R.A. Black

Dynamic Mechanical Conditioning of Collagen-Gel Blood Vessel Constructs Induces Remodeling In Vitro

AbstractDynamic mechanical conditioning is investigated as a means of improving the mechanical properties of tissue-engineered blood vessel constructs composed of living cells embedded in a collagen-gel scaffold. This approach attempts to elicit a unique response from the embedded cells so as to reorganize their surrounding matrix, thus improving the overall mechanical stability of the constructs. Mechanical conditioning, in the form of cyclic strain, was applied to the tubular constructs at a frequency of 1 Hz for 4 and 8 days. The response to conditioning thus evinced involved increased contraction and mechanical strength, as compared to statically cultured controls. Significant increases in ultimate stress and material modulus were seen over an 8 day culture period. Accompanying morphological changes showed increased circumferential orientation in response to the cyclic stimulus. We conclude that dynamic mechanical conditioning during tissue culture leads to an improvement in the properties of tissue-engineered blood vessel constructs in terms of mechanical strength and histological organization. This concept, in conjunction with a proper biochemical environment, could present a better model for engineering vascular constructs.

Flow patterns in the radiocephalic arteriovenous fistula: an in vitro study.

A significant number of late failures of arteriovenous fistulae for haemodialysis access are related to the progression of intimal hyperplasia. Although the aetiology of this process is still unknown, the geometry of the fistula and the local haemodynamics are thought to be contributory factors. An in-vitro study was carried out to investigate the local haemodynamics in a model of a Cimino-Brescia arteriovenous (AV) fistula with a 30 degrees anastomotic angle and vein-to-artery diameter ratio of 1.6. Flow patterns were obtained by planar illumination of micro-particles suspended in the fluid. Steady and pulsatile flow studies were performed over a range of flow conditions corresponding to those recorded in patients. Quantitative measurements of wall shear stress and turbulence were made using laser Doppler anemometry. The flow structures in pulsatile flow were similar to those seen in steady flow with no significant qualitative changes over the cardiac cycle. This was probably the result of the low pulsatility index of the flow waveform in AV fistulae. Turbulence was the dominant feature in the vein, with relative turbulence intensity > 0.5 within 10 mm of the suture line decreasing to a relatively constant value of about 0.10-0.15 between 40 and 70 mm from the suture line. Peak and mean Reynolds shear stress of 15 and 20 N/m2, respectively, were recorded at the suture line. On the floor of the artery, peak values of temporal mean and oscillating wall shear stress of 9.22 and 29.8 N/m2, respectively. In the vein, both mean and oscillating wall shear stress decreased with distance from the anastomosis.

Modelling the lymphatic system: challenges and opportunities

The lymphatic system is a vital part of the circulatory and immune systems, and plays an important role in homeostasis by controlling extracellular fluid volume and in combating infection. Nevertheless, there is a notable disparity in terms of research effort expended in relation to the treatment of lymphatic diseases in contrast to the cardiovascular system. While similarities to the cardiovascular system exist, there are considerable differences in their anatomy and physiology. This review outlines some of the challenges and opportunities for those engaged in modelling biological systems. The study of the lymphatic system is still in its infancy, the vast majority of the models presented in the literature to date having been developed since 2003. The number of distinct models and their variants are few in number, and only one effort has been made thus far to study the entire lymphatic network; elements of the lymphatic system such as the nodes, which act as pumps and reservoirs, have not been addressed by mathematical models. Clearly, more work will be necessary in combination with experimental verification in order to progress and update the knowledge on the function of the lymphatic system. As our knowledge and understanding of its function increase, new and more effective treatments of lymphatic diseases are bound to emerge.

Computer aided design and fabrication of models for in vitro studies of vascular fluid dynamics

Abstract An integrated computer aided design/computer aided manufacture system has been used to model the complex geometry of blood vessel anastomoses. Computer models are first constructed with key dimensions derived from radiological images of bypass grafts, and from casts of actual blood vessel anastomoses. Physical models are then fabricated in one of two ways: the surface geometry data can be used to control the movement of a three-axis milling machine; alternatively, the same data can be exported in a form that can be interpreted by a stereolithography apparatus. Both methods produce geometrically defined solid investments that can be used in a multistep casting process that yields high-quality physical models for vascular fluid dynamic studies. This technique is useful for parametric studies.

Development of a simulator for endovascular repair of abdominal aortic aneurysms

AbstractThe design and development of a simulator for endovascular repair of abdominal aortic aneurysm (AAA) is described. The simulator consists of an interchangeable model of a human AAA based on computed tomography data and is produced by means of computer-aided design and manufacture (CAD/CAM) techniques. The model has renal, iliac, and femoral arteries, and is perfused with a temperature controlled blood–analog fluid under simulated physiological flow conditions. “Fluoroscopic imaging” is simulated by a computerized imaging system that uses visible light. A movable video camera relays images in the antero–posterior and lateral planes of the AAA to a monitor. The imaging system allows “arteriography” and “road-mapping” to be performed so as to facilitate accurate deployment of endovascular stent-grafts. The system has been used for teaching and demonstrating endovascular techniques to clinicians, as well as the evaluation of new stent-graft devices. Its successful incorporation into endovascular workshops has demonstrated its role in the training of clinicians in endovascular repair of AAA.

Surface functionalization of polyurethane for the immobilization of bioactive moieties on tissue scaffolds

Segmented polyurethanes are widely used in medical devices because of their desirable physical and chemical properties and proven biocompatibility. While polyurethane is in many respects an ideal tissue scaffold, its performance is no better than other synthetic polymers, which is due in part to its surface properties. Here, we describe a method for the functionalization of polyurethane scaffolds that involves physically incorporating another polymer (poly(ethyleneimine)) such that the surface integrity and bulk properties are retained; the primary amine groups thus incorporated into the polyurethane surface enable subsequent coupling with dextran and recombinant peptides by means of reductive amination. The efficacy of the surface functionalization of a medical grade aliphatic poly(ether)urethane is verified by surface analysis (secondary ion mass spectrometry) and fluorescence and spectrophotometric assays adapted specifically for this purpose. Further assessment of the surfaces by direct cell contact and analysis of the cellular response in terms of cell coverage and morphology before and after modification with the specific peptide sequences GRGDSPK and recombinant Fibrillin-1 fragment PF9.

Formation and travel of vortices in model ventricles: application to the design of skeletal muscle ventricles

Vortex-ring production was studied in axisymmetric elastomeric ventricles designed to stimulate flow in a cardiovascular assist device. A flow visualization technique was used to investigate the effects of reducing the inlet diameter and predilating the ventricle on vortex travel in two ventricles of different shape and size. In most cases, vortex rings formed during the filling phase. They were bounded by the incoming jet of fluid and the ventricular wall. The velocity of their centres during the filling period was proportional to the inflow velocity. During filling, vortex velocity was substantially independent of the shape and diameter of the two ventricles studied. It was dependent mainly on orifice diameter: a narrower inlet led to greater inflow velocities and proportionately greater vortex velocities. At the end of the filling phase, each vortex increased in size to occupy the full radial extent of the ventricle. This process was associated with a decrease in the axial velocity and strength of the vortex. At low flow rates, these losses resulted in the arrest of the vortex at end filling. Vortex motion in ventricles is particularly important in the design of a cardiovascular device such as the skeletal muscle ventricle (SMV), where small ejection fractions may leave blood at the apex of the ventricle relatively undisturbed. It is suggested that inlet diameter could be selected to favour the formation and travel of vortices, with a resultant reduction in apical residence time and hence a reduced risk of thrombus formation.

Vascular prostheses: performance related to cell-shear responses

BACKGROUND This work concerned the endothelialization of vascular prostheses and subsequent improvement of functionality with respect to tissue engineering. The aim of the study was to investigate the initial, pre-shear stress cellular behavior with respect to three vascular biomaterials to explain subsequent cellular responses to physiological shear stresses. MATERIALS AND METHODS Expanded polytetrafluoroethylene (ePTFE), polyethyleneterephthalate (polyester; Dacron; PET), and electrostatically spun polyurethane (PU) (all pre-impregnated with collagen I/III) were cell-seeded with L929 immortalized murine fibroblasts or human umbilical vein endothelial cells (HUVECs). Cytoskeletal involvement, cell height profiles, and immunohistochemistry were examined after 7 d static culture. RESULTS All three vascular biomaterials demonstrated different structures. Cell behavior varied both between the materials and the two cell types: cytoskeletal involvement was greater for the HUVECs and the more fibrous surfaces; height profiles were greater for the L929 and PET, and lowest on PU. Immunohistochemistry of HUVEC samples also showed differences: PU revealed the greatest expression of intercellular adhesion molecule-1 and E-selectin (PET and ePTFE the lowest, respectively); ePTFE produced the greatest for vascular cell adhesion molecule-1 (PET the lowest). CONCLUSIONS Material substrate influenced the cellular response. Cells demonstrating firm adhesion increased their cytoskeletal processes and expression of cell-substratum and inter-cellular adhesion markers, which may explain their ability to adapt more readily to shear stress. The fibrous PU structure appeared to be most suited to further shear stress exposure. This study demonstrated the potential of the underlying vascular material to affect the long-term cellular functionality of the prosthesis.

α2(VIII) Collagen Substrata Enhance Endothelial Cell Retention Under Acute Shear Stress Flow via an α2β1 Integrin–Dependent Mechanism An In Vitro and In Vivo Study

Background— Essential to tissue-engineered vascular grafts is the formation of a functional endothelial monolayer capable of resisting the forces of blood flow. This study targeted &agr;2(VIII) collagen, a major component of the subendothelial matrix, and examined the ability of and mechanisms by which endothelial cells attach to this collagen under static and dynamic conditions both in vitro and in vivo. Methods and Results— Attachment of human endothelial cells to recombinant &agr;2(VIII) collagen was assessed in vitro under static and shear conditions of up to 100 dyne/cm2. &agr;2(VIII) collagen supported endothelial cell attachment in a dose-dependent manner, with an 18-fold higher affinity for endothelial cells compared with fibronectin. Cell attachment was significantly inhibited by function-blocking anti-&agr;2 (56%) and -&bgr;1 (98%) integrin antibodies but was not RGD dependent. Under flow, endothelial cells were retained at significantly higher levels on &agr;2(VIII) collagen (53% and 51%) than either fibronectin (23% and 16%) or glass substrata (7% and 1%) at shear rates of 30 and 60 dyne/cm2, respectively. In vivo studies, using endothelialized polyurethane grafts, demonstrated significantly higher cell retention rates to &agr;2(VIII) collagen-coated than to fibronectin-coated prostheses in the midgraft area (P<0.05) after 24 hours’ implantation in the caprine carotid artery. Conclusions— These studies demonstrate that &agr;2(VIII) collagen has the potential to improve both initial cell attachment and retention of endothelial cells on vascular grafts in vivo, which opens new avenues of research into the development of single-stage endothelialized prostheses and the next generation of tissue-engineered vascular grafts.

Collagen–nanofiber hydrogel composites promote contact guidance of human lymphatic microvascular endothelial cells and directed capillary tube formation

Collagen and fibronectin matrices are known to stimulate migration of microvascular endothelial cells and the process of tubulogenesis, but the physical, chemical, and topographical cues for directed vessel formation have yet to be determined. In this study, growth, migration, elongation, and tube formation of human lymphatic microvascular endothelial cells (LECs) were investigated on electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic-co-D-lactic acid) (PLDL) nanofiber-coated substrates, and correlated with fiber density and diameter. Directed migration of LECs was observed in the presence of aligned nanofibers, whereas random fiber alignment slowed down migration and growth of LECs. Cell guidance was significantly enhanced in the presence of more hydrophobic PLDL polymer nanofibers compared to PLGA (10:90). Subsequent experiments with tube-forming assays reveal the ability of resorbable hydrophobic nanofibers >300 nm in diameter to promote cell guidance in collagen gels without direct cell-fiber contact, in contrast to the previously reported contact-guidance phenomena. Our results show that endothelial cell guidance is possible within nanofiber/collagen-gel constructs that mimic the native extracellular matrix in terms of size and orientation of fibrillar components.