Graft Luminal Surface Research Articles

Development of tissue-engineered vascular grafts from decellularized parsley stems.

Cardiovascular diseases are mostly associated with narrowing or blockage of blood vessels, and it is the most common cause of death worldwide. The use of vascular grafts is a promising approach to bypass or replace the blocked vessels for long-term treatment. Although autologous arteries or veins are the most preferred tissue sources for vascular bypass, the limited presence and poor quality of autologous vessels necessitate seeking alternative biomaterials. Recently, synthetic grafts have gained attention as an alternative to autologous grafts. However, the high failure rate of synthetic grafts has been reported primarily due to thrombosis, atherosclerosis, intimal hyperplasia, or infection. Thrombosis, the main reason for failure upon implantation, is associated with damage or absence of endothelial cell lining in the vascular graft's luminal surface. To overcome this, tissue-engineered vascular grafts (TEVGs) have come into prominence. Alongside the well-established scaffold manufacturing techniques, decellularized plant-based constructs have recently gained significant importance and are an emerging field in tissue engineering and regenerative medicine. Accordingly, in this study, we demonstrated the fabrication of tubular scaffolds from decellularized parsley stems and recellularized them with human endothelial cells to be used as a potential TEVG. Our results suggested that the native plant DNA was successfully removed, and soft tubular biomaterials were successfully manufactured via the chemical decellularization of the parsley stems. The decellularized parsley stems showed suitable mechanical and biological properties to be used as a TEVG material, and they provided a suitable environment for the culture of human endothelial cells to attach and create a pseudo endothelium prior to implantation. This study is the first one to demonstrate the potential of the parsley stems to be used as a potential TEVG biomaterial.

In Vivo Evaluation of PCL Vascular Grafts Implanted in Rat Abdominal Aorta.

Electrospun tissue-engineered grafts made of biodegradable materials have become a perspective search field in terms of vascular replacement, and more research is required to describe their in vivo transformation. This study aimed to give a detailed observation of hemodynamic and structural properties of electrospun, monolayered poly-ε-caprolactone (PCL) grafts in an in vivo experiment using a rat aorta replacement model at 10, 30, 60 and 90 implantation days. It was shown using ultrasound diagnostic and X-ray tomography that PCL grafts maintain patency throughout the entire follow-up period, without stenosis or thrombosis. Vascular compliance, assessed by the resistance index (RI), remains at the stable level from the 10th to the 90th day. A histological study using hematoxylin-eosin (H&E), von Kossa and Russell–Movat pentachrome staining demonstrated the dynamics of tissue response to the implant. By the 10th day, an endothelial monolayer was forming on the graft luminal surface, followed by the gradual growth and compaction of the neointima up to the 90th day. The intense inflammatory cellular reaction observed on the 10th day in the thickness of the scaffold was changed by the fibroblast and myofibroblast penetration by the 30th day. The cellularity maximum was reached on the 60th day, but by the 90th day the cellularity significantly (p = 0.02) decreased. From the 60th day, in some samples, the calcium phosphate depositions were revealed at the scaffold-neointima interface. Scanning electron microscopy showed that the scaffolds retained their fibrillar structure up to the 90th day. Thus, we have shown that the advantages of PCL scaffolds are excellent endothelialization and good surgical outcome. The disadvantages include their slow biodegradation, ineffective cellularization, and risks for mineralization and intimal hyperplasia.

Development of Small-Diameter Elastin-Silk Fibroin Vascular Grafts.

Globally, increasing mortality from cardiovascular disease has become a problem in recent years. Vascular replacement has been used as a treatment for these diseases, but with blood vessels <6 mm in diameter, existing vascular grafts made of synthetic polymers can be occluded by thrombus formation or intimal hyperplasia. Therefore, the development of new artificial vascular grafts is desirable. In this study, we developed an elastin (EL)–silk fibroin (SF) double-raschel knitted vascular graft 1.5 mm in diameter. Water-soluble EL was prepared from insoluble EL by hydrolysis with oxalic acid. Compared to SF, EL was less likely to adhere to platelets, while vascular endothelial cells were three times more likely to adhere. SF artificial blood vessels densely packed with porous EL were fabricated, and these prevented the leakage of blood from the graft during implantation, while the migration of cells after implantation was promoted. Several kinds of 13C solid-state NMR spectra were observed with the EL–SF grafts in dry and hydrated states. It was noted that the EL molecules in the graft had very high mobility in the hydrated state. The EL–SF grafts were implanted into the abdominal aorta of rats to evaluate their patency and remodeling ability. No adverse reactions, such as bleeding at the time of implantation or disconnection of the sutured ends, were observed in the implanted grafts, and all were patent at the time of extraction. In addition, vascular endothelial cells were present on the graft's luminal surface 2 weeks after implantation. Therefore, we conclude that EL–SF artificial vascular grafts may be useful where small-diameter grafts are required.

Collagen-Based Tissue Engineering Strategies for Vascular Medicine.

Cardiovascular diseases (CVDs) account for the 31% of total death per year, making them the first cause of death in the world. Atherosclerosis is at the root of the most life-threatening CVDs. Vascular bypass/replacement surgery is the primary therapy for patients with atherosclerosis. The use of polymeric grafts for this application is still burdened by high-rate failure, mostly caused by thrombosis and neointima hyperplasia at the implantation site. As a solution for these problems, the fast re-establishment of a functional endothelial cell (EC) layer has been proposed, representing a strategy of crucial importance to reduce these adverse outcomes. Implant modifications using molecules and growth factors with the aim of speeding up the re-endothelialization process has been proposed over the last years. Collagen, by virtue of several favorable properties, has been widely studied for its application in vascular graft enrichment, mainly as a coating for vascular graft luminal surface and as a drug delivery system for the release of pro-endothelialization factors. Collagen coatings provide receptor–ligand binding sites for ECs on the graft surface and, at the same time, act as biological sealants, effectively reducing graft porosity. The development of collagen-based drug delivery systems, in which small-molecule and protein-based drugs are immobilized within a collagen scaffold in order to control their release for biomedical applications, has been widely explored. These systems help in protecting the biological activity of the loaded molecules while slowing their diffusion from collagen scaffolds, providing optimal effects on the targeted vascular cells. Moreover, collagen-based vascular tissue engineering substitutes, despite not showing yet optimal mechanical properties for their use in the therapy, have shown a high potential as physiologically relevant models for the study of cardiovascular therapeutic drugs and diseases. In this review, the current state of the art about the use of collagen-based strategies, mainly as a coating material for the functionalization of vascular graft luminal surface, as a drug delivery system for the release of pro-endothelialization factors, and as physiologically relevant in vitro vascular models, and the future trend in this field of research will be presented and discussed.

Site-specific thrombolytic and anticoagulant biomaterials

Thrombolytic drugs are used in medicine to dissolve blood clots in a procedure termed thrombolysis. To prevent occlusion, patients are treated with an antiplatelet or antithrombotic drug, or a combination of both. However, the traditional thrombolytic therapies have often been associated with the risk of severe bleeding. We have incorporated low molecular weight heparin (LMWH) and/or streptokinase to biodegradable polyvinyl alcohol (PVA) and gelatine biomaterials in a form of films. These biomaterials will be used to decorate inner surfaces of experimental vascular grafts to evaluate the potential of enhancing their thromboresistance by controlled release of one or both the active compounds. Solvent casting method was used to produce the PVA and crosslinked gelatine films. High initial burst release of LMWH has been observed during our in vitro experiments, followed by a slow stable release during subsequent hours. Biomaterials with LMWH incorporated in the vascular graft luminal surface can be used to prevent thrombus formation shortly after the surgery graft implantation and decrease it in the following hours. Slow stable streptokinase release can be used to attenuate excessive fibrin deposition inside the graft.

Heparin-bonded expanded polytetrafluoroethylene femoropopliteal bypass grafts outperform expanded polytetrafluoroethylene grafts without heparin in a long-term comparison

Expanded polytetrafluoroethylene (ePTFE) grafts have become an acceptable prosthetic alternative to autologous vein for infrainguinal arterial reconstructions. Recently, heparin bonding to the graft's luminal surface has been used as an adjunctive method of improving graft patency. We retrospectively evaluated a prospective experience with heparin-bonded ePTFE (HePTFE) vs the results of a prior experience with standard ePTFE (SePTFE) to compare patency rates in above-knee (AK) and below-knee (BK) femoropopliteal bypass through the 5-year follow-up. Prosthetic femoropopliteal bypasses performed by our four-surgeon group were constructed using 6-mm SePTFE grafts before August 2007. Subsequently, all such bypasses were constructed using 6-mm HePTFE (Propaten graft; W. L. Gore & Associates, Flagstaff, Ariz). The surgical technique used in all cases throughout the study was similar irrespective of surgeon or graft material. Loss of primary patency was defined by graft thrombosis; duplex scan confirmed graft failure. Descriptive estimation of patency rates was carried out using standard Kaplan-Meier methods. Cox proportional hazards regression was used to analyze relationships between predictors and time to loss of patency. There were 192 AK (87 Propaten, 105 SePTFE) and 60 BK (42 Propaten, 18 SePTFE) grafts. Overall primary patency for Propaten was statistically better than SePTFE at years 1 (93.6% vs 84.2%), 2 (79.7% vs 73.8%), 3 (79.7% vs 69.5%), 4 (74.5% vs 59.9%) and 5 (74.5% vs 56.2%; log-rank test, P= .036). Five-year primary patency was superior for Propaten AK grafts (85.2% vs 59.3%; log-rank test, P= .028) and for Propaten BK grafts (59.6% vs 0.0/undeterminable; log-rank test, P= .016). Variables significantly associated with loss of patency for Propaten and SePTFE were claudication (hazard ratio [HR], 0.41), age (HR, 0.95), isolated popliteal artery (HR, 3.1-4.4), and ankle-brachial index after the procedure (HR, 0.10). Controlling for conduit effect, clopidogrel use did not significantly affect patency (P= .076). No grafts were associated with heparin-induced thrombocytopenia. These data show that the Propaten HePTFE graft offered significantly better long-term patency over the SePTFE graft, suggesting Propaten as the prosthetic graft of choice for bypasses to the femoropopliteal artery when autologous vein is unavailable or inappropriate.

Fabrication and Short-Term in Vivo Performance of a Natural Elastic Lamina-Polymeric Hybrid Vascular Graft.

Although significant advances have been made in the development of artificial vascular grafts, small-diameter grafts still suffer from excessive platelet activation, thrombus formation, smooth muscle cell intimal hyperplasia, and high occurrences of restenosis. Recent discoveries demonstrating the excellent blood-contacting properties of the natural elastic lamina have raised the possibility that an acellular elastic lamina could effectively serve as a patent blood-contacting surface in engineered vascular grafts. However, the elastic lamina alone lacks the requisite mechanical properties to function as a viable vascular graft. Here, we have screened a wide range of biodegradable and biostable medical-grade polymers for their ability to adhere to the outer surface of the elastic lamina and allow cellular repopulation following engraftment in the rat abdominal aorta. We demonstrate a novel method for the fabrication of elastic lamina-polymeric hybrid small-diameter vascular grafts and identify poly(ether urethane) (PEU 1074A) as ideal for this purpose. In vivo results demonstrate graft patency over 21 days, with low thrombus formation, mild inflammation, and the general absence of smooth muscle cell hyperplasia on the graft's luminal surface. The results provide a new direction for developing small-diameter vascular grafts that are mass-producible, shelf-stable, and universally compatible due to a lack of immune response and inhibit the in-graft restenosis response that is common to nonautologous materials.

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.

Development of tissue-engineered self-expandable aortic stent grafts (Bio stent grafts) using in-body tissue architecture technology in beagles.

In this study, we aimed to describe the development of tissue-engineered self-expandable aortic stent grafts (Bio stent graft) using in-body tissue architecture technology in beagles and to determine its mechanical and histological properties. The preparation mold was assembled by insertion of an acryl rod (outer diameter, 8.6 mm; length, 40 mm) into a self-expanding nitinol stent (internal diameter, 9.0 mm; length, 35 mm). The molds (n = 6) were embedded into the subcutaneous pouches of three beagles for 4 weeks. After harvesting and removing each rod, the excessive fragile tissue connected around the molds was trimmed, and thus tubular autologous connective tissues with the stent were obtained for use as Bio stent grafts (outer diameter, approximately 9.3 mm in all molds). The stent strut was completely surrounded by the dense collagenous membrane (thickness, ∼150 µm). The Bio stent graft luminal surface was extremely flat and smooth. The graft wall of the Bio stent graft possessed an elastic modulus that was almost two times higher than that of the native beagle abdominal aorta. This Bio stent graft is expected to exhibit excellent biocompatibility after being implanted in the aorta, which may reduce the risk of type 1 endoleaks or migration.

Luminal Surface Engineering, ‘Micro and Nanopatterning’: Potential for Self Endothelialising Vascular Grafts?

New technologies are being explored to meet the clinical need for an ‘off-the-shelf’ small diameter vascular graft with superior or at least equivalent properties to autologous vessel. The field of nanotechnology and fabrication promises major advances in biomaterial design and wall structure to deliver biomimetic grafts. This review brings together recent work on this topic. A literature search was conducted of PubMed and ISI Web of Knowledge using relevant keywords. Articles published after January 2005 were given preference. Personal communications and PhD theses were also used as sources. An evolving focus on surface patterning of biomaterials has been found to carry great potential. Influencing cellular behaviour on prosthetic grafts using graft luminal surface modulation at the micro- and nano-levels is the basis of this recent concept in vascular graft development. This technology may deliver small diameter grafts with the potential for spontaneous in situ endothelialisation without the need for prior ‘seeding’, with the potential to open a new chapter in vascular graft development.

Invited commentary

A major obstacle limiting the success of vascular conduit placement remains the limited ability to obtain endothelial coverage of the graft luminal surface. Among the beneficial functions of a confluent endothelium are thromboresistance, selective permeability, and interactions with circulating blood elements and underlying smooth muscle. Clinical and laboratory results show that large-diameter high-flow systems tolerate the use of conduits that lack an endothelial lining. That is not the case for small caliber systems and systems that have slower flow. Conduits with slow blood flow and smaller-caliber conduits perform poorly due to thrombotic complications, possibly due to the lack of nitric oxide and prostacyclin and the longer interaction of the blood elements with the nonendothelialized luminal surface. This study and others like it focus on defining methods for creating a conduit that has a confluent luminal endothelial lining that tolerates placement in the circulation. The scaffold, matrix or material of the graft, and the source and conditions under which the endothelial cells are harvested and placed on the conduit are important in determining the degree of success in achieving endothelial confluence and the durability of the endothelial layer when challenged with flow conditions. The authors have demonstrated clearly that, in the short term, the more complex culture conditions of cyclic shear stress produce an endothelial graft lining that functions in a manner superior to that produced by conditions of steady shear stress at low or high levels. Improvement in endothelial function with cyclic shear stress was noted in terms of maintenance of integrity of the endothelial layer and inhibition of adhesion of circulating blood elements to the luminal surface of the graft. The authors observed by Western blotting increased expression of endothelial nitric oxide synthase and prostaglandin I synthase in the cyclic shear stress conduits compared with the conduits created under other conditions. Defining the role of these or other biologic molecules in conferring favorable graft function will require formal investigation. The functional performance characteristics of the endothelial lining of a conduit likely are the result of many factors, one of which appears to be the type of shear stress used when creating the endothelial monolayer. Other factors may include the scaffold, the origin of the endothelial cells and their culture conditions, both biologic and mechanical. The ideal conduit for certain applications may also include smooth muscle cells along with endothelial cells to allow for a more durable structure or potentially for biologic molecule delivery via vascularized structures that have genetically engineered cells designed to deliver physiologic regulatory molecules locally or to the circulation. Patterns of molecular expression that may be expected to yield favorable endothelial performance characteristics might possibly include robust eNOS expression and decreased expression of tissue factor and other unfavorable candidate molecules. Additional work, including longer-term studies and studies in arterial-arterial grafts is required to adequately address the challenge of adequate conduit design. The fate of an endothelium layer after preconditioningJournal of Vascular SurgeryVol. 51Issue 1PreviewA strategy in minimizing thrombotic events of vascular constructs is to seed the luminal surface with autologous endothelial cells (ECs). The task of seeding ECs can be achieved via bioreactors, which induce mechanical forces (shear stress, strain, pressure) onto the ECs. Although bioreactors can achieve a confluent layer of ECs in vitro, their acute response to blood remains unclear. Moreover, the necessary mechanical conditions that will increase EC adhesion and function remain unclear. We hypothesize that preconditioning seeded endothelium under physiological flow will enhance their retention and function. Full-Text PDF Open Archive

Expression of fibrinolysis activators and their inhibitor in neointima of polyester vascular grafts

The aim of the study was to evaluate dynamic changes in the expression of fibrinolytic system components in neointima forming in polyester vascular grafts. The study was carried out on 18 mongrel dogs divided into three groups, that underwent replacement of abdominal aorta with a polyester double velour prosthesis. Grafts were removed at 1, 4 and 12 months. The specimens were fixed according to AMeX method. Immunohistochemical labeling for von Willebrand factor (vWf), tissue plasminogen activator (t-PA), urokinase (u-PA), its receptor (u-PAR), plasminogen activator inhibitor type 1 (PAI-1) and D-dimer (DD) was performed. Increasing intensity of vWf expression on the graft luminal surface was found in successive periods of the study. A light positive t-PA and u-PA staining was shown in neointima at 1 month and its intensity was significantly increased at 4 and 12 months. Expression of u-PAR appeared at 4 months. A light positive PAI-1 and DD staining was demonstrated in neointima in all periods of the study. The results demonstrated increasing expression of fibrinolysis activators in neointima of polyester vascular grafts. Intensive expression of plasminogen activators, when compared to their inhibitor may reduce thrombotic properties of graft neointima particularly in the late period after prosthesis implantation.

Luminal surface microgeometry affects platelet adhesion in small-diameter synthetic grafts

One of the major problems when using small-diameter vascular grafts in arterial reconstruction is the development of platelet-rich thrombi as a consequence of blood contact with artificial surfaces. The degree of occlusion is certainly affected by the thrombogenicity of the internal surface that seems to play a key role in patency and long-term wound healing of grafts. In this study, the blood compatibility of Cardiothane ® (CA) vascular grafts was investigated. The CA material, a blend of polyurethane and polydimethylsiloxane that has shown relatively good physical and biocompatibility properties, was manufactured into vascular grafts by the instrument named “spray-machine”. Grafts with different luminal surface porosity were produced using increasing CA concentrations by the “spray-machine” and the blood compatibility was evaluated in vitro by a circulation system in which the human blood was allowed to interact with the material in a well-controlled setting. The samples of circulating blood were collected at different times of circulation and platelet adhesion and activation were studied. Grafts with a highly porous luminal surface induced a lower adhesion and activation of platelets in vitro than the low-porosity ones. These results underlined the importance of the microgeometry of the graft luminal surface in the interaction with blood.

Improvements in GORE-TEX® vascular graft performance by Carmeda® bioactive surface heparin immobilization

Objectives: a performance improvement in small-diameter bypass grafts remains a clinical objective. The purpose of the present investigation was to evaluate the potential of enhancing the thromboresistance of ePTFE grafts using a bioactive heparinized graft luminal surface in a canine model. Material and Methods: this study investigated the utility of heparin immobilization onto expanded polytetrafluoroethylene using Carmeda® BioActive Surface technology (CBAS-ePTFE) as a means of improving vascular graft thromboresistance. Graft luminal surfaces were covered uniformly with the stably bound, end-point immobilized heparin. Results: acute canine (5 greyhounds) interposition experiments comparing CBAS-ePTFE grafts to control ePTFE grafts showed that CBAS-ePTFE grafts remained patent and had significantly greater thrombus-free luminal surface (p < 50.05). In a chronic canine (16 greyhounds) interposition experiment, significantly improved patency (p < 50.05) was observed with CBAS-ePTFE grafts compared to controls. Long-term in vivo heparin bioactivity was demonstrated on CBAS-ePTFE grafts explanted between 1 and 12 weeks. On all CBAS-ePTFE grafts, heparin activity levels ranged from 15-25 pmol/cm2 and did not differ significantly (p > 40.05). Discussion: these results support the conclusion that a stable, CBAS-ePTFE surface provides improved thromboresistance and improved patency in canine interposition models. Maintenance of heparin catalytic activity on the graft surface in vivo likely contributes to this outcome and holds promise for the utility of this graft surface for clinical applications.Eur J Vasc Endovasc Surg 25, 432-437 (2003)

Endothelial cell seeding of a 4-mm I.D. polyurethane vascular graft.

We evaluated the extent (luminal coverage) of the endothelial cell (EC) lining/neointimal development and the thromboresistance of electrostatically EC seeded small diameter ChronoFlex-polyurethane vascular grafts. The evaluation consisted of harvesting autologous, canine jugular vein ECs, electrostatically seeding the polyurethane grafts (4-mm I.D., length = 6 cm) with the harvested ECs, implanting the grafts in a canine femoral artery model for four to six weeks, and excising the grafts for histological and scanning electron microscopy evaluations. Results of the histological evaluation (mid-graft region only) indicated that electrostatic EC seeding led to neointimal development and to minimal to no thrombus formation within the EC seeded grafts. The unseeded control grafts resulted in no neointimal development and substantial thrombus formation on the graft luminal surfaces. Scanning electron microscopy examination demonstrated a mature, confluent endothelium with a "cobblestone" appearance on the EC seeded graft luminal surface after six weeks. We conclude that electrostatic EC seeding enhanced the development of a neointima and reduced the incidence of thrombosis in polyurethane grafts implanted in a canine femoral artery model.

Progenitor endothelial cells on vascular grafts: an ultrastructural study.

The morphology of progenitor endothelial cells on vascular graft surfaces is addressed in this report. Such cells were seen to attach to intima-expressed CD34 and Flk-1 antigen and showed positive 5-bromo-2-deoxyuridine (BrdU) uptake. We examined CD34 and Flk-1 antigen-expressing endothelial progenitor cells three-dimensionally using confocal laser scanning microscopy (CLSM). Under detailed CLSM observation, through an ameboid-form cell, these progenitor endothelial cells changed from a globular to a flattened form. We also investigated these morphological changes using scanning electron microscopy. From these results, progenitor endothelial cells were observed not only near the advancing edge of endothelium, but also around the developing intimal site. Their form also changed from globular to flattened as observed in the CLSM results. These morphological changes were seen more frequently near the advancing edge and around the developing intimal site. They attached directly to vascular prosthesis fibers and likewise covered the graft luminal surface. Progenitor endothelial cells in any form had a common surface structure. We conclude from our results that progenitor endothelial cells can attach to graft fibers directly without clotting and directly cover the graft luminal surfaces.

Electrostatic endothelial cell transplantation within smalldiameter (

This article presents a novel, clinically relevant electrostatic endothelial cell transplantation (seeding/sodding) device (U.S. & Foreign Patent Protections Pending) for smalldiameter (<6 mm) vascular prostheses. The prototype apparatus was designed and built to tissue engineer 4.0 mm, I.D. GORE-TEX® (W.L. Gore & Associates, Inc.) standard wall graft segments varying in length from 4 to 12 cm. The prototype electrostatic endothelial cell transplantation apparatus is composed of an external and internal conductor, aluminum base, end supports, pillow blocks, filling apparatus, electric motor drive system, and a voltage source. The cylindrical capacitor arrangement of the device along with an electrical potential applied across the internal and external conductors creates the unique feature of this endothelial cell transplantation technique, an electric field within the cylindrical capacitor (within the graft lumen) which in turn induces a temporary positive surface charge on the graft (dielectric material) luminal surface. Multiple studies have shown that a positively charged substrate is more conducive to endothelial cell adhesion and morphological maturation (flattening) (1,2, 7,8,10,13–15). This induced positive surface charge dissipates immediately upon removal from the electrostatic endothelial cell transplantation device. Thus, after endothelial cell adhesion the graft luminal surface reverts back to its natural (nonthrombogenic) negative surface charge.

Electrostatic Endothelial Cell Transplantation within Small-Diameter (&lt;6 MM) Vascular Prostheses: A Prototype Apparatus and Procedure

This article presents a novel, clinically relevant electrostatic endothelial cell transplantation (seeding/sodding) device (U.S. & Foreign Patent Protections Pending) for small-diameter (<6 mm) vascular prostheses. The prototype apparatus was designed and built to tissue engineer 4.0 mm, I.D. GORE-TEX (W.L. Gore & Associates, Inc.) standard wall graft segments varying in length from 4 to 12 cm. The prototype electrostatic endothelial cell transplantation apparatus is composed of an external and internal conductor, aluminum base, end supports, pillow blocks, filling apparatus, electric motor drive system, and a voltage source. The cylindrical capacitor arrangement of the device along with an electrical potential applied across the internal and external conductors creates the unique feature of this endothelial cell transplantation technique, an electric field within the cylindrical capacitor (within the graft lumen) which in turn induces a temporary positive surface charge on the graft (dielectric material) luminal surface. Multiple studies have shown that a positively charged substrate is more conducive to endothelial cell adhesion and morphological maturation (flattening) (1,2, 7,8,10,13-15). This induced positive surface charge dissipates immediately upon removal from the electrostatic endothelial cell transplantation device. Thus, after endothelial cell adhesion the graft luminal surface reverts back to its natural (nonthrombogenic) negative surface charge.

Phosphorylcholine Coating of ePTFE Grafts Reduces Neointimal Hyperplasia in Canine Model

This study attempts to prevent neointimal hyperplasia by coating the graft luminal surface with a derivative of phosphorylcholine (PC), thereby providing a biocompatible surface with the assumption of limiting pannus tissue ingrowth from the graft anastomoses. Bilateral carotid artery bypass grafts were placed in six dogs using expanded polytetrafluoroethylene (ePTFE). In each animal, one carotid arterial-arterial conduit was constructed using a graft having a PC coating over the entire luminal surface of the graft. On the contralateral side, uncoated graft served as a control. The processed specimens were analyzed for graft neointimal area and neointimal thickness. Cell proliferation was assessed by staining for bromodeoxyuridine (BrdU) incorporation. All grafts were patent except one control graft that was occluded at 4 weeks. There was a significant reduction in the anastomotic graft neointimal area between the treated and control groups (0.27 +/- 0.17 mm2 versus 0.53 +/- 0.13 mm2, respectively; p = 0.008). Furthermore, the BrdU labeling index in the graft neointimal tissues was significantly smaller (p < 0.001) in the treated group (2.64 +/- 0.77%) as compared with the control group (5.07 +/- 0.83%). These data demonstrate that PC coating of ePTFE significantly reduces graft neointimal hyperplasia and cell proliferation in a canine carotid artery bypass model. The application of PC within the ePTFE graft effectively blocks tissue ingrowth from the adjacent native vessel, thereby preserving the anastomosis luminal diameter.

A New Cardiac Wall Substitute Made of Dacron Fabric Prosthesis Sealed with Autologous Tissue Fragments: Comparative Studies of Neointima Formation and Bleeding Resistant Property Against Fibrinolysis Phenomenon with Those of a Preclotted Sealing Graft

A cardiac wall substitute made of a Dacron fabric prosthesis sealed with minced-autologous tissue fragments was developed. An animal study showed rapid neointima formation on the graft luminal surface and no bleeding problem under load test with tissue plasminogen activator (tPA) administration. To evaluate the efficacy of the sealing method, a special substitute that was sealed with tissue fragments in one half and preclotted with fresh blood in the other half was prepared. The grafts were implanted into the right ventricular outflow tract in 14 dogs. During a load test with administration of tPA intravenously, the preclotted area bled quite remarkably; however, the area sealed with tissue fragments did not bleed. Two weeks after implantation, red thrombus adhered on the preclotted area, while a white, glistening surface was noticed on the tissue fragments area. Light microscopical observation showed complete neointima formation in the tissue fragmented area but a thrombus layer in the preclotted area. These results indicated that the tissue fragmentation was a superior method compared with preclotting for creating a cardiac wall substitute.