Development of mechanically robust functionalised chitosan-based hydrogels for tissue engineering applications in cartilaginous tissues

Background: Age-related diseases of cartilaginous tissues, such as intervertebral disc (IVD) degeneration (a major cause of low back pain [LBP]), pose an increasing global socioeconomic problem as the population ages and increases. There are currently no successful long-term treatments, although cell-based tissue engineering offers huge potential, by allowing regeneration of tissues damaged through disease. This includes restoring mobility to diseased joints and offering long-term pain relief. Adult stem cells, known as mesenchymal stem cells (MSCs), present in bone marrow offer great potential for tissue engineering as they can be isolated easily, grow quickly and can form (differentiate into) the cells found in cartilaginous tissues. Indeed the Manchester team have shown that MSCs can form IVD cells, making them ideal for IVD regeneration. However, tissue engineering requires a biomaterial to support cells and aid tissue regeneration. The centre of the IVD is gel-like; therefore hydrogels (gels with a high water content) are the most suitable. Hydrogels can also be injected in to the IVD, thus avoiding invasive surgery. The Manchester team have previously shown that MSCs seeded into chitosan/glycerophosphate hydrogels become IVD cells and produce an IVD-like tissue. However, currently these gels lack the mechanical strength required to exist within the loaded environment of the human spine. Therefore our objectives are to: 1. Develop novel chitosan-based hydrogels which can withstand the loads experienced within the human spine, whilst allowing MSCs to differentiate into IVD cells and regenerate a functional tissue; 2. To convert these new mechanically robust hydrogels into thermosensitive hydrogels (liquids at room temperature and gel at body temperature) which can be injected into the IVD thus minimising complex surgery and aftercare; 3. Add specific function to the mechanically robust, thermosensitive hydrogels through the addition of nutrients (such as glucose) to enhance cell survival, or the addition of factors to prevent further damage to the tissue through future disease. To achieve this the Warwick team will employ state-of-the-art chemistry to produce mechanically robust hydrogels, based around the same biocompatible materials previously evaluated. To do this highly efficient covalent cross-linking chemistries will be investigated. The development of mechanically robust materials that undergo gelation at temperatures close to that of the human body will be addressed with the use of selectively reversible protection of the active gelation sites and will result in a paradigm shift in hydrogel materials for tissue engineering. The Manchester team will screen these hydrogels at each stage to ensure they support MSC survival and differentiation to IVD cells. They have identified genes specific to IVD cells, which can be used to ensure MSCs have indeed become IVD cells. Additionally, they have also developed a 'bioreactor', which uses human IVD tissue obtained from cadavers (which have been donated for research) into which MSC-seeded hydrogels are injected. The injected cell-seeded hydrogels can then be cultured within the 'bioreactor', which accurately mimics conditions in the human spine, including the mechanical loads experienced during daily movements, making this a more relevant system than current animal models. The data obtained will allow identification of the most suitable hydrogel for IVD regeneration. Expected outcomes and potential clinical benefit: Our ability to combine expertise in chemistry and MSC-based tissue engineering, together with our unique testing system, will help advance the translation of MSC-based tissue engineering therapies for IVD degeneration to clinic. In so doing we have the ability to eradicate disc degeneration and thus improve the quality of life for millions of people and save billions of pounds for healthcare systems and the wider economies around the globe.

Tissue engineering offers a new generation of treatments for a range of age-related musculoskeletal conditions, such as intervertebral disc (IVD) degeneration; the leading cause of low back pain (LBP). The nucleus pulposus (NP) of the IVD is a highly-hydrated, proteoglycan-rich tissue, containing chondrocyte-like NP cells and it is this region which is predominantly affected during degeneration. For IVD regeneration, repair of the NP is essential, although degenerate NP cells are not a suitable cell source. Mesenchymal stem cells offer great potential and can be differentiated to cells with an NP-like phenotype. Successful tissue engineering requires a biomaterial with properties similar to the native gel-like NP tissue. Hydrogels, such as thermosensitive chitosan/glycerophosphate hydrogels can support MSC differentiation to NP-like cells, but do not have the mechanical properties required for survival within the loaded environment of the spine. Thus our aim is to use state-of-the-art chemistries, combined with expertise in MSC biology and tissue engineering to develop novel chitosan-based hydrogels, which are thermosensitive, mechanically robust and functionalised to enhance cell survival within the spine and prevent future disease reoccurrence. Initially, using innovative chemistry a new generation of chitosan-based hydrogels with improved mechanical properties will be developed and evaluated for their ability to support MSC survival and differentiation. Successful gels will then be converted using Diels-Alder chemistry to thermosensitive injectable hydrogels and tested using a novel human whole disc explant model of IVD degeneration housed within a loading bioreactor. Finally, mechanically robust thermosensitive hydrogels will be functionalised through addition of factors to promote cell survival or prevent further tissue degeneration. Future application of this technology will revolutionise treatment of a range of age-related musculoskeletal disorders.

Who will benefit from this research? The following groups will benefit from this research: - The main beneficiaries of this research will be members of the public who suffer with disc degeneration and consequent low back pain (LBP) as it will offer a long-term treatment, relief of pain and increased mobility - There will also be a huge benefit to the NHS and as it will offer a long-term treatment, therefore saving huge sums over current treatments - There will be wider benefits to the UK society and economy through increased wellbeing in the community and thus increased productivity due to fewer individuals suffering LBP - NHS practitioners e.g. neurosurgeons and orthopaedic surgeons, involved in treating intervertebral disc (IVD) degeneration and low back pain - Academic research groups interested in IVD regeneration - Academic groups interested in the application of Diels-Alder chemistry, such as medicinal chemistry and polymer chemists - Academic research groups and companies involved in developing tissue engineering strategies for a wide range of soft and hard tissues - Specialist medical companies involved in treating IVD degeneration e.g. Replication Medical, Arthro Kinetics Plc, SpineWave and DePuy Spine Inc - The application of thermally-activated chemistryto easily attach small molecules, such as anti-inflammatory compounds and growth factors has a wide range of potential application and will be of interest to a wide range of academic researchers and companies - Successful commercialisation of the proposed treatment will also create employment within the UK - Success of the research will also highlight the UK as a worldwide centre of excellence for tissue engineering and chemistry research How will they benefit from this research? The research will focus on development of a viable cell-based tissue engineering strategy for treatment of IVD degeneration and LBP; future application of which would benefit patient groups within the UK and around the world through by offering a successful long-term treatment and thereby increasing general wellbeing within the population. Healthcare systems would therefore benefit through reduced in-patient and long-term treatment costs, as would the wider UK economy through increased productivity within the population, and through potential development of a spin-out company. Other academic research will also benefit through the application of the technologies developed in other areas of biomedical research e.g. soft and hard tissue engineering, drug delivery etc. What will be done to ensure that they benefit from this research? To commercialise the research the investigators will actively seek to identify and work with relevant clinical (surgeons) and commercial partners (e.g. Replication Medical, Arthro Kinetics Plc, SpineWave and DePuy Spine Inc). Research findings will be disseminated through publication in the scientific literature and presentation at relevant academic conferences, and the investigators will actively encourage and seek interaction and collaboration with other researchers to apply the research in a wide range of tissue engineering and other applications. The impact of this will be immediate and highlight the UK as a centre of excellence for both chemistry and stem cell-based tissue engineering research. Information from the research will also be disseminated via public engagement activities, such as A-level study days for students interested in bioscience and at other organised events, as well as via radio and television interviews and on both University websites. This will inform the general public, identify user groups (potential patients) and highlight the importance and potential of this type of research to future scientists. All the investigators involved have demonstrated a clear track record of collaboration within academia and industry, public engagement activities and dissemination of findings through a variety of media.