Kirstie Andrews

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.

Upregulation of matrix and adhesion molecules induced by controlled topography

Electrostatic spinning is receiving increasing attention in the field of tissue engineering, due to its ability to produce 3-dimensional, multidirectional, microfibrous scaffolds. These structures are capable of supporting a wide range of cell growth; however, there is little knowledge relating material substrates with specific cellular interactions and responses. The aim of this research was to investigate if electrostatically spun scaffolds, with controlled topographical features, would affect the adhesion mechanisms of contacting cells. A range of electrostatically spun Tecoflex® SG-80A polyurethane scaffolds was characterized in terms of inter-fibre separation, fibre diameter, surface roughness, void fraction and fibre orientation. Human embryonic lung fibroblasts and human vein endothelial cells were cultured on these scaffolds for 7, 14, 28 days, and analysed for their expression of extracellular matrix and adhesion molecules using image analysis and laser scanning confocal microscopy. There were significant differences in adhesion mechanisms between scaffolds, cell types and culture periods. Fibroblast-scaffolds were stimulated and oriented to a greater degree, and at earlier cultures, by the controlled topographical features than the endothelial cells. These conclusions confirm that cellular behaviour can be influenced by the induced scaffold topography at both molecular and cellular levels, with implications for optimum application specific tissue engineering constructs.

Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro

One of the major obstacles to obtaining human cells of a defined and reproducible standard suitable for use as medical therapies is the necessity for FCS (fetal calf serum) media augmentation in routine cell culture applications. FCS has become the supplement of choice for cell culture research, as it contains an array of proteins, growth factors and essential ions necessary for cellular viability and proliferation in vitro. It is, however, a potential route for the introduction of zoonotic pathogens and makes defining the cell culture milieu impossible in terms of reproducibility, as the precise composition of each batch of serum not only changes but is in fact extremely variable. The present study determined the magnitude of donor variations in terms of elemental composition of FCS and the effect these variations had on the expression of a group of proteins associated with the antigenicity of primary human umbilical-vein endothelial cells, using a combination of ICPMS (inductively coupled plasma MS) and flow cytometry. Statistically significant differences were demonstrated for a set of trace elements in FCS, with correlations made to variations in antigenic expression during culture. The findings question in detail the suitability of FCS for the in vitro supplementation of cultures of primary human cells due to the lack of reproducibility and modulations in protein expression when cultured in conjunction with sera from xenogeneic donors.

Developing smaller-diameter biocompatible vascular grafts

There is a serious clinical need for the development of a replacement blood vessel, particularly with regard to the smaller vasculature structures. This chapter examines the requirements for small-calibre vascular grafts (<6 mm diameter) and discusses the development to date in achieving patent replacement vessels. The different research methodologies are investigated, including the materials and cells used, modifications (both physical and chemical) made to the grafts and the ranging experimental conditions tested. The chapter includes a focus on the technique of electrostatic spinning which shows significant potential in the areas of controlled graft production and cell–material interactions.

In vivo mechanical behaviour of the anterior cruciate ligament: A study of six daily and high impact activities

The anterior cruciate ligament (ACL) plays a key role in the stability of the knee joint restricting the rotation and anterior tibial translation. However, there is a lack of knowledge of the in vivo ACL mechanical behaviour during high impact manoeuvres. The motion of 12 young participants with healthy knees was captured while they performed the following activities: walking, running, cross-over cutting, sidestep cutting, jumping and jumping with one leg. The in vivo ACL length and strain were estimated using experimental kinematic data and three degree of freedom (DOF) knee model. The in vivo ACL tensile forces were determined with a well-established force/strain relationship obtained through ACL tensile tests. Statistical regression models between ACL length with respect to angles for each activity have been performed in order to better understand the ACL failure mechanisms. The maximum ACL tensile force was observed during jumping vertically at maximum effort with two legs (1.076±0.113 N/BW). Surprisingly, the peak tensile ACL force for all subjects during crossover cutting (0.715±0.2647 N/BW) was lower than during walking (0.774±0.064 N/BW). Regression coefficients for crossover cutting indicated that excessive knee rotation and abduction angles contribute more significantly to the ACL elongation than in activities such as walking or running. These findings suggested that the ACL is subjected to multidirectional loading; further studies will be performed to investigate torsion, tensile and shear force on the ligament.

Enhancing Endothelialisation of Artificial/Engineered Blood Vessels Using Structural Cues

There is a clinical need for the replacement or repair of damaged and diseased blood vessels; hence, artificial materials are engineered to form replacement tissue analogues. In order to achieve functionality of these grafts, the process of endothelialisation is required. This forms a natural confluent barrier between the artificial substrate and the contacting blood flow, preventing immune responses, plaque formation or stenosis. Endothelialisation is a difficult vascular biology technique to achieve; this chapter focuses on the use of structural cues from the underlying material to enhance the functionality of the engineered vessel. We introduce the use of artificial vessels, describe the importance of the endothelialisation process and explain the options to achieve this. Methodologies to examine the effects of these structures upon the endothelial cell responses are detailed, including microscopy, image analysis, and staining for cell marker expression of endothelial cell-material samples. A focus on the use of electrospinning combined with these techniques, and sample results, is also provided.

Vascular Flow Modelling Using Computational Fluid Dynamics

Computational Fluid Dynamics (CFD) is a technique to analyse fluid flow, heat transfer and associated phenomena, using computer-based simulation. CFD has recently shown great potential for calculation of haemodynamic parameters and correlation to various cardiovascular diseases, particularly atherosclerosis and aneurysms. Consequently, the clinical community has taken an interest in different aspects of CFD simulations. The increasing power-to-cost ratio of computers and the advent of methods for subject-specific modelling of cardiovascular mechanics have further improved the application of CFD to biomedical problems. This chapter introduces the CFD technique and its applications to cardiovascular problems and describes the main merits and limitations of this tool. The main steps involved in a typical CFD process are then explained in detail and useful information is provided on some key CFD stages including pre-processing, meshing, solving and post-processing. Furthermore, three of the most common haemodynamic parameters including Wall Shear Stress, Oscillatory Shear Index and Relative Residence Time are introduced and relevant equations are explained.