The main aim of this project was to enhance our knowledge on angiogenesis by developing systems that enable us to study and promote angiogenesis in tissue engineering and regenerative medicine applications.
Over the last 30 years, there have been significant advances in the production of tissue-engineered (TE) materials suitable for use in the clinic. However, one of the key challenges is to ensure rapid neovascularisation into these constructs in order for them to survive post-transplantation. While relatively thin simple TE constructs can survive on well-vascularised wound beds, thicker constructs (>200 µm) usually fail to engraft due to lack of oxygen and nutrients in vivo. Both prevascularisation and scaffold functionalisation strategies with the use of angiogenic factors are viewed as promising approaches to accelerate vascular ingrowth into TE constructs to circumvent slow vascularisation after implantation.
Angiogenesis is a tightly regulated process and the majority of the current strategies for promoting rapid neovascularisation focus either on the addition of proangiogenic factors to TE constructs or adding laboratory expanded proangiogenic cells such as endothelial cells, endothelial progenitors or stem cells to tissue engineering scaffold systems prior to implantation.
For functionalisation of the TE constructs with the proangiogenic factors, vascular endothelial growth factor (VEGF) is recognised to be the most well-studied angiogenic factor due to occupying a key role in the angiogenic cascade. VEGF has been proven to have important roles in different steps of the angiogenic process in vivo: vasodilation and permeability, destabilisation of vessels and degradation of extracellular matrix (ECM), proliferation and migration of endothelial cells, and lumen formation and vessel stabilisation. However, VEGF acts as part of a well-regulated process, and its actions are highly dose-dependent, and a range of studies show that VEGF addition can lead to excessively leaky, permeable and haemorrhagic vessels such as those that are found in tumorigenesis. Therefore, seeking alternatives to VEGF is inevitable.
Accordingly, I have investigated the angiogenic potential of 2-deoxy-D-Ribose (2dDR) using well-established in vitro and in vivo models. For the in vitro assessments, the proliferation, migration and tube formation of human aortic endothelial cells (HAECs) in response to different concentrations of 2dDR were assessed. The angiogenic activity of 2dDR was further assessed in vivo using two well-established angiogenesis systems; ex-ovo chick chorioallantoic membrane (CAM) assay and diabetic rat wound healing model.
In vitro and in vivo angiogenesis models are important tools to explore the newly discovered pro-angiogenic or and anti-angiogenic agents. Although in vivo assays are the most representative and reliable models for the evaluation of angiogenesis, they are also expensive, technically difficult, time-consuming, and ethically questionable. On the other hand, in vitro angiogenesis assays are inexpensive, quick, technically simple, and reproducible, but they are usually based on evaluating only one aspect of angiogenesis (for example, only proliferation, migration or differentiation), and they may produce false results due to the unspecific reaction of cells. Moreover, most of the in vitro angiogenesis assays are limited to static, 2D cell culture systems where culturing cells on stiff and flat substrates is a simplified method and does not represent the dynamic and highly complex tissue systems. 2D culture of cells distorts cell-cell and cell-matrix interactions which affects cell proliferation, migration and differentiation, where 3D in vitro models better represent the in vivo in a cost-effective way and with no ethical concerns. To date, none of the studies demonstrated any in vitro models that allow researchers to evaluate angiogenesis assessing both proliferation and migration of ECs in a 3D environment. Thus, in this project, three different systems made of synthetic and natural polymers were developed to be used as in vitro platforms to study and promote angiogenesis.
First angiogenesis model was a poly-(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) synthetic vascular scaffold developed by combining electrospinning and 3D printing. Nanofibrous PHBV channels were capable of supporting human dermal microvascular endothelial cells (HDMEC) to adhere and create an endothelium monolayer within the channels. The use of the developed system as an in vitro platform to study angiogenesis was evaluated using Matrigel. The outgrowth of HDMECs was further analysed using a reconstructed human skin model which was also developed in the scope of this project.
The second model was developed via decellularisation of baby spinach leaves. The natural vascular structure of the leaves was then repopulated with HDMECs in the presence of helper human dermal fibroblasts, and their potential to promote angiogenesis was investigated using ex-ovo CAM assay. The decellularised spinach leaves were further assessed for their potential use as an in vitro angiogenesis model where outgrowth of HDMECs was analysed in response to VEGF and 2dDR.
The third synthetic model was developed via a combination of electrospinning and emulsion templating techniques. The developed 3D dynamic model was made of electrospun PHBV tube and polycaprolactone (PCL) polymerised high internal phase emulsion (PolyHIPE). The model was capable of supporting HAECs to form an endothelium-like monolayer within the tubular channel, and the outgrowth of HAECs was investigated in response to 2dDR and VEGF under static and dynamic conditions.
Then, as an alternative to the use of pro-angiogenic agents, I assessed the effectiveness of prevascularisation (use of pro-angiogenic cells) technique for promoting angiogenesis in ex-ovo CAM assay. I fabricated basic electrospun PHBV scaffolds and cellularised them with a combination of endothelial cells and fibroblasts to evaluate the potential of these cells to induce angiogenesis in vivo.
Finally, in collaboration with my colleague, Betül Aldemir, we investigated an alternative use of the bilayer scaffolds manufactured by combining electrospinning and emulsion templating techniques, which was found very rapid and effective route to produce TE scaffolds that have the potential to be used in various tissue engineering and regenerative medicine applications. To explore the use of these scaffolds as a bilayer barrier membrane for guided tissue regeneration (GTR) applications, we explored the cell-occlusiveness of electrospun layer and the bone promoting properties of the PolyHIPE layer.
