Characterisation of cellular responses and drug delivery capabilities within mineralised hydrogel scaffolds

There is an extensive clinical need for novel interventions addressing the burden placed by degenerative musculoskeletal diseases on healthcare systems worldwide. The healthcare challenge of osteoarthritis (OA) of the knee joint, in particular, which is affecting 8.75 million people in the UK alone, accounts for nearly 7% of the global burden of disease. This is only set to increase in incidence due to an ageing population. OA leads to the destruction of complex osteochondral tissue interfaces and requires effective innovative solutions as current treatment options frequently fail to provide functional results. To provide a safe and cost-effective solution, this project aims to develop cell-free, biocompatible hydrogel materials containing biomimetic anisotropic gradients of different phosphate-salt nanoparticles. In addition to the development of novel synthesis and nanoparticles gradient precipitation approaches in a parallel CDT-TERM PhD project, the current projects aims at studying the biological responses of clinically relevant adult stem cells (dental pulp stem cells, bone-marrow derived mesenchymal stem cells and synovial cells) to such matrices with the aim to improve cell invasion, retention and differentiation through matrix modification as the effective recruitment of endogenous cells is paramount to any cell-free materials approach.
The research challenge of tailoring several candidate matrix-nanoparticulate systems developed in a parallel CDT-TERM PhD study for enhanced cell compatibility and control over cell behaviour through minimal manipulation will be essential for the translation of these systems. Fine tuning of nanoparticle composition, concentration and the addition of further extracellular matrix components such as fibronectin are expected to improve biocompatibility, cell attachment and retention whilst additionally influencing cellular differentiation through the biological activity of the employed cations. Therefore, this project encompasses a multi-disciplinary approach, studying how material properties of advanced matrix systems that can influence cellular behaviour and differentiation to osteogenic or chondrogenic phenotypes. Biocompatibility testing will be performed following industry standards and guidelines (supported by the advisory industrial partner) and differentiation will be assessed using state of the art reporter gene assays specific for cellular differentiation in conjunction with gene expression studies. As the technology is a platform technology for advanced biomaterial devices but also amenable drug delivery, modification of the system with therapeutic nucleic acids will also be investigated. Through this multi-disciplinary approach focusing on the parallel development of a medical device that can alternatively also be used in conjunction with ATMPs this project will therefore have wide ranging applications in the TERM field including tissue interface engineering and regenerative approaches in dentistry.

This project aims at providing preclinical cell compatibility, drug delivery and cellular differentiation data on promising agarose candidate systems for future translation of gradient biomaterials as devices to improve tissue regeneration and osteochondral regeneration by:

- Establishing drug delivery and release (ion release, nucleic acid release)
- Studying cell-interaction and guided differentiation in vitro by culturing clinically relevant MSCs (mesenchymal stem cells from bone marrow, dental pulp stem cells and synovial cells) on the developed materials.
- Assessment of biocompatibility and differentiation using a range of methods (advanced CLSM, reporter gene assays, qRT-PCR, immunohistochemistry).
- Engineering novel molecular engineered payloads based on DNA origami (in collaboration with Dr. Matteo Castronovo, University of Leeds) and linear MIDGE DNA vectors specifically for the electrophoretic platform system.

Regenerative Medicine been defined as "an interdisciplinary approach, spanning tissue
engineering, stem cell biology, gene therapy, cellular therapeutics, biomaterials (scaffolds and matrices),nanoscience, bioengineering and chemical biology that seeks to repair or replace damaged or diseased human cells or tissues to restore normal function, (UK Strategy for Regenerative Medicine). CDT TERM will focus on acellular therapies, scaffolds,autologous cells and regenerative devices, which can be delivered to patients as class three device interventions, thus reducing the time and cost of translation and which provide an opportunity to deliver economic growth and benefits to health in the next decade. The primary beneficiaries of CDT TERM are patients, the health service, UK industry, as well as the academic community and the students themselves. Recognising that the impact and benefit from CDT TERM will arise in the future, the statements describing impact below are supported by evidence of actual impact from our existing research and training.

Patients will benefit from regenerative interventions, which address unmet clinical needs, have improved safety and reliability, have been stratified to meet patients needs and manufactured in a cost effective manner. An example of impact arising from previous students work is a new acellular scaffold for young adult heart valve repair, which has demonstrated improved clinical outcomes at five years.

The Health Service will benefit from collaborations on research, development and evaluation of technologies, through existing partnerships with National Health Service Blood and Transplant NHSBT and the Leeds Biomedical Musculoskeletal Research Unit LMBRU. NHSBT will benefit through collaborative projects, through technology transfer, through enhancement of manufacturing processes, through pre-clinical evaluation of products and supply of trained personnel. We currently collaborate on heart valves, skin, ligaments and arteries, have licensed patents on acellular bioprocesses, and support product and process developments with pre-clinical testing and simulation. LMBRU and NHS clinicians will benefits from our collaborative research and training environment and access to our research expertise, facilities and students. Existing collaborative projects include, delivery devices for minimally manipulated stem cells and applied imaging for early OA.

Industry will benefit from supply of highly trained multidisciplinary engineers and scientists, from collaborative research and development projects, from creation and translation of IP, creation of spinout companies and through access to unique equipment, facilities and expertise. We have demonstrated: successful spin outs in form of Tissue Regenix and Credentis; successful commercialisation of a novel biological scaffolds for vascular patch repair; sustainable long term R and D and successful licensing of technology with DePuy; collaborative research with Invibio, partnering with Simulation Solutions to develop new pre-clinical simulation systems, which been adopted by regulatory agencies such as China FDA. Our graduates and researchers are employed by our industry partners.

The academic community will benefit through collaborative research and access to our facilities. We have funded collaborations with over 30 academic institutions in UK and internationally. The CDT TERM will support these collaborations and the academic partners will support student research and training. The CDT students will benefit from enhanced integrated multidisciplinary training and research, a cohort experience focused on research innovation and translation, access to our research partners, industry and clinicians. Feedback from existing students has identified the benefit of the multidisciplinary experience, the depth and breadth of excellence in our research base, the outstanding facilities and the added value of the cohort training.