Modulation of dermal microvascular endithelial cell responses to growth factors and haemostatic factors in the presence of vitronectin

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

Modelling of some biological materials using continuum mechanics

Continuum mechanics provides a mathematical framework for modelling the physical stresses experienced by a material. Recent studies show that physical stresses play an important role in a wide variety of biological processes, including dermal wound healing, soft tissue growth and morphogenesis. Thus, continuum mechanics is a useful mathematical tool for modelling a range of biological phenomena. Unfortunately, classical continuum mechanics is of limited use in biomechanical problems. As cells refashion the �bres that make up a soft tissue, they sometimes alter the tissue's fundamental mechanical structure. Advanced mathematical techniques are needed in order to accurately describe this sort of biological `plasticity'. A number of such techniques have been proposed by previous researchers. However, models that incorporate biological plasticity tend to be very complicated. Furthermore, these models are often di�cult to apply and/or interpret, making them of limited practical use. One alternative approach is to ignore biological plasticity and use classical continuum mechanics. For example, most mechanochemical models of dermal wound healing assume that the skin behaves as a linear viscoelastic solid. Our analysis indicates that this assumption leads to physically unrealistic results. In this thesis we present a novel and practical approach to modelling biological plasticity. Our principal aim is to combine the simplicity of classical linear models with the sophistication of plasticity theory. To achieve this, we perform a careful mathematical analysis of the concept of a `zero stress state'. This leads us to a formal de�nition of strain that is appropriate for materials that undergo internal remodelling. Next, we consider the evolution of the zero stress state over time. We develop a novel theory of `morphoelasticity' that can be used to describe how the zero stress state changes in response to growth and remodelling. Importantly, our work yields an intuitive and internally consistent way of modelling anisotropic growth. Furthermore, we are able to use our theory of morphoelasticity to develop evolution equations for elastic strain. We also present some applications of our theory. For example, we show that morphoelasticity can be used to obtain a constitutive law for a Maxwell viscoelastic uid that is valid at large deformation gradients. Similarly, we analyse a morphoelastic model of the stress-dependent growth of a tumour spheroid. This work leads to the prediction that a tumour spheroid will always be in a state of radial compression and circumferential tension. Finally, we conclude by presenting a novel mechanochemical model of dermal wound healing that takes into account the plasticity of the healing skin.