Small-diameter Grafts Research Articles

Suture-reinforced electrospun polydioxanone–elastin small-diameter tubes for use in vascular tissue engineering: A feasibility study

This study characterizes the cross-linking of electrospun elastin and the mechanical properties of suture-reinforced 1.5 mm internal diameter electrospun tubes composed of blended polydioxanone (PDO) and soluble elastin. Several tube configurations were tested to assess the effects of reinforcement on tube mechanical properties. Between the electrospun layers of each double-layered prosthetic, zero, one or two 6-0 sutures were wound, maintaining 1 mm spacing with a pitch of 9°. Single-layered tubes without suture were also examined. Samples were cross-linked and tested for compliance and burst strength. Compliance decreased significantly ( p < 0.05) and burst strength significantly increased ( p < 0.01) with reinforcement. Uncross-linked tubes were also tested to determine the effects of cross-linking. Results demonstrated that cross-linking significantly decreases burst strength ( p < 0.01), while decreases in compliance for cross-linked tubes were not significant. Cross-linked suture-reinforced PDO–elastin tubes had burst pressures more than 10 times greater than normal systolic pressures and exhibited a range of compliance values, including those matching native artery. These tubes display many characteristics of the “ideal” small-diameter graft, having mechanical properties that can be tailored to match those desired in vascular replacement applications.

Tissue Regeneration of a Hybrid Vascular Graft Composed of Biodegradable Layers and Non-Biodegradable Layer by Static and Pulsatile Flows

Clinical applications of expanded polytetrafluoroethylene (ePTFE) as a small diameter graft have been limited due to its limited patency rates, even though its demands are high. After fabricating the biodegradable PLGA layers on both the inside and outside of ePTFE, long-term in vitro smooth muscle cell culture was performed on the luminal scaffold surface. The fabricated hybrid ePTFE scaffolds were designed to have three distinctive layers and porous structures in the biodegradable layers generated by gas-foaming of the ammonium bicarbonate porogens, i.e. two layers of poly(lactide-co-glycolide) (PLGA) as biodegradable layers for tissue engineering and an ePTFE layer in the middle as a non-biodegradable layer. We evaluated the regenerated vascular tissues after applying either static or pulstile flow on a smooth muscle cells-seeded hybrid scaffold. Analysis of the engineered tissues was performed with SEM for morphological observation and H&amp;E staining for observation of tissue development dependent upon a mode of culture system, flow patterns and scaffold species.

The use of mild trypsinization conditions in the detachment of endothelial cells to promote subsequent endothelialization on synthetic surfaces

A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for 5 min and seeded onto glass pretreated with fibronectin had close to 100% cell retention when shear stresses as high as 200 dyn/cm 2 were applied for 2 min. An equivalent level of cell retention was observed on fibronectin coated Teflon-AF™ for shear stresses up to 60 dyn/cm 2 applied for 4 h. Using 0.025% trypsin, initial cell spreading and cell surface α 5 β 1 integrins were increased relative to cells treated with 0.5% trypsin. After 1 h of attachment, focal adhesions formed when low trypsin concentrations were used but were less evident with high trypsin concentrations. These results showed that low trypsin concentrations produced faster spreading, a higher number of intact integrins, and rapid focal adhesion formation.

Histopathologic Changes of Acellularized Xenogenic Carotid Vascular Grafts Implanted in a Pig-to-Goat Model

The present study was designed to determine the in vivo patency and recellularization pattern of acellularized small-diameter xenogenic arterial grafts. We implanted acellularized porcine carotid arteries in bilateral carotid arteries of goats and microscopically analyzed the recellularization pattern of these grafts with the recipient's cells over time. Carotid arteries of pigs weighing 30-40 kg were harvested and decellularized with hypertonic saline followed by sodium dodecyl sulfate. Acellularized porcine carotid vascular xenografts (0.4-0.5 cm in diameter) were prepared into 4 cm-long segments and implanted bilaterally in the carotid arteries of 10 black-haired goats. The in vivo patency of the implanted acellularized xenogenic grafts was evaluated at regular intervals by color Doppler ultrasonography. The goats were sacrificed at predetermined intervals (1 week, 1 month, 3 months, 6 months, 12 months after implantation), two animals at each interval. Upon retrieval, visual inspections and histopathologic examinations of the grafts were performed. To identify smooth muscle cells and functioning endothelial cells, immunohistochemical staining for alpha-smooth muscle actin and von Willebrand factor were also performed. All experimental animals survived the observation period. Nineteen out of 20 implanted grafts showed patency with no thrombi. Microscopic analysis revealed that the grafts were completely covered with the hosts' endothelial cells, beginning from anastomotic sites. The grafts were gradually recellularized with recipients'cells including fibroblasts, myofibroblasts and smooth muscle cells. In conclusion, this study suggested that acellularized xenogenic vascular grafts can be a good alternative for the small-diameter vascular graft.

The effect of shear stress on human endothelial cells seeded on cylindrical viscoelastic conduits: an investigation of gene expression

The present study assesses the effect of physiological shear stress on gene expression from human ECs (endothelial cells) seeded on a small-diameter cylindrical bypass graft constructed from nanocomposite based on poly(carbonate-silsesquioxane-bridge-urea)urethane. ECs were seeded on to 5-mm-diameter conduits, placed in a physiological flow circuit and exposed to 1 or 4 h of shear stress at 1.4+/-0.3 Pa. Subsets of conduits were incubated at 37 degrees C and 5% CO2/95% O2 for a further 4 h to determine if gene expression returned to basal levels. PCR was conducted for glyceraldehyde-3-phosphate dehydrogenase, TGFbeta-1 (transforming growth factor beta-1), COL-1 (collagen-1) and PECAM-1 (platelet/EC adhesion molecule-1). Increases in gene expression were seen following flow in nanocomposite conduits. These were significant at 4 h for TGFbeta-1, COL-1 and PECAM-1. After a 4 h recovery period, there were no significant differences in gene intensity, suggesting that this change is transient. These data prove that mRNA can be obtained from ECs seeded on tubular conduits and exposed to shear stress and that gene-expression studies can be successfully carried out. We believe this is a substantial improvement on studies based on flat sheets.

The effect of gradually graded shear stress on the morphological integrity of a huvec-seeded compliant small-diameter vascular graft

The premature endothelialization of tissue-engineered grafts had often induced cellular detachment at an early period of implantation in arterial circulation, resulting in occlusion at an early period of implantation. This study was aimed to determine whether gradually increased shear stress applied ex vivo improves cell retention and tissue morphological integrity including cell shape and alignment, actin fiber alignment and expression of vascular endothelial (VE) cadherin. Tissue-engineered grafts used for this study were human umbilical vein endothelial cell (HUVEC)-seeded compliant small-diameter grafts made of poly(L-lactide-co-epsilon-caprolactone) fiber meshes fabricated by electrospinning. The shear stresses applied to grafts, generated using a custom-designed mock circulatory apparatus, were 3.2, 8.7 and 19.6 dyn/cm(2). The grafts completely monolayered prior to shear stress exposure exhibited a polygonal cobblestone morphology with randomly distributed actin fibers and VE cadherin at the continuous peripheral region of adjacent cells. The 24-h-loading of high shear stresses (8.7 and 19.6 dyn/cm(2)) equivalent to those of the arterial circulatory system resulted in severe cellular damage resulting in the complete loss of cells. However, a gradually increased graded exposure from a low (3.2 dyn/cm(2)) to a high shear stress (19.6 dyn/cm(2)) resulted in a markedly reduced cell detachment, a highly elongated cell shape, and orientation or alignment of both cells and actin fibers, which were parallel to the direction of flow. Although VE-cadherin expression was not detected yet, a higher degree of tissue integrity was achieved, which may greatly improve the performance particularly at an early period of implantation.

Small-Diameter Artificial Arteries Engineered In Vitro

Although the need for a functional arterial replacement is clear, the lower blood flow velocities of small-diameter arteries like the coronary artery have led to the failure of synthetic materials that are successful for large-diameter grafts. Although autologous vessels remain the standard for small diameter grafts, many patients do not have a vessel suitable for use because of vascular disease, amputation, or previous harvest. As a result, tissue engineering has emerged as a promising approach to address the shortcomings of current therapies. Investigators have explored the use of arterial tissue cells or differentiated stem cells combined with various types of natural and synthetic scaffolds to make tubular constructs and subject them to chemical and/or mechanical stimulation in an attempt to develop a functional small-diameter arterial replacement graft with varying degrees of success. Here, we review the progress in all these major facets of the field.

Review:Application of Stem Cells for Vascular Tissue Engineering

As the prevalence of vascular disease has continued to expand, the need for a suitable arterial replacement has prompted researchers to look beyond synthetic and autologous grafts toward the field of tissue engineering. Advances in vascular tissue engineering have utilized both mesenchymal and hematopoietic stem cells as a cell source in an attempt to create a fully engineered small-diameter graft. Stem cells offer enormous potential as a cell source because of their proliferative and growth potential, and the application of stem cell technology has far-reaching implications for future applications. The innovative use of stem cells for vascular tissue engineering has opened new possibilities for a fully engineered blood vessel. The purpose of this review is to summarize the current perspective on the use of stem cells for vascular tissue engineering. It focuses principally on the classes of stem cells used, techniques for differentiation scaffolding technology, and the successes and failures of models.

Endothelial Progenitor Cells, Neointimal Hyperplasia, and Hemodialysis Vascular Access Dysfunction

Vascular stenosis as a result of neointimal hyperplasia is a major clinical problem that has an impact on multiple and diverse disciplines, including cardiology (coronary restenosis), cardiothoracic and vascular surgery (saphenous vein and polytetrafluoroethylene [PTFE] graft failure), neurology (carotid stenosis), nephrology (dialysis access dysfunction), and transplant medicine (chronic allograft rejection in hearts and kidneys). The traditional response to injury hypothesis on the pathogenesis of neointimal hyperplasia focuses on the migration of medial smooth muscle cells from the media into the intima.1 Recently, there has been a great deal of excitement about the role of circulating smooth muscle progenitor cells in the pathogenesis of neointimal hyperplasia. These cells have been identified in a variety of experimental models of vascular injury,2 and interventions that reduce the number of these cells can attenuate neointimal hyperplasia. In marked contrast to the deleterious effects of smooth muscle progenitor cells on neointimal hyperplasia, circulating endothelial progenitor cells (EPCs) are believed to play an important role in vascular repair and in the inhibition of neointimal hyperplasia.3 See p 12 In this issue of Circulation , Rotmans and colleagues have attempted to achieve the “holy grail” for a vascular access procedure: rapid and complete endothelialization.4 Their experiments are based on a newly developed technique that can coat the surface of stent and graft material with antibodies against CD34 (Orbus Medical Technologies). CD34 is a marker for hematopoietic stem cells, and previous research has demonstrated that coronary stents coated with anti-CD34 have a significant increase in endothelialization as early as 1 hour after deployment because of the binding of circulating CD34+ cells, which then differentiate into endothelial cells.5 Placement of anti-CD34–coated grafts in an arteriovenous model of PTFE graft stenosis by Rotmans et al resulted in almost complete endothelialization of the grafts (both …

Implantation of Novel Small-Diameter Polyurethane Vascular Prostheses Interposed in Canine Femoral and Carotid Arteries

Objectives: The performance of small-diameter (3–5-mm) vascular grafts still poses a challenge in the field of vascular surgery. We present here our preliminary experience with implanting unique small-sized polycarbonate urethane vascular grafts in 7 dogs. Material and Methods: Each animal was implanted with 4 interposition grafts, 2 femoral and 2 carotid. No anti-thrombotic medication was administered. Doppler sonography was performed at 3-month intervals to examine for patency and flow characteristics. Animals were sacrificed electively at 3, 6 and 12 months. Results: At 3 months, all grafts were patent. After 6 months, 3 grafts occluded and at 1 year a further 6 grafts occluded. Hence 9 of 28 grafts occluded (67.9% patency). During the study, no correlation could be established between flow velocity or resistance index and occlusion. Histopathology showed intimal hyperplasia to be the cause of occlusion. Conclusions: Compared to literature data on small-diameter grafts in the same position, ADIAM’s Biomechanical grafts performed clearly better. Compliance data suggest a correlation between elastic compliance and patency.

Explant pathology study of decellularized carotid artery vascular grafts.

The purpose of this study was to evaluate the morphologic findings in small-diameter freeze-dried decellularized carotid artery grafts implanted in goats as carotid artery interposition grafts for 6-7 months. Unimplanted decellularized carotid artery grafts did not contain intact cells; however, remnants of smooth muscle cells were present in the media. The extracellular matrix was well preserved. All decellularized grafts were patent at explant, without significant dimensional changes or aneurysm formation. Their luminal surfaces were lined by a thin neointima, consisting of myofibroblasts, collagen, and a discontinuous layer of endothelial cells. Histologic evidence of calcification within the explants was not observed; however, electron microscopy showed calcification of minute remnants of cell membranes. Inflammatory cells were not present in the graft wall. Host cell migration was greatest in the adventitia along the length of the graft. Migration of host cells into the media was more apparent close to the anastomoses, forming cellular nests rich in extracellular proteoglycans, whereas cell migration into areas subjacent to the lumen was minimal. Ingrowth of host blood vessels was not observed. These results demonstrate satisfactory structural and morphologic features of a decellularized carotid artery small-diameter graft implanted for up to 7 months.

Endothelial cell seeding of polymeric vascular grafts.

Pioneers in the field of small diameter graft development sought to promote graft endothelialization and, thereby, increase patency by transplanting a varying degree of autologous endothelial cells onto vascular grafts prior to implantation. This process has become known as endothelial cell seeding. The underlying hypothesis is quite simple; that is, by promoting the establishment of the patient's own endothelial cells on the blood contacting surface of a vascular prosthesis, a "normal" endothelial cell lining and associated basement membrane, together known as the neointima, will form on the graft and counteract the rheologic, physiologic, and biomaterial forces working synergistically to promote graft failure. After 30 years of research in this area, this simple hypothesis has proven to be deceptively naive. The purpose of this review is to summarize the historic context and current base of knowledge regarding many of the technical issues relevant to the endothelial cell seeding process. Special attention is given to electrostatic endothelial cell seeding, the latest research methodology designed specifically to accelerate endothelial cell adhesion and morphological maturation onto expanded poly(tetrafluoroethylene) (e-PTFE) small diameter vascular grafts.

Endothelialization of Small-Diameter Vascular Prostheses

In the field of arterial vascular reconstructions there is an increasing need for functional small-diameter artificial grafts (inner diameter <6 mm). When autologous replacement vessels are not available, for example because of the bad condition of the vascular system in the patient, the surgeon has no other alternative than to implant a synthetic polymer-based vessel. After implantation the initial major problem concerning these vessels is the almost immediate occlusion, due to blood coagulation and platelet deposition, under the relatively low flow conditions. As the search for the perfect bio-inert polymer has not revealed a material with suitable properties for this application, improved performance of small-diameter artificial blood vessels is now being sought in the biological field. The poor blood-compatibility of an artificial vascular graft is not simply because of its coagulation-stimulating or platelet-activating properties, but more due to its inability to actively participate in the prevention of blood coagulation and platelet deposition. As these functions are naturally performed by endothelial cells, the utilization of these cells seems inevitable for the construction of a functional small-diameter artificial blood vessels. This review describes the current status of the use of endothelial cells to improve the performance of artificial vascular prostheses.

Predicting iliac limb occlusions after bifurcated aortic stent grafting: Anatomic and device-related causes

Graft limb occlusion may complicate endovascular abdominal aortic aneurysm repair. The precise etiologic factors that contribute to the development of these graft limb thromboses have not been defined. We evaluated our experience with bifurcated aortic endografts to determine factors that may predict subsequent limb thrombosis. The management of the thrombosed limbs and the results after treatment were also investigated. During a 4-year period, 351 patients with aortic aneurysms underwent treatment with bifurcated endografts (702 graft limbs at risk). These 351 bifurcated devices included AneuRx (Medtronic, Minneapolis, Minn; n = 35), Ancure (Guidant, Menlo Park, Calif; n = 8), Gore (W.L. Gore & Associates, Sunnyvale, Calif; n = 25), Talent (World Medical, Sunrise, Fla; n = 255), Teramed (Teramed, Minneapolis, Minn; n = 10), and Vanguard (Boston Scientific Vascular, Natick, Mass; n = 18). Details regarding the type of device, mechanism of deployment, and aortoiliac artery anatomy were collected prospectively and analyzed. Graft limbs were analyzed for diameter, use of additional endograft iliac extensions, deployment in the external iliac artery, and endograft to vessel oversizing. Follow-up included physical examination, duplex ultrasonography, and spiral computed tomographic scans at 1 month, 6 months, and 12 months and annually thereafter. The follow-up period ranged from 2 to 54 months, with a mean follow-up period of 20 months. Twenty-six of 702 limbs (3.7%) had an occlusion develop. The risk of limb thrombosis was associated with a smaller limb diameter. Mean graft limb diameter was 14 mm in the occluded population, and patent limbs had a mean diameter of 16 mm. Thrombosis occurred in 16 of 291 limbs (5.5%) that were 14 mm or less and in 10 of 411 limbs (2.4%) that were greater than 14 mm (P =.03). Extension of a graft to the external iliac artery was performed in 96 of the 702 limbs. Eight of these 96 limbs (8.3%) had thrombosis develop as compared with 18 of 606 (3.0%) that extended to the common iliac artery (P =.01). No significant association was present between limb thrombosis and the contralateral or ipsilateral side of a device, the configuration of the iliac graft limb end (closed web, open web, or bare spring), or the degree of iliac graft limb oversizing. AneuRx, Ancure, Vanguard, and Talent grafts each sustained limb occlusions, with no occlusions seen among the Gore and Teramed devices. No significant increased risk of graft limb thrombosis was observed in unsupported grafts (1/16; 6.3%) versus supported grafts (25/686; 3.6%; P = not significant). Thromboses occurred between 1 day and 23 months after surgery. Thirteen of the 26 thromboses (50%) occurred within 30 days of surgery. Presenting symptoms were mild to moderate claudication in eight patients (30.8%), severe claudication in 16 patient (61.5%), and paresthesia and rest pain in two patients (7.7%). Eighteen of 26 patients (69.2%) eventually needed intervention to reestablish flow to the occluded limb, including thrombolysis and stenting in two patients (7.7%), axillary femoral bypass in one patient (3.8%), femoral-femoral bypass in 13 patients (50.0%), and axillary-bifemoral bypass in two patients (7.7%). All patients with mild to moderate symptoms under observation had improvement in symptoms with no further interventions necessary. All revascularizations were successful in relieving symptoms. Graft limb occlusion is a recognized complication of endovascular treatment of abdominal aortic aneurysms that may be associated with smaller graft limb diameter and extension to the external iliac artery. Occlusions usually necessitate additional intervention for resolution of ischemic symptoms. The use of small diameter grafts should be avoided when possible to reduce the risk of graft limb occlusions.

Effective hemodynamic diameter: an intrinsic property of vein grafts with predictive value for patency.

Conduit size and quality are major determinants of the long-term success of infrainguinal autologous vein grafting. However, accurate measurement of the internal diameter of vein grafts is difficult given their variable wall thickness and taper. The purpose of this study was to define the "effective" internal diameter of a vein graft according to its hemodynamic properties and to determine its significance for graft patency. Sixty infrainguinal bypass grafts performed on 57 patients were evaluated intraoperatively. Proximal and distal graft pressure and blood flow (Q(meas)) were measured with fluid-filled catheter transduction and ultrasonic transit-time flowimetry, respectively, after unclamping. Waveforms were recorded digitally at 200 Hz under baseline conditions and after stimulation with 60 mg of papaverine. According to Fourier transformation of the measured pressure gradient (DeltaP), the Womersley solution for fluid flow in a straight rigid tube was used to calculate theoretical flow waveforms (Q(calc)) for a range of graft diameters. The theoretical waveforms were then compared with the measured flow waveforms and the best-fit diameter chosen as the "effective hemodynamic diameter" (EHD). Only grafts in which the correlation coefficient of Q(calc) versus Q(meas) was more than 0.90 were accepted (n = 47) to assure validity of the hemodynamic model. After a mean follow-up of 12.5 months (range, 0.1-43.9 months), patency was determined by the life table method. Hemodynamic and clinical variables were tabulated, and their effect on patency determined the use of univariate and multivariate Cox regression. Mean EHD was 4.1 +/- 0.1 mm with a range of 2.5 to 5.7 mm. Administration of papaverine caused profound changes in DeltaP (+78% +/- 17%) and Q(meas) (+71% +/- 12%) as expected, but had no effect on EHD (+0.05% +/- 0.1%). Univariate regression identified five variables associated with decreased secondary patency (P <.10): low EHD, conduit source other than the greater saphenous vein, high baseline DeltaP(mean), female sex, and redo operation. Of these, only low EHD was significant after multivariate analysis (P =.03). Patency of small diameter grafts (EHD < 3.6 mm; n = 11) was compared with patency of larger grafts (EHD > 3.6 mm; n = 36) to test a frequently espoused clinical guideline. Grafts with an EHD less than 3.6 mm exhibited significantly lower secondary patency compared with larger grafts (P =.0001). The positive and negative predictive values for an EHD less than 3.6 mm for secondary graft failure for grafts with at least 1 year follow-up were 86% and 88%, respectively. An EHD is a unique parameter that quantifies conduit size and has a significant impact on vein graft patency. An EHD less than 3.6 mm portends graft failure.

Remodeling of an acellular collagen graft into a physiologically responsive neovessel.

Surgical treatment of vascular disease has become common, creating the need for a readily available, small-diameter vascular graft. However, the use of synthetic materials is limited to grafts larger than 5-6 mm because of the frequency of occlusion observed with smaller-diameter prosthetics. An alternative to synthetic materials would be a biomaterial that could be used in the design of a tissue-engineered graft. We demonstrate that a small-diameter (4 mm) graft constructed from a collagen biomaterial derived from the submucosa of the small intestine and type I bovine collagen has the potential to integrate into the host tissue and provide a scaffold for remodeling into a functional blood vessel. The results obtained using a rabbit arterial bypass model have shown excellent hemostasis and patency. Furthermore, within three months after implantation, the collagen grafts were remodeled into cellularized vessels that exhibited physiological activity in response to vasoactive agents.

Small-Diameter Vascular Graft Prostheses: Current Status

In contrast to large-diameter vascular grafts (i.e. larger than 5 mm) which remain excellent for more than 10 years after implantation, small-diameter vascular grafts of both Dacron and Teflon occlude rapidly upon implantation. In this overview article, the strategies used to improve the patency of these small-diameter grafts, the current status in clinical trials, and further perspectives in the field of artificial vascular graft development are reviewed. It is concluded that, in view of recent developments in tissue engineering approaches, the future of small-diameter vascular prostheses looks promising.

On matching compliance between canine carotid arteries and polyurethane grafts.

Compliance mismatch between a host artery and an implanted graft has been suggested as a contributing factor to a small diameter graft failure. In this study, static compliance and dynamic compliance were defined and measured in vitro and in vivo for canine carotid arteries and 2 types of polyurethane grafts. Based on these compliance values, the circumferential modulus (E[theta]) and longitudinal modulus (Ez) were calculated. It was shown that grafts have constant moduli over a wide range of pressure while the moduli of carotid arteries increase significantly with increasing pressure (dynamic E[theta] from 0.20 to 1.32 MPa). Polyurethane grafts are nearly isotropic, with the modulus almost identical in each direction, while carotid arteries are anisotropic (E[theta]/Ez = 2-3). The dynamic moduli are generally higher than static values and are especially pronounced for arteries. Due to these different inherent characteristics, the compliance of a synthetic graft may match that of the host artery only in the circumferential direction and within a small pressure range. A stated limitation is therefore given for complete compliance matching. The results provide a rationale for identifying the degree of compliance match. These efforts may lead to better designed vascular grafts.

Healing Comparison of Small Intestine Submucosa and ePTFE Grafts in the Canine Carotid Artery

Continuing investigation of small-diameter vascular graft materials suggests that unacceptable graft complications continue and that the ideal material has not yet been found. We compared healing of xenogeneic small diameter grafts (3.5 to 5.0 mm diameter) made from porcine small intestine submucosa (SIS) implanted in the carotid artery to expanded polytetrafluorethylene (ePTFE) in the contralateral carotid in 8 dogs. Two dogs were sacrificed for graft evaluation at 7, 28, 90, and 180 days after surgery. Only one SIS graft was occluded at 28 days and the other 7 were patent. Six of 8 ePTFE grafts were occluded with thrombi. One was patent at 7 and one at 90 days. At 7 days postimplant, the luminal surface of the SIS graft was covered by a thick (30 μm), compact fibrin meshwork. By 28 days endothelial cells were seen completely covering the fibrin meshwork which stained for FVIII-related antigen. Smooth muscle cells were observed in the neointima. Most ePTFE grafts had fibrin on the luminal surface which formed fibrin thrombi with platelets and numerous red blood cells. Complete endothelial coverage of the ePTFE grafts was not observed by 180 days. There was not a pronounced neointima seen on the luminal surface of the graft. The vasa vasorum was present in the fibrous capsule surrounding the ePTFE graft, but it did not penetrate into the graft as seen in the SIS graft. At 90 days the SIS vascular graft had the histological appearance similar to a normal artery. The SIS graft patency and healing characteristics were superior to the synthetic ePTFE graft and warrant further investigation.

Synthetic vascular grafts seeded with genetically modified endothelium in the dog: Evaluation of the effect of seeding technique and retroviral vector on cell persistence in vivo

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial Neo R gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts ( n = 3) preclotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated ( n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 × 10 3 to 1.5 × 10 5 cells/cm 2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 μg/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the Neo R gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.