombin inhibited thrombin/thrombomodulin-mediated TAFI activation in a concentration-dependent manner. The ability of E-WE to enhance tPA induced clot lysis was also evaluated in vitro. When both E-WE thrombin and tPA were incorporated within plasma clots, lysis was accelerated by up to 74% compared with the addition of tPA alone. We then tested the ability of E-WE thrombin to interrupt arterial-type experimental thrombus formation in baboons when combined with a standard interventional dose of tPA (1mg/kg). Thrombosis was initiated in the baboons by interposing 4mm internal diameter collagen coated ePTFE vascular grafts within an arterio/venous shunt. Thrombus formation was monitored by real-time gamma camera imaging of autologous 111In-labelled platelet accumulation in the grafts for a total of 90 min. Fibrin deposition was determined by direct endpoint measurement of incorporated 125I-labelled fibrinogen. Antithrombotic interventions were injected intravenously at 30 min after graft deployment into the shunt. Treatment with tPA (1mg/kg, iv) reduced fibrin deposition by 57%, but did not reduce graft-associated platelet accumulation compared with controls (n=8 and 9, respectively). E-WE thrombin, at doses ranging from 2-10µg/kg (n=8), interrupted thrombus growth within 10 min of treatment, and reduced platelet and fibrin deposition by 53-70% and 45-58% at 90 min, respectively, compared with controls. When E-WE thrombin (2µg/kg) was co-administered with tPA (1mg/kg), a profound 91% reduction in thrombus fibrin content was observed (n=1), with platelet deposition being reduced by 34%. Bleeding time and volume were also assessed during the studies, with E-WE treated animals showing no increased bleeding compared with controls. However, tPA caused ecchymoses that were not observed with E-WE thrombin treatment. The combination therapy showed no overt anti-hemostatic effects beyond tPA administration alone. These data suggest that E-WE thrombin can inhibit TAFI activation, and co-administration of E-WE thrombin with tPA may improve the efficacy of thrombolysis without additional hemostasis impairment. DisclosuresTucker:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Markway:Aronora, Inc: Employment, Equity Ownership. Wallisch:Aronora, Inc: Employment, Equity Ownership. Verbout:Aronora, Inc: Employment, Equity Ownership. Lorentz:Aronora, Inc: Employment, Equity Ownership. Carris:Aronora, Inc: Employment, Equity Ownership. Gruber:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Elastic mismatch between ePTFE and PLLA vascular grafts in relation to femoral and carotid arteries in humans: in vivo, in vitro and in silico assessment

Many factors have been held responsible for the elevated failure rates of vascular substitutes. Several studies have shown that the lower the mismatches regarding mechanical or functional properties between the native vessel and its vascular substitute, the lower the hemodynamic alterations. From a theoretical point of view, the best substitutes have similar characteristics to the tissue or organ to be replaced. In the present work, samples of electrospun nanofibrous Poly(L-lactic acid) (PLLA) and expanded polytetrafluoroethylene (ePTFE) vascular grafts were subjected to pulsated pressure conditions using an experimental setup. Graft dynamic response was obtained from experimental measurements applying an ultrasonic gold standard method (to measure graft diameter) and a high frequency response pressure transducer (to obtain intraluminal pressure). Similarly, femoral and carotid arteries of normotensive male control subjects and ambulatory male patients with mild to moderate hypertension were explored in vivo in order to relate their mechanical performance to that observed in the synthetic grafts. The obtained results verified the existence of important biomechanical differences between vascular grafts and human arteries. Under normal physiological conditions, the pressure-strain modulus (106dyn/cm2) of ePTFE and PLLA (1095 ± 21.5 and 12.4 ± 1.47, respectively) resulted significantly higher (p < 0.05) than the carotid and femoral modulus values (4.47 ± 1.36 and 5.9 ± 1.42, respectively). During the hypertensive condition (higher pressure levels) ePTFE and PLLA increased their elasticity values to 1136 ± 21.6 and 15.97 ± 2.6, respectively, with respect to an increase of 9.27 ± 3.59 and 15.70 ± 5.96 of carotid and femoral arteries. Additionally, the elastic mismatch generated by the interposition of both grafts between native arteries (carotid or femoral) was quantified by means of computational simulations. PLLA tubular structures manifested a mechanical behavior that mimics the response to pulsatile hypertensive pressure regimes (the lowest reflection coefficient was observed) which constitutes a highly desired property for vascular tissue-engineered scaffold production.

Co-expression of fibulin-5 and VEGF165 increases long-term patency of synthetic vascular grafts seeded with autologous endothelial cells

Small caliber synthetic vascular grafts are commonly used for bypass surgery and dialysis access sites but have high failure rates because of neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. However, EC detachment following exposure to blood flow still remains a major obstacle in the development of biosynthetic grafts. We tested the hypothesis that induced expression by the seeded EC, of vascular endothelial growth factor165 (VEGF165) and of fibulin-5, an extracellular matrix glycoprotein that has a crucial role in elastin fiber organization and increase EC adherence to surfaces, may improve long-term graft patency. Autologous ECs were isolated from venous segments, and were transduced with retroviral vectors expressing fibulin-5 and VEGF165. The modified cells were seeded on expanded polytetrafluoroethylene (ePTFE) grafts and implanted in a large animal model. Three months after transplantation, all grafts seeded with modified EC were patent on a selective angiography, whereas only a third of the control grafts were patent. Similar results were shown at 6 months. Thus, seeding ePTFE vascular grafts with genetically modified EC improved long-term small caliber graft patency. The biosynthetic grafts may provide a novel therapeutic modality for patients with peripheral vascular disease and patients requiring vascular access for hemodialysis.

Dual biofunctional polymer modifications to address endothelialization and smooth muscle cell integration of ePTFE vascular grafts.

Expanded polytetrafluoroethylene (ePTFE) grafts were coated on the luminal surface with a cell-adhesive fluorosurfactant (FSP) polymer to promote endothelialization, followed by ethanol hydration to degas the pores and subsequent cell-adhesive, enzymatically degradable poly(ethylene glycol)-based hydrogel incorporation into the graft interstices to accommodate potential smooth muscle cell integration in the graft wall. The FSP coating on ePTFE was stable as demonstrated by a significantly reduced receding water contact angle on FSP-coated ePTFE (14.5 ± 6.4°) compared to uncoated ePTFE (105.3 ± 4.5°, P < 0.05) after ethanol exposure. X-ray photoelectron spectroscopy analysis of the same surfaces confirmed FSP presence. Localization of the FSP and hydrogel within the ePTFE graft construct was assessed using fluorescently labeled polymers, and demonstrated hydrogel infiltration throughout the thickness of the graft wall, with FSP coating limited to the lumen and adventitial surfaces. FSP at the luminal surface on dual-coated grafts was able to bind endothelial cells (EC) (98.7 ± 23.1 cells/mm(2) ) similar to fibronectin controls (129.4 ± 40.7 cells/mm(2) ), and significantly higher than uncoated ePTFE (31.6 ± 19 cells/mm(2,) P < 0.05). These results indicate that ePTFE grafts can be simultaneously modified with two different polymers that have potential to directly address both endothelialization and intimal hyperplasia. Such a construct is a promising candidate for an off-the-shelf synthetic graft for small-diameter graft applications.

Enabling non-invasive assessment of an engineered endothelium on ePTFE vascular grafts without increasing oxidative stress

Magnetic resonance imaging (MRI) in combination with contrast enhancement is a potentially powerful tool to non-invasively monitor cell distribution in tissue engineering and regenerative medicine. The most commonly used contrast agent for cell labeling is super paramagnetic iron oxide nanoparticles (SPIONs). However, uptake of SPIONs triggers the production of reactive oxygen species (ROS) in cells often leading to a pro-inflammatory phenotype. The objective of this study was to develop a labeling system to non-invasively visualize an engineered endothelium in vascular grafts without creating excessive oxidative stress. Specifically, we investigated: (1) chitosan-coated SPIONs (CSPIONs) as an antioxidant contrast agent for contrast enhancement, and (2) poly(1,8-octamethylene citrate) (POC) as an antioxidant interface to support cell adhesion and function of labeled cells on the vascular graft. While SPION-labeled endothelial cells (ECs) experienced elevated ROS formation and altered cell morphology, CSPION-labeled ECs cultured on POC-coated surfaces mitigated SPION-induced ROS formation and maintained EC morphology, phenotype, viability and functions. A monolayer of labeled ECs exhibited sufficient contrast with T2-weighed MR imaging. CSPION labeling of endothelial cells in combination with coating the graft wall with POC allows non-invasive monitoring of an engineered endothelium on ePTFE grafts without increasing oxidative stress.

In vivo evaluation of biomimetic fluorosurfactant polymer-coated expanded polytetrafluoroethylene vascular grafts in a porcine carotid artery bypass model

The objective of this study was to evaluate the potential for biomimetic self-assembling fluorosurfactant polymer (FSP) coatings incorporating heptamaltose (M7-FSP) to block nonspecific protein adsorption, the cell adhesive RGD peptide (RGD-FSP), or the endothelial cell-selective CRRETAWAC peptide (cRRE-FSP) to improve patency and endothelialization in small-diameter expanded polytetrafluoroethylene (ePTFE) vascular graft implants. ePTFE vascular grafts (4mm in diameter, 5cm in length) were coated with M7-FSP, RGD-FSP, or cRRE-FSP by dissolving FSPs in distilled water and flowing solution through the graft lumen for 24hours. Coatings were confirmed by receding water contact angle measurements on the lumen surface. RGD-FSP and cRRE-FSP grafts were presodded invitro with porcine pulmonary artery endothelial cells (PPAECs) using a custom-designed flow system. PPAEC coverage on the lumen surface was visualized with epifluorescent microscopy and quantified. Grafts were implanted as carotid artery interposition bypass grafts in seven pigs for 33± 2days (ePTFE, n= 3; M7-FSP, n= 4; RGD-FSP, n= 3; cRRE-FSP, n= 4). Patency was confirmed immediately after implantation with duplex color flow ultrasound and at explantation with contrast-enhanced angiography. Grafts were sectioned for histology and stained: Movat pentachrome stain to outline vascular layers, immunofluorescent staining to identify endothelial cells (anti-von Willebrand factor antibody), and immunohistochemical staining to identify smooth muscle cells (anti-smooth muscle α-actin antibody). Neointima to lumen area ratio was determined to evaluate neointimal hyperplasia. Receding water contact angle measurements on graft luminal surfaces were significantly lower (P< .05) on FSP-coated ePTFE surfaces (M7-FSP, 40± 16 degrees; RGD-FSP, 25± 10 degrees; cRRE-FSP, 33± 16 degrees) compared with uncoated ePTFE (126± 2 degrees), confirming presence of the FSP layer. Invitro sodding of PPAECs on RGD-FSP and cRRE-FSP grafts resulted in a confluent monolayer of PPAECs on the luminal surface, with a similar cell population on RGD-FSP (1200± 187 cells/mm(2)) and cRRE-FSP (1134± 153 cells/mm(2)) grafts. All grafts were patent immediately after implantation, and one of three uncoated, two of three RGD-FSP, two of four M7-FSP, and two of four cRRE-FSP grafts remained patent after 1month. PPAEC coverage of the lumen surface was seen in all patent grafts. RGD-FSP grafts had a slightly higher neointima to lumen area ratio (0.53± 0.06) compared with uncoated (0.29±0.15), M7-FSP (0.20± 0.15), or cRRE-FSP (0.17± 0.09) grafts. Biomimetic FSP-coated ePTFE grafts can be used successfully invivo and have potential to support endothelialization. Grafts modified with the M7-FSP and cRRE-FSP showed lower intimal hyperplasia compared with RGD-FSP grafts.

Biological effects of anti-CD34-coated ePTFE vascular grafts. Early in vivo experimental results.

Aim of the studyTo assess the biological activity of anti-CD34 antibody-coated ePTFE vascular prostheses.Material and methodsIndium111-labeled autologous thrombocytes were administered to 5 anesthetized pigs after the placement of femoral arterial and venous catheters. An arterio-venous fistula, created by the random interposition of 4 different ePTFE grafts (A = dry control, B = dry anti-CD34, C = wet control, D = wet anti-CD34), was blood perfused for 0, 10, 30, 60 and 120 minutes. Radioactivity of each graft was measured and expressed in cpm/mg. Morphological studies were performed to assess intraluminal deposition.ResultsThe median radioactivity of graft B was significantly higher than that of graft A after 60 min (1074 vs. 18; p = 0.021) and 120 min (1990 vs. 25; p = 0.043) of perfusion. Similarly, graft D was significantly more active than graft C (60 min: 1388 vs. 26; p = 0.021 and 120 min: 2780 vs. 23; p = 0.021). Histological and SEM results confirmed the radio-labeling in-vivo studies by showing significantly more protein/cell and platelet depositions (p = 0.012).ConclusionsAnti-CD34-coated ePTFE grafts bound significantly more platelets/cells and proteins than their uncoated counterparts, confirming the bioactivity of the antibody. This process is time-dependent and matches the morphological results. The anti-CD34 coating may enhance temporal and spatial endothelialization of vascular grafts and, thus, possibly improve clinical results by providing direct endothelial progenitor cell (EPC) adhesion/entrapment or by creating a biocompatible protein-thrombocyte/cell layer that indirectly enhances migration and further proliferation of EPCs.

Characterization of silk sponge in the wet state using13C solid state NMR for development of a porous silk vascular graft with small diameter

The 13C solid state NMR spectra of the sponge prepared from Bombyx mori silk fibroin with different 13C labeling sites, [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala were observed in dry and wet states. Here the silk fibroin sponge was prepared using poly(ethylene) glycol diglycidyl ether (PGDE) as porogen. Two broad peaks assigned to β-sheet and random coil were observed for each 13C-labeled site in the dry state. Moreover, in the wet state, one additional sharp peak assigned to random coil with high mobility due to the presence of water was observed. When the silk fibroin aqueous solution and PGDE was mixed in the sponge preparation process, the slow structural change from random coil to β-sheet structure occurs together with decrease in the motion of the silk fibroin backbone chain. The silk fibroin graft with small diameter was prepared with a knitted silk fiber tube coated with this silk fibroin sponge. The high porosity and suture retention strength which are comparable to an ePTFE vascular graft were attained. The excellent results, high patency and high tissue ingrowth, were obtained with the porous silk fibroin graft with small diameter by a rat abdominal aorta implantation experiment.

Rapid Interruption Of Occlusive Thrombus Formation By The Protein C Activator Enzyme EWE Thrombin (ProCase) In Primates

A hemostatically safe antithrombotic treatment with rapid onset could significantly improve the chances for survival during acute thrombotic events such as heart attack and ischemic stroke. Current thrombolytic treatment with tissue plasminogen activator (tPA) is effective at halting and reversing the progression of arterial thrombi, but bleeding side-effects significantly limit its usefulness. EWE thrombin (ProCase) is an investigational selective protein C activator thrombin analog under development for treating acute thrombotic emergencies. We therefore tested the ability of EWE thrombin to interrupt arterial-type thrombus formation in baboons compared to a standard interventional dose of tPA (1mg/kg, iv). Thrombosis was initiated by interposing 2mm or 4mm internal diameter (ID) collagen coated ePTFE vascular grafts within an arterio/venous shunt. Platelet thrombus formation was monitored by gamma camera imaging of autologous 111In-labelled platelets for a total of 90 min in the 4mm grafts, or until occlusion time in the 2mm grafts. Fibrin deposition was determined by direct measurement of 125I-labelled fibrinogen. All interventions were given systemically, starting 30 min or 15 min after thrombus initiation for the 4mm grafts and 2mm grafts, respectively. In the 4mm devices, treatment with tPA reduced graft-associated platelet accumulation by 17% and fibrin deposition by 61% compared with controls (n=6 each). A negative platelet accumulation rate, which is an indication of thrombolysis, occurred between 40-50 min after initiating tPA treatment. EWE thrombin, at doses ranging from 2-10µg/kg iv bolus rapidly interrupted thrombus development and reduced platelet deposition by 43-65% (n=4). Fibrin deposition was also reduced by 36-49% compared with controls. A negative platelet accumulation rate occurred between 10-15 min after EWE thrombin treatment, compared with continuous platelet accumulation in all control experiments. In the 2mm ID grafts, 0/6 control group devices remained patent with an average occlusion time of 25±2 min. By comparison, EWE thrombin (10µg/kg iv bolus given at 15 min) successfully interrupted occlusive thrombus formation in 40% of the devices (2/5) during the 60 min study, and significantly prolonged the occlusion time to an average of 43±8 min. These data suggest that EWE thrombin can effectively interrupt occlusive arterial-type thrombus development at very low doses, and appears to be more effective and act more rapidly than tPA at limiting experimental platelet-rich thrombus accumulation. We conclude that EWE thrombin may be a more effective and safer alternative to tPA for treating acute thrombotic emergencies. Disclosures:Tucker:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Wallisch:Aronora, Inc: Employment. Leung:Aronora, Inc: Employment, Equity Ownership. Gruber:Aronora, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Verbout:Aronora, Inc: Employment, Equity Ownership.

Cross‐reactivity of cell‐selective CRRETAWAC peptide with human and porcine endothelial cells

We report on the cross-reactivity of the cell adhesive peptide CRRETAWAC between human and porcine endothelial cells (ECs). CRRETAWAC is a phage display derived peptide which has been shown to bind the α5 β1 receptor on human ECs, but does not bind platelets and thus could be incorporated into a coating for cardiovascular biomaterials that resists platelet adhesion and thrombosis, while allowing for endothelialization. To determine the cross-reactivity of the peptide, attachment and growth of human and porcine ECs on CRRETAWAC fluorosurfactant polymer (FSP) coated surfaces was explored. CRRETAWAC FSP was synthesized and characterized by mass spectrometry, NMR, and IR spectroscopy. pEC attachment and growth on CRRETAWAC FSP was similar to the positive controls, human fibronectin and RGD FSP, achieving confluence in 72 h. Initial adhesion on CRRETAWAC FSP was also similar for porcine and human ECs. Blocking with soluble CRRETAWAC peptide reduced adhesion to CRRETAWAC coated surfaces by over 50%, indicating that the pECs specifically bind CRRETAWAC peptide. With this study, we have demonstrated that CRRETAWAC peptide coated surfaces are capable of binding porcine ECs in a specific manner and supporting a confluent layer of pECs. In vitro validation of the porcine model was critical for ensuring the best chance of success for the in vivo testing of CRRETAWAC coated ePTFE vascular grafts.

Hyperplasia of neointima in small-caliber decellularized matrix vascular grafts at 4 weeks after transplantation

Objective To explore neointimal hyperplasia in small-caliber decellularized matrix vascular grafts at 4 weeks after transplantation.Methods 17 male dogs were randomly divided into 2 groups.All the animals underwent right common carotid artery interposition with DCM (n =9) or ePTFE (n =8) vascular grafts.Color Doppler Ultrasound was applied to detect peak systolic velocity (PSV),end diastolic velocity (EDV),resistance index (RI),and inner diameter of anastomosis and midportion of the grafts at 1 and 4 weeks after implantation.All the grafts were harvested and evaluated by HE staining and light microscopy.Results All implanted grafts remained patent within 4 weeks.The difference of RI (△ RI) at the distal anastomosis between 1 and 4 weeks differed significantly in the two groups (P＜0.05).The difference of other hemodynamic parameters at anastomosis and midportion of the grafts did not differ significantly between the two groups (P＞0.05 for all comparisons).The difference of inner diameter (△ D) at anastomosis and midportion was significantly smaller in DCM group than in ePTFE group (P＜0.05 for all comparisons).Conclusions The degree of neointimal hyperplasia is smaller in small-caliber DCM vascular grafts than in ePTFE vascular grafts within 4 weeks after ttransplantation. Key words: Small-caliber, Vascular grafts; Decellularized matrix; ePTFE; Color Doppler ultrasound

Immobilization of Actively Thromboresistant Assemblies on Sterile Blood‐Contacting Surfaces

Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.

Adipose Stromal Vascular Fraction Cells Isolated Using an Automated Point of Care System Improve the Patency of Expanded Polytetrafluoroethylene Vascular Grafts

We evaluated the use of an automated, point-of-care instrument to derive canine adipose stromal vascular fraction cells, and the subsequent deposition of these cells onto the luminal surface of an expanded polytetrafluoroethylene (ePTFE) vascular graft for use as a bypass graft. The hypothesis evaluated was that an instrument requiring minimal user interface will provide a therapeutic dose of cells to improve the patency of synthetic vascular grafts in an autologous animal model of graft patency. The stromal vascular fraction (SVF) cells were isolated using an automated adipose tissue processing and cell isolation system and cells sodded onto the surface of an ePTFE vascular graft. Control grafts, used off-the-shelf without cell treatment were used as a control to assess patency effects. Each animal received a control, untreated graft implanted in one carotid artery, and the cell-treated graft implanted in the carotid artery on the contralateral side. The grafts were implanted for 6 months utilizing 12 animals. Results indicate a fully automated adipose tissue processing system will consistently produce functional autologous cells for immediate use in the operating room. Cell-sodded polymeric grafts exhibited improved patency compared to control grafts after 6 month implantation in the canine carotid artery model.

The blood and vascular cell compatibility of heparin-modified ePTFE vascular grafts

Prosthetic vascular grafts do not mimic the antithrombogenic properties of native blood vessels and therefore have higher rates of complications that involve thrombosis and restenosis. We developed an approach for grafting bioactive heparin, a potent anticoagulant glycosaminoglycan, to the lumen of ePTFE vascular grafts to improve their interactions with blood and vascular cells. Heparin was bound to aminated poly(1,8-octanediol-co-citrate) (POC) via its carboxyl functional groups onto POC-modified ePTFE grafts. The bioactivity and stability of the POC-immobilized heparin (POC–Heparin) were characterized via platelet adhesion and clotting assays. The effects of POC–Heparin on the adhesion, viability and phenotype of primary endothelial cells (EC), blood outgrowth endothelial cells (BOECs) obtained from endothelial progenitor cells (EPCs) isolated from human peripheral blood, and smooth muscle cells were also investigated. POC–Heparin grafts maintained bioactivity under physiologically relevant conditions in vitro for at least one month. Specifically, POC–Heparin-coated ePTFE grafts significantly reduced platelet adhesion and inhibited whole blood clotting kinetics. POC–Heparin supported EC and BOEC adhesion, viability, proliferation, NO production, and expression of endothelial cell-specific markers von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin). Smooth muscle cells cultured on POC–Heparin showed increased expression of α-actin and decreased cell proliferation. This approach can be easily adapted to modify other blood contacting devices such as stents where antithrombogenicity and improved endothelialization are desirable properties.

Study on vascular transplantation with small-caliber decellularized matrix vascular grafts

Objective To explore the feasibility of smal-caliber decellularized matrix (DCM) vascular grafts in vascular transplantation.Methods 40 male dogs were randomly divided into three groups,and underwent right common carotid artery interposition with DCM (group Ⅰ,n =16),expanded polytetrafluoroethylene (ePTFE) (group Ⅱ,n =14 ) vascular grafts or autologous external jugular vein (group Ⅲ,n =10).Postoperative Doppler Ultrasound were done to detect the patency.Grafts were explanted at 4 and 8 weeks after implantation.The retrieved specimens were evaluated by morphology,hematoxylin and eosin (HE) stain,immunohistochemistry stain,and scanning electron microscopy.Results The patency rates at 1 week among the three groups (75.0％,64.3％,100.0％ respectively) were of no difference of significance (P ＞0.05).4 and 8 week patency rates of group Ⅲ ( 100.0％,88.9％ )are both higher than group Ⅰ (56.3％,26.7％ ) and group Ⅱ (57.1％,23.1％,P ＜0.05 ),however,no differences of significance were gained between the latter two (P ＞ 0.05).The thrombus formation area of DCM grafts was smaller than that of ePTFE grafts.DCM grafts achieved better endothelialization in the stomas neointima than ePTFE grafts within 8 weeks.Conclusion Compared with ePTFE grafts,DCM grafts show superiority in reducing thrombogeneity,and improving endothelialization. Key words: Small-caliber; Vascular grafts; Decellularized matrix; Expanded polytetrafluoroethylene; External jugular vein