Engineering of bypass conduits to improve patency.

For patients with severe coronary artery and distal peripheral vascular disease not amenable to angioplasty and lacking sufficient autologous vessels there is a pressing need for improvements to current surgical bypass options. It has been decades since any real progress in bypass material has reached mainstream surgical practice. This review looks at possible remedies to this situation. Options considered are methods to reduce prosthetic graft thrombogenicity, including endothelial cell seeding and developments of new prosthetic materials. The promise of tissue-engineered blood vessels is examined with a specific look at how peptides can improve cell adhesion to scaffolds.

The mechanical behavior of vascular grafts: a review.

The development of intimal hyperplasia (IH) near the anastomosis of a vascular graft to artery is directly related to changes in the wall shear rate distribution. Mismatch in compliance and diameter at the end-to-end anastomosis of a compliant artery and rigid graft cause shear rate disturbances that may induce intimal hyperplasia and ultimately graft failure. The principal strategy being developed to prevent IH is based on the design and fabrication of compliant synthetic or innovative tissue-engineered grafts with viscoelastic properties that mirror those of the human artery. The goal of this review is to discuss how mechanical properties including compliance mismatch, diameter mismatch, Young's modulus and impedance phase angle affect graft failure due to intimal hyperplasia.

Cellular engineering of vascular bypass grafts: role of chemical coatings for enhancing endothelial cell attachment.

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5-6mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patient's own cells. Currently, to be effective, high-density seeding regimens have to be undertaken. This is because endothelial cells (ECs) are washed off the graft lumen once exposed to physiological blood flow. EC attachment has been shown to be significantly improved by pre-coating with substances known to attach ECs selectively. The review examines the various types of coating and bonding technology used to date to enhance endothelial cell attachment onto the surface of prosthetic vascular bypass grafts.

Synthesis and evaluation of amphiphilic RGD derivatives: uses for solvent casting in polymers and tissue engineering applications.

Derivatives containing arginine-glycine-aspartic acid (RGD) inhibit fibrinogen binding to activated platelets and promote endothelial and smooth muscle cell attachment. An amphiphilic derivative of RGD that can be dissolved in an organic solvent has potential in the development of non-thrombogenic biomaterials. Such a derivative, LA-GRGD, was synthesised by coupling glycine-arginine-glycine-aspartic acid (GRGD) with lauric acid (LA). Its solubility and antithrombotic, cytotoxic and cell-binding effects were then evaluated in comparison with heparin (which is used clinically) and a fibronectin-engineered protein polymer (FEPP). Thromboelastography (TEG) was used to measure blood clotting time using fresh whole blood from healthy volunteers. Tissue factor (TF) activity was measured using plasma with a standard prothrombin time assay (PT). Cytotoxicity was assessed on human umbilical cord endothelial cells (HUVECs) using an Alamar blue assay. Solubility of the conjugate was assessed in a co-solvent. These techniques were used to study LA-GRGD, using heparin and FEPP as controls. The amphiphilic property of LA-GRGD was dependent on the feed mole ratio of GRGD to LA. LA-GRGD was soluble in acetone:water and water. LA-GRGD inhibited TF by >90% and prolonged TEG-r by 8.2+/-3.3 min (200 microg ml(-1)). Heparin inhibited TF by >90%, but prolonged TEG-r by 97.4+/-1.6 min (1 U ml(-1)); FEPP inhibited TF by >90% (100 microg ml(-1)) and prolonged TEG-r by 73.7+/-8.4 min (10 microg ml(-1)). Heparin had no cytotoxic effect on EC metabolism and viability at the concentrations studied (0.1-100 U ml(-1)). No significant cytotoxic effect was produced by LA-GRGD or FEPP at concentrations ranging from 0.1 microg ml(-1) to 50 microg ml(-1), but, at higher concentrations (100 microg ml(-1) and 200 microg ml(-1)), a detrimental effect was observed. Cell binding studies showed that LA-GRGD bound 29% of ECs compared with FEPP (60%) and heparin (22%). This new approach for synthesising amphiphilic RGD and its analogues has potential as a drug delivery system for the manufacture of new polymer formulations for use in bypass grafts and other tissue-engineered devices.

Dynamic protein adsorption at the polyurethane copolymer/water interface.

Polyurethanes (PU) and their polymeric derivatives are widely used in the manufacturing of medical devices. It is important to understand how protein adsorbs onto PU materials as this molecular process directly implicates surface biocompatibility. In this work, we compared protein adsorption at the PU film surfaces with that from the hydrophilic silicon oxide. Two PU polymers were used, a commercial polyurethane (PUA) and a novel poly(carbonate-urea)urethane matrix containing silsesquioxanes (PU4). AFM imaging revealed micro-domain segregation on both PU surfaces, but the incorporation of pendent silsesquioxanes made the PU4 surface much rougher, with the outer surface comprised of soft upper PU segments and lower PU-silsesquioxane hard segments. It appeared that fibrinogen was preferable to adsorb onto the upper soft PU segments. The spectroscopic ellipsometry (SE) measurements at the PU film/solution interface showed that human serum albumin (HSA) adsorption was little affected by surface chemistry whilst fibrinogen adsorption was much greater on the two PU surfaces indicating a strong surface effect. Further studies revealed that HSA adsorption was reversible on hydrophilic SiO(2) against changes in pH from 5 to 7, but irreversible on the two PU surfaces. In contrast, fibrinogen adsorption against the same pH cycling was found to be irreversible on all three surfaces. The different extent of irreversibility was clearly indicative of different interfacial interactions. Sequential protein adsorption revealed that the PU4 surface shared similar physiochemical properties to the SiO(2) surface, demonstrating the success in incorporating the siloxane pendant nanocages. The knowledge of protein surface structure and behaviour may lead to the development of effective means to control surface biocompatibility.

The mechanical properties of infrainguinal vascular bypass grafts: their role in influencing patency.

When autologous vein is unavailable, prosthetic graft materials, particularly expanded polytetrafluoroethylene are used for peripheral arterial revascularisation. Poor long term patency of prosthetic materials is due to distal anastomotic intimal hyperplasia. Intimal hyperplasia is directly linked to shear stress abnormalities at the vessel wall. Compliance and calibre mismatch between native vessel and graft, as well as anastomotic line stress concentration contribute towards unnatural wall shear stress. High porosity reduces graft compliance by causing fibrovascular infiltration, whereas low porosity discourages the development of an endothelial lining and hence effective antithrombogenicity. Therefore, consideration of mechanical properties is necessary in graft development. Current research into synthetic vascular grafts concentrates on simulating the mechanical properties of native arteries and tissue engineering aims to construct a new biological arterial conduit.

Tissue engineering of blood vessels.

BACKGROUND: Tissue engineering techniques have been employed successfully in the management of wounds, burns and cartilage repair. Current prosthetic alternatives to autologous vascular bypass grafts remain poor in terms of patency and infection risk. Growing biological blood vessels has been proposed as an alternative. METHODS: This review is based on a literature search using Medline, PubMed, ISIS and CAS of original articles and reviews, and unpublished material and abstracts. RESULTS AND CONCLUSIONS: Complete incorporation into host tissues and the maintenance of a viable and self-renewing endothelial layer are the fundamental goals to be achieved when developing a tissue-engineered blood vessel. Sourcing of cells and modulating their interaction with extracellular matrix and supporting scaffold have been the focus of intense research. Although the use of tissue-engineered blood vessels in humans is so far limited, advances in our knowledge of stem cell precursors and the development of new biomaterials should enable this technology to reach routine clinical practice within a decade.

Nanocomposite containing bioactive peptides promote endothelialisation by circulating progenitor cells: an in vitro evaluation.

OBJECTIVE: The formation of an endothelial cell layer on the luminal surface of cardiovascular devices, especially bypass grafts, is an important attribute in order to improve their patency. Endothelial progenitor cells (EPCs) have a potential role in the endothelialisation of bypass grafts. We hypothesised that a novel approach to improve endothelialisation of bypass grafts by EPCs would be the creation on the graft lumen of a microenvironment that supports EPC adhesion and differentiation. METHODS: A new generation of nanocomposite based on silsesquioxane in the form of polyhedral oligomeric silsesquioxane (POSS) nanocages which incorporate bioactive peptides (RGD) was made into sheets. Peripheral blood mononuclear cells (PBMCs) containing EPCs isolated from six consenting young, healthy, adult volunteers were then plated both on (1) sheets of the nanocomposite with the bioactive peptide, (2) sheets of the nanocomposite without the bioactive peptide, (3) culture dishes as control and then cultured in presence of vascular endothelial growth factor (VEGF). Confirmation of endothelial and EPCs markers was carried out using fluorescence-activated cell sorter (FACS) analysis, reverse transcription polymerase chain reaction (RT-PCR) and immunostaining. RESULTS: One to two percent of PBMCs expressed CD34 as determined by FACS analysis. Cells were demonstrated to express mRNA for the EPC markers CD34, platelet-endothelial cell adhesion molecule-1 (CD31), CD133 and vascular endothelial growth factor receptor-2(FlK-1/KDR). Endothelial cell-colony forming units were formed between day 5 and day 7 after plating. Colonies were confirmed to be endothelial like cells by immunostaining. There were significantly greater numbers of EPC colonies on the bioactive nanocomposites as compared to the nanocomposite alone and the uncoated dishes. CONCLUSION: We report a new nanocomposite based biomaterial that has been demonstrated, in vitro, to promote endothelialisation from PBMCs containing EPCs.

Evidence that the reactions of nickel in the presence of vitamin C do not produce toxic oxygen intermediates such as hydroxyl but ascorbate and carbon radicals.

A variety of reactions containing different amounts of nickel ions together with vitamin C were prepared and used in spin trapping experiments designed to show nickel is not a Fenton active metal. Carbon based radicals were observed at low ratios of ascorbate to nickel, and ascorbate radical was observed at high ratios of ascorbate to nickel. No buffer effects were observed. There was no evidence for oxygen intermediates as products of the reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), seeming to indicate nickel is not a Fenton metal. To test this hypothesis further trapping studies were run with the singlet oxygen trap 2,2,6,6-tetramethyl-4-piperidone hydrochloride, in which dioxygen was found not to affect the formation of carbon radicals. These results help to explain the damage to DNA observed in the presence of vitamin C and nickel in vitro.

Evidence for a relationship between the generation of reactive intermediates and the physicochemical characteristics of nickel oxides.

Spin trapping experiments have been carried out on solutions generated by the dissolution of nickel oxides, all of which contained NiO (bunsenite) as the predominant crystalline phase. Samples containing nickel (III), of low crystallinity, with small particle size and high surface areas can in the presence of H2O2, cause the generation of the hydroxyl radical (as detected by spin trapping with 5,5-dimethyl-1-pyrroline N-oxide or DMPO) and/or another highly reactive species capable of cleaving the C-S bond in dimethyl sulphoxide. In contrast samples which predominantly contain nickel (II), of high crystallinity, with a large particle size and low surface area cause the generation of alkyl and alkoxyl radicals, or another highly reactive species capable of causing the decomposition of lipid peroxides, but did not generate significant quantities of the hydroxy radical type. These findings help to explain the varied ability of nickel containing solids to cause DNA lesions in vivo and in vitro.

Design of a small peptide-based proteinase inhibitor by modeling the active-site region of barley chymotrypsin inhibitor 2.

A synthetic peptide-based proteinase inhibitor was constructed by modeling the regions responsible for inhibition in barley chymotrypsin inhibitor 2 (CI-2). The 18-residue peptide was designed by molecular modeling, based on the crystal structure of CI-2. The amino acid sequences that interact with the proteinase were preserved, as well as residues that maintain the structure of the inhibitory loop. A disulfide bridge was introduced to force the peptide to adopt a cyclic structure. Kinetic studies on binding of the cyclic peptide to subtilisin BPN', subtilisin Carlsberg, chymotrypsin, and pancreatic elastase show that the cyclic peptide retains both the inhibition properties, the kinetic mechanism, and the specificity of the original protein inhibitor. Formation of a cyclic structure was found to be essential, and activity was abolished by reduction of the disulfide. As with CI-2, tightest binding is found to subtilisin BPN', where the Ki value for the cyclic peptide was 28 x 10(-12) M, compared with 29 x 10(-12)M for CI-2 under identical conditions. This remarkable result shows that it is possible to use a short synthetic peptide to model the molecular recognition properties of the intact protein, in this case obtaining full functionality with just 18 residues instead of 83 for CI-2.

Evidence that the reactions of cadmium in the presence of metallothionein can produce hydroxyl radicals.

A variety of Cu reconstituted metallothioneins (MTs) containing different amounts of copper ions together with Cd7-MT free of copper were prepared and used in spin trapping experiments designed to show cadmium is not a Fenton active metal. A significant increase of the DMPO/.OH adduct was observed, with increased concentrations of the copper containing MTs, H2O2 enhanced DMPO/.OH adduct formation, catalase and the Cu (I) specific chelating agent bathocuproine, reduced DMPO/.OH adduct formation. These results suggest that Cu (I) and H2O2 both have important roles in the production of active species in these systems and cause DMPO/.OH formation. However, Cd7-MT showed no ability to cause generation of DMPO adducts with H2O2 seeming to indicate cadmium is not a Fenton metal. To test this hypothesis further trapping studies were run with added sulphite and lipid peroxide using both commercial MT and Cd7-MT since cadmium causes peroxidation in vivo. Commercial MT generates radicals with added sulphite and peroxide, Cd7-MT does not, demonstrating that cadmium is not a Fenton metal. These results help to explain the oxidative damage to DNA observed in the presence of MT and cadmium in vitro.

A new technique for measuring the cell growth and metabolism of endothelial cells seeded on vascular prostheses.

For the improvement of vascular graft patency, an endothelial cell (EC) lining is desirable. It is essential that the EC remains viable after being seeded onto the prosthetic graft. The aim of this study was to adapt an Alamar redox assay (ABRA) as a technique to monitor the viability of ECs seeded on prosthetic grafts. To test the graft types, we seeded human umbilical vein ECs on compliant polyurethane (CPU), expanded polytetrafluoroethylene, and Dacron at a density of 2 x 10(5) cell/cm(2). After 24 h of incubation, ABRA was added, and the absorbance was measured at 4, 8, and 24 h. To assess seeded cell concentrations on grafts, we seeded CPU at densities ranging from 1 x 10(5) to 8 x 10(5) cell/cm(2). The validity of the test was assessed with sodium azide and mitomycin C, known physiological perturbators. ABRA reduction demonstrated that ECs were viable and functional postseeding on the prosthetic grafts. A significant correlation was observed with ABRA reduction and cell concentrations (p < 0.001). The acid phosphatase assay demonstrated enzyme activity in the cells, but they were not maintained under normal physiological conditions. The ABRA bioreduced product was soluble, stable, and noncytotoxic over 24 h. The assay is independent of the geometry or physiochemistry of the graft type. The technique allows the continuous assessment of the metabolism and viability of seeded cells, is simple to perform, and does not destroy the cells or graft materials.

Tissue engineering of vascular bypass grafts: role of endothelial cell extraction.

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5-6 mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patients' own endothelial cells. This review examines the various techniques and technologies used to date in order to extract endothelial cells for such graft engineering.

Compliance properties of conduits used in vascular reconstruction.

BACKGROUND: Compliance mismatch between native artery and prosthetic graft used for infrainguinal bypass is implicated in the aetiology of graft failure. The aim was to quantify the elastic properties of a new compliant poly(carbonate)polyurethane (CPU) vascular graft, and to compare the compliance properties of grafts made from CPU, expanded polytetrafluoroethylene (ePTFE), Dacron and human saphenous vein with that of human muscular artery. METHODS: A pulsatile flow phantom was used to perfuse vessel and prosthetic graft segments at physiological pulse pressure and flow. Intraluminal pressure was measured using a Millar Mikro-tip catheter transducer and vessel wall motion was determined with duplex ultrasonography using an echo-locked wall-tracking system. Diametrical compliance and a stiffness index were then calculated for each type of conduit over mean pressures ranging from 30 to 100 mmHg by 10-mmHg increments. RESULTS: The compliance values of CPU and artery (mean over the pressure range) were similar (mean(s.d.) 8.1(0.4) and 8.0(5.9) per cent per mmHg x 10(-2) respectively), although the elastic behaviour of artery was anisotropic unlike CPU, which was isotropic. Dacron and ePTFE grafts had lower compliance values (1.8(1.2) and 1.2(0.3)per cent per mmHg x 10(-2) respectively, averaged over the pressure range). In both these cases, compliance and stiffness differed significantly from that of artery over a mean pressure range of 30-90 mmHg. Human saphenous vein exhibited anisotropic behaviour and, although compliant at low pressure (30 mmHg), was markedly incompliant at higher pressures. CONCLUSION: Compliant polyurethane grafts offer a greater degree of compliance match than either ePTFE or Dacron.

A hybrid compliant vascular graft seeded with microvascular endothelial cells extracted from human omentum.

We report the development of a hybrid vascular graft using an innovative compliant poly(carbonate-urea)urethane unlike any previous polyurethane MyoLink as a permanent scaffold. The engineered graft has a hierarchical arterial structure: a monolayer of oriented microvascular endothelial cells (MVECs) and 3-D matrix (human collagen Type 4/dermatan sulfate) bonded onto MyoLink. The grafts' clinical feasibility was evaluated by determining optimal MVEC seeding density, incubation time, viability, and adhesion of these cells when exposed to arterial shear stress. MVECs from human omentum were isolated by a new centrifugation protocol, radiolabeled and seeded onto hybrid graft 2 to 18 x 105 cells/cm(2) for 24 h at 37 degrees C and for 1 to 5 h at 6 x 105 cells/cm(2), washed 3 times, and gamma counted. Qualitative assessment of seeding density/incubation was also undertaken with scanning electron microscopy (SEM) and viability tested with a modified Alamar Blue assay. A pulsatile flow phantom was used to subject the hybrid graft (200 mm length, 5 mm internal diameter) seeded with radiolabeled MVECs (6 x 105 cells/cm(2) at 2 h) to arterial shear and dynamic scintigraphy images acquired in real time using a nuclear medicine gamma camera system during 14 h of perfusion (n = 6). The optimal seeding density was 6 x 105 cells/cm(2), and qualitative SEM confirmed this. Incubating cells for 2 h produced significantly greater cell attachment than was seen for 1 h incubation (p < 0.05), and there was no significant difference in adhesion between cells incubated over the 2 h. Exposure of grafts to acute shear stress resulted in significantly higher proportions of initial cells attached to hybrid grafts compared to native MyoLink grafts (67 +/- 3% versus 55 +/- 2%, p < 0.001). As shown here, tissue engineering of native Myolink grafts significantly reduces the seeding density and incubation time to produce a monolayer onto which cells adhere to better.

In vivo femoropopliteal arterial wall compliance in subjects with and without lower limb vascular disease.

OBJECTIVE: The purpose of this study was to further the development of a compliant vascular graft with a preliminary assessment of the elastic properties of the femoropopliteal artery in subjects with and without lower limb peripheral vascular disease. METHODS: This prospective controlled study was set in a university department of surgery. Using an ultrasound scan wall tracking system with the simultaneous measurement of brachial blood pressure, measurements of femoropopliteal artery wall motion were undertaken in 11 patients with peripheral vascular disease (group 1), in 11 older control subjects who were matched for blood pressure, age, and sex (group 2), and in 12 younger control subjects (group 3). Diametrical compliance and stiffness index were determined for the common femoral artery, the proximal superficial femoral artery, the distal superficial femoral artery (DSFA), and the midgenicular popliteal artery. RESULTS: All the arterial segments in group 1 showed a trend towards increased stiffness and less compliance than the group 2, age-matched control vessels, with significantly lower distensibility noted at the common femoral artery (mean compliance of 6.2% vs 14.1% mm Hg(-1) x 10(-2), respectively; P <.05) and the DSFA (mean compliance of 2.2% vs 1.9% mm Hg(-1) x 10(-2), respectively; P <.05). The popliteal artery segment in group 3 proved to be more compliant and less stiff than did the same vessel in group 2 (8.5% vs 4.7% mm Hg(-1) x 10(-2), respectively; P <.01). In all three study groups, the DSFA was consistently noted to be the least distensible vessel segment. CONCLUSION: Lower limb peripheral vascular disease is associated with a reduction in femoropopliteal artery elasticity. Age alone appears to have a minimal effect on the compliance of the proximal half of the femoropopliteal segment. The elastic properties of the femoropopliteal vessel are subject to marked variation along its course. To minimize compliance mismatch, the degree of elasticity engineered into a vascular graft must reflect that observed in vivo.

Cellular engineering of conduits for coronary and lower limb bypass surgery: role of cell attachment peptides and pre-conditioning in optimising smooth muscle cells (SMC) adherence to compliant poly(carbonate-urea)urethane (MyoLink) scaffolds.

OBJECTIVE: We are developing a hybrid arterial bypass graft of compliant poly(carbonate-urea)urethane (MyoLink), endothelial and smooth muscle cells (SMCs). To enhance adhesion of SMCs we assessed various attachment factors and the effect of pre-conditioning on cell retention. METHODS: MyoLink segments were coated with either RGD, superfibronectin, fibronectin, fibronectin-like engineered polymer protein (FEPP), FEPP plus or type 1 collagen overnight. (111)Indium-radiolabelled SMCs were placed onto MyoLink segments for 48 h before being aspirated, then lavaged off. All grafts, aspirates and lavages were counted in a gamma counter. SMC viability on the MyoLink segments was also assessed for viability using the Alamar blue redox assay. Separately, MyoLink grafts lined with radiolabelled SMCs were divided into a pre-conditioned group, exposed to subarterial pulsatile flow whilst another group were held in static culture. After 1-week, grafts were exposed to arterial pulsatile flow whilst radioactivity was assessed using a gamma camera. RESULTS: Only FEPP plus significantly enhanced SMC attachment: mean of 32+/-6% cell attachment compared to 21+/-5% for uncoated control. Cell viability was enhanced by all attachment factors except fibronectin. Pre-conditioning was shown to significantly enhance the retention of SMCs onto the MyoLink once exposed to pulsatile arterial flow: the final attachment was 57+/-7% for the static and 76+/-7% for the pre-conditioned group. CONCLUSIONS: FEPP plus enhances SMC attachment to MyoLink. We believe this is because of its repeating sequences of RGD and its positive charge. Pre-conditioning enhances the retention of SMCs to MyoLink once exposed to pulsatile arterial flow.