Hypoxic pre-conditioning of seeded tissue engineered scaffold to improve in vivo neovascularisation

We are currently facing an aging population with more chronic diseases. This increases the risk of organ failure or loss as the general population live longer to face more of such health issues, hence adding further pressures of the health care system in delivering care to these affected individuals. There is increasing need for organ transplants with a growing problem of organ donor shortage. Finding other means to meet the population's growing demands is becoming ever more crucial. With advancement in technology, it is now possible for scientist to use tissue regenerative techniques to engineer new organs via the use of biological or synthetically created scaffolds to host the cells which make up the target organ. However, one of the major obstacles for growing new artificial organs is the provision of adequate blood supply to these vital cells during the period of initial implantation into the body. Lack of blood supply prevents the implanted organ from fully integrating into the body and ultimately failure of the tissue engineered structure as a whole. Hence, understanding and improving angiogenesis underpins the future success of tissue engineering of organs.

The aim of this research is to increase the understanding of how we can improve the growth of blood vessels during this crucial time of integration and to be able to apply this knowledge into the development of a tissue engineered scaffold which will enable rapid growth of blood vessels to sustain its survival once implanted. In our project, we will focus on artificial windpipes as the tissue engineered scaffold model as this has been previously used in patients successfully. We have found that cells placed in a low oxygen environment will produce multiple factors which have been shown to accelerate the growth of new blood vessels. We intent to harness this unique characteristic of the cells by first implanting them into the tissue engineered windpipes scaffolds and placing them in low oxygen environments. This will trigger the release of factors which will increase the growth of new blood vessels. We will aim to see if we can demonstrate this feature when this tissue engineered construct is subsequently transplanted into living animals. It is hoped that this will show that the transplanted construct will generate and release its own factors in response to the low oxygen environment and therefore accelerate the growth of new blood vessels.

The outcome from this research will help scientist gain further understanding on how to improve new blood vessel growth into tissue engineered constructs. In the long term, it may also help in the development of off-the-shelf tissue engineered products for clinical use by improving their survival in the body. It will also provide vital knowledge in achieving long-term survivorship of other artificial organs for researchers in other fields of regenerative medicine.

Aim: To determine if hypoxic pre-conditioning of seeded decellularised trachea scaffold will promote the release of pro-angiogenic cytokines and accelerate angiogenesis.

Objectives:
1. Optimising hypoxic pre-conditions - Determine if hypoxic preconditioning of seeded cells promotes pro-angiogenic factors which accelerates neoangiogenesis
2. Testing hypoxic pre-conditioning response in in vivo study - Using heterotopic transplant of seeded scaffolds on mice model to demonstrate accelerated functional angiogenesis in vivo

Methodology:
1. Determine if hypoxic preconditioning enhances angiogenesis of seeded scaffolds by using CAM assay to delineate the best conditions
2. Perform cytokine analysis of conditioned medium to interrogate for pro-angiogenic factors
3. Ascertain functional characteristics of angiogenesis by using in vivo murine model for heterotopic transplant of scaffold accompanied by histological and SEM analysis of explanted scaffolds
4. Establish if a novel photoacoustic imaging technique can provide non-invasive analysis of microvasculature

Scientific and medical opportunities:
1. Improve the understanding of angiogenesis process in implanted trachea scaffolds in vivo
2. Improve angiogenesis in seeded scaffolds by determining the right hypoxic environment for MSC production of pro-angiogenic factors
3. Potential amalgamation of this research with other on-going parallel research on optimisation of tissue engineered trachea in vivo which will facilitate the development of GMP grade scaffold for clinical use
4. Validation of the use of photoacoustic images to monitor angiogenesis in vivo will allow for reduction in the use of more invasive methods and possible pre-clinical applications

Tissue Engineering/ Regenerative Medicine Researchers
Understanding and improving angiogenesis underpins the future of tissue engineering. Hence my research will have significant impact on the future of tissue engineering. By studying the effect of hypoxic preconditioned mesenchymal stem cells (MSCs) on neoangiogenesis , this project will determine if seeding such cells will enable the production of pro-angiogenic factors autonomously within tissue engineered scaffolds. This will have a number of research outputs such as the validation MSCs as a source of cytokine release, establishing if MSCs are responsive to the local environment thereby enabling cells to have feedback mechanisms for cytokine release and understanding the fate of MSCs once implanted. These findings will greatly improve current understanding of MSC hypoxic behaviour within scaffolds and enable scientist to exploit these features further in other tissue engineered applications. This will also have significant impact on the use of these MSCs as therapeutic source of pro-angiogenic factors for regenerative medicine purposes such as such as wound healing in diabetic ulcers and tissue repair of damaged cartilage and bone.

Medical Physicist/Collaborator's Impact
Part of my project will be a collaborative study with the UCL Medical Physics and Biomedical Engineering Department on use of Photoacoustic (PA) Imaging technology in assessing angiogenesis in three-dimensional decellularised trachea in vivo. Research output from this study will most definitely have an impact on the wider scientific community, for both Medical Physicist and Regenerative Medicine Scientist. Dissemination of the results will be via publication in literature as well as in academic presentations. In addition, the PA imaging data may help support the use of this technology in the observation of tissue integration and angiogenesis of tissue engineered scaffolds in clinical applications. This will no doubt have a significant impact on the future of this technology. In the initial stages, any positive results from this preliminary research will enable further funding for a larger scale pre-clinical research on the use of PA technology in tissue engineering. Given this is quite a novel application, there may be potential intellectual assets from this study.

Patients and potential healthcare benefits
By improving our understanding of the process of angiogenesis in vivo, we hope to be able to extrapolate these findings to predict what will happen to these tissue engineered scaffolds once implanted into the patients. With advancing technology, I anticipate this will have significant impact on the translatability of this research. The delivery of a rapidly vascularised scaffold will greatly improve the survivorship of tissue engineered constructs. This will have significant benefit patients who require urgent tissue engineered organs where survival of the cells within the construct is crucial to its success. There will be numerous clinical applications to other tissue engineered organs, such as liver, kidneys and heart. Collectively, this will help accelerate such research towards their clinical application, ultimately benefiting future patients on the receiving end.

Industrial/Economic Impact
The delivery of scaffolds with enhanced neovascularisation properties has implications on the manufacturing of GMP scaffolds for clinical use. As I will aim to focus on the potential molecular and cellular response within tissue engineered scaffolds as part of my research study, any positive research findings has a potential for larger scale clinical application of improved cellular therapies and techniques. This may in turn have significant industrial impact via pharmaceutical interest in the products of research.