Elucidating and modulating macrophage and stem cell responses to bioactive nanoclays for bone regeneration

Medical advances have led to a welcome increase in life expectancy. However, this progress presents new challenges: increases in age-related diseases, and associated reductions in quality of life. There are over 9 million bone fractures per year, worldwide, of which approximately 5%-10% are associated with delayed healing or non-union. The field of regenerative medicine seeks new therapies that harness the potential of stem cells to regenerate damaged or diseased tissue. These therapies can involve stem cell transplantation, or using drugs and materials to recruit and stimulate stem cells already present in adult tissues.

Clays offer surprisingly rich possibilities for regenerative medicine. This is largely due to their ability to adsorb and bind biological molecules; a property that has for a long time been used in the design of tablets to control the release and action of certain drugs. The UK research group have developed synthetic nano-sized (1 millionth of a millimetre) clay particles that form injectable gels that set in the body. Gelation occurs spontaneously as the clay particles bind and interlock with proteins adsorbed from blood. These protein-rich 'nanoclay gels' create environments favourable for stem cells to colonise. By mixing-in proteins called growth factors, we can recruit and stimulate stem cells to remodel the nanoclay gel into new bone tissue. Given nanoclay gels bind and do not release growth factors, these potent stem cell stimulating molecules can be used in the future to fuse bones and repair non-healing fractures with greater precision, safety and efficiency.

While very promising, there is a lot about this repair process that we do not yet understand. For example, we know that the response of cells of the immune-system will play a vital role in determining how the body responds to nanoclay in the first hours and days after injection. The Japanese research group are world-leaders in understanding and controlling this immune response to implanted materials. For example the group have shown that it is possible to release drugs in a highly controlled manner at the site of repair to attract and stimulate immune cells to limit inflammation and promote regeneration.

This UK-Japan collaboration will therefore study how immune cells function to clear away nanoclay particles and simultaneously attract stem cells for bone formation. In partnership, we will develop new gel technologies that combine nanoclay gels with gelatin to facilitate controlled release of a drug that influences immune cells towards regeneration. The drug we will test, pioglitazone, is already used in the treatment of diabetes and so, if successful, has a good chance of being adopted for regenerative medicine in the future. We will test our approach both 'in vitro', on cells grown in a petri dish and 'in vivo', within the body by injecting the gels under the skin of mice. In vitro experiments will employ high powered microscopes to explore the fate of clay nanoparticles after they are engulfed by the immune cells. We will also measure the biochemical signals released by immune cells in response to nanoclay and test whether cell signalling can be modified to promote regeneration by pioglitazone. 'In vivo', we will rely on state of the art live animal imaging techniques to track immune cell responses to nanoclay gels over time and correlate these responses to new bone formation via simultaneous micro CT (3D X-Ray). This exciting approach allows us to track responses in a single animal over time which substantially reduces the number of mice needed for experiments. Finally, having developed and optimised our new approach we will test the ability of the technology to accelerate bone repair in a surgically prepared bone defect in a rat cranium. These experiments are well established and essential to test our approach and justify subsequent research investment towards clinical translation for patient benefit.

Nanoclay gels offer new possibilities for regenerative medicine given their ability to assemble gels, promote cellular ingrowth and bind biological molecules. By incorporating growth-factors such as BMP2, we have shown the potential to achieve bone induction at the lowest dose published in the literature to date. We propose to utilise this strategy in spinal fusion and fracture repair to induce bone formation with greater precision and safety.
Immune cells such as macrophages, are key in mediating the colonisation of an implanted biomaterial. There is strong evidence to indicate that nanoclay gels are successfully colonised and remodelled by stem cells, but the mediating role of macrophages, particularly in relation to stem cell recruitment and nanoclay degradation and clearance remains unclear. Previous work by the Kyoto group has shown the potential of dual drug release strategies from hydrogels to achieve efficient macrophage polarisation and enhance regenerative responses.
In this collaboration we will explore the early macrophage response to nanoclay gels and develop a drug release strategy utilising the peroxisome proliferator-activated receptor gamma agonist pioglitazone (PIO) - a well-known modulator of inflammatory responses currently licensed for treatment of type II diabetes - to explore the effect of macrophage polarisation on nanoclay hosted BMP2 bone induction. This work will build on a successful micelle based approach developed in Kyoto to solubilise pioglitazone for hydrogel incorporation and controlled release.
In vitro studies will examine macrophage phagocytosis and signalling in response to nanoclay and characterise macrophage-stem cell cross talk. In vivo ectopic bone and calvarial defect assays will harness unique in vivo radiolabelling (Kyoto) and cell and clay tracking (Soton) capabilities to dissect the dynamics of drug release, clay degradation, macrophage and stem cell recruitment and bone induction.

This collaboration has strong potential to impact UK and Japanese healthcare by developing new IP to underpin a promising regenerative medicine technology. The project addresses several of the strategic priorities identified in the "Strategy for UK Regenerative Medicine" developed by the MRC and other research councils, by providing a new approach to controlling cellular differentiation, inducing endogenous responses, and developing biologically inductive biomaterials. Japanese government also held up the development of regenerative medicine as one of growth strategies since the early 2010s and has intensively infused research funds for the field of regenerative medicine. This project will not only meet the Japanese government strategy but also provide a strong impact to the future strategy.
This project seeks to generate insight into the ways in which clay nanoparticles interact with immune cells and stem cells to modulate regenerative responses. Unusually for such a project, the background for this investigation is not a toxicological or acute inflammatory response to nanoparticles, but rather, strong evidence for nanoclay bioactivity and regeneration.

Understanding why nanoclays are bioactive is of interest both for the development of biocompatible and biofunctional materials, and for understanding the key interactions between immune and stem cells that modulate inflammation and repair. Knowledge gained through this research program will therefore underpin safer and more effective regenerative medicine strategies. The direct beneficiaries of this work will therefore be the increasing, aging demographic for whom current reparative options have proved inadequate. Additional beneficiaries will include the UK and Japanese tax payer through reducing cost of, for example, revision surgery and bed stay, and industry by feeding the private biomedical technology sector (as seen through the founding of Renovos Biologics Ltd. and MedGel in Japan).

As well as direct socio-economic impact, the proposal will benefit emerging research talent within the University of Southampton and Kyoto University by immersing graduate students and a postdoctoral researcher (PDRA) in a rigorous multidisciplinary research program, dynamic public engagement and expanding commercialisation activity.

Educational benefit will continue to be pursued through the development of a cutting edge public engagement program further extending the impact of our award winning "Stem Cell Mountain" exhibit first launched at the BBSRC Great British Bioscience Festival. We recognise that continued public engagement around the themes of stem cells and regenerative medicine is vital to maintain public confidence and strengthen the case for investment in both the UK and Japan. We aim to expose both groups to one another's public outreach activities - with a joint public engagement activity targetting IF-Oxford Science and Ideas Festival in Autumn 2021 and regularly held joint meetings of academia, industry, and government in Kyoto. This cross-fertilisation will extend and enhance the public and commercial impact of this program throughout the time frame of the grant, and beyond, to achieve broad public impact.