Stem cell metabolomics for bone therapies and tissue engineering

Like a scene from an Indiana Jones movie, imagine the scenario where an anthropologist, exploring the lost civilisation of the Mayans, comes across skulls that appear to have an almost full set of false teeth. Were these teeth inserted post mortem to embellish their way to the after-life, or, had they been inserted during their time on Earth? Closer inspection showed that they were indeed pieces of shell, fashioned into individual teeth that had been inserted into the jaw bone to replace teeth that had been lost through natural causes. Remarkably, detailed analysis showed that these shell dentures had totally integrated into the jaw bone. In other words, the jaw bone had happily accepted the shell implants - this feature is called osteo-integration. Further work showed that, not only was the shell integrated into the bone - it was also osteo-inductive, i.e. encouraged bone formation. It also did this without apparent rejection of the implant.
What is even more intriguing is that here we have an invertebrate system (mollusc shell) enabling bone formation in a vertebrate (human). Where does the connection lie? Unravelling this connection is an important aspect of this project. Not just the quest to determine how and why it happens, but to unlock and exploit the mechanism of osteo-induction and use this to provide material for bone implants.
The shell used by the Mayans, as in most invertebrates, is composed of calcium carbonate, or chalk. Normally, calcium carbonate exists in one of two forms, called polymorphs: calcite and aragonite. Aragonite is also called nacre or Mother of Pearl. Although these polymorphs have the same chemical composition they have different structures.
Our preliminary data confirm that one of these invertebrate polymorphs, nacre, does indeed induce bone formation. The other polymorph, calcite, appears to hold a different function in that it encourages cells to avoid specialisation into specific cell types such as fat- or bone-producing cells. We aim to understand these responses so that we can design and construct materials that can be used to elicit desirable and predictable cellular responses. This is important for the following reasons:
By 2031 more than 50% of people in the UK will be over 65. This demographic scenario will place extreme pressure on orthopaedic surgeons with a rise in the required number of hip and knee replacements. Simultaneously, the population is living longer and thus, further intervention may be required later in life. Currently, a more sports active younger population are now presenting with sport injuries with a high potential for subsequent osteoarthritis. The problem is that in younger and more active people, implants tend to fail more rapidly and revision operations are less successful than the original replacement surgery. In trauma clinics, facial reconstructive surgery demands large areas of intact bone; usually painfully sequestered from convenient sites. These examples present major challenges for clinicians, and also for scientists and engineers who need to rapidly drive new technologies on a suitably large scale in order to meet these demands by supplying novel materials for joint implantation.
In a separate clinical issue, the supply of high-quality non-specialised cells to clinic would advance direct regenerative therapy and aid tissue engineering. The current problem is that cells grown in culture tend to become specialised almost immediately. The fact that calcite encourages cells to remain non-specialised offers the possibility of providing sufficient quantities of cells for tissue therapies.
The timing could not be better. We are only now in a position to understand and exploit bone formation and supply of cells, with such a rapidly ageing population, there is urgent need for such transformative approaches.

The route to meet the objectives is as follows:
- Detailed scanning electron microscopy characterisation of natural biomineral surfaces in terms of polymorph (electron backscatter diffraction, EBSD), structure, nano- and micro-topography and crystallographic orientation (EBSD).
- Benchmarking the response of MSCs to the well-characterised natural material surfaces above. Cellular response quantified in terms of attachment, spreading and production of markers for formation of bone, cartilage, fat and muscle as well as cell growth and stem cell renewal.
- Use mineral-free mimics of nacre and calcite to investigate topographical influence of nacre and calcite and quantify the response of mesenchymal cells to these mineral-free topographies in comparison to the response to natural nacre and calcite.
- Quantify cellular response to topography-free nacre and calcite chemistry using XPS in a strategy that isolates mineral chemistry from the chemistry of the intervening organic layers.
- Develop techniques to study stem cell differentiation via metabolomics using mass spectrometry and novel bioinformatics.
- Quantify metabolomic response to scientist-designed templates (NSQ50 and SQ), natural biominerals and mimics. (Note that the deliberate slight disorder in the nano-pattern of NSQ50 promotes bone formation while the strict order of SQ promotes retention of multipotency).
- Produce bio-inspired templates for predictable, tuneable stem cell responses.

Ultimate beneficiaries
While this project aims to provide the fundamental knowledge required to exploit the phenomenon of bone formation induced by nacre and stem cell renewal promoted by calcite, the ultimate beneficiaries are very clear even if they are not immediate. The final beneficiaries fall into several categories: the elderly, younger sports-active members of the population, patients requiring reconstructive surgery or tissue therapy and ultimately the surgeons performing the necessary procedures.
The alarming demographic scenario of the rapidly ageing UK population brings with it increasing demand for hip and knee replacements. In trauma clinics, facial reconstructive surgery demands large areas of intact bone; usually painfully sequestered from convenient sites. The large quantity of bone material required in many procedures is a constant problem for orthopaedic surgeons. In all of these cases, tuneable bone therapies would alleviate these problems. The knowledge acquired in this project will be exploited in controlled bone therapies with the potential to transform the treatment of bone and joint disease, addressing a major societal issue and helping the health and wellbeing of the population by overcoming the major problems outlined above.
The parallel, potentially even more exciting, aspect of this project is that of developing robust techniques in metabolomics to understand and therefore control stem cell behaviour. This adventurous approach will address a separate clinical issue; the supply of high-quality autologous stem cells to clinic. This supply would advance direct regenerative therapy and aid tissue engineering. Mesenchymal stem cells (MSCs) are an excellent source of autologous stem cells. However, due to MSCs rapid and spontaneous differentiation (usually to fibroblasts) on tissue culture surfaces it is currently impossible to meet the growing demand to expand autologous MSCs for therapaeutic use. By understanding the aspect of calcite that encourages MSCs to grow without differentiation, we will be able to design novel cell culture substrata to serve the demand for tissue engineering and regenerative therapy. Additionally, the exploration of metabolomics as a means of understanding stem cell behaviour will inform the design of substrates that induce specific cell differentiations.

Immediate beneficiaries
From the early stages of this project we will make advances within individual academic and clinical disciplines as well as across disciplines with the PDRA benefiting from the high level of training built into this programme. The academics will benefit from collaborations across discipline boundaries, crossing from academia to application. By bringing together these disciplines, the advances made in this project will have major impact on the field of clinical therapies, metabolomics, biomaterials, biomineralisation, cell engineering, and materials science. With such a range of disciplines, it is particularly important that dissemination of findings occurs at international conferences and in high impact journals across all of these subjects to ensure that academic and clinical impacts are maximised and that findings are not restricted to discipline silos.
The project is set up to maximise the links and feedback loops between academics and clinical practitioners. With the orthopaedic surgeon, Mr Dominic Meek (DM), associated with this project, we are in a very strong position to ensure that our approach meets the needs of practitioners. The Glasgow Orthopaedic Research Initiative (GLORI) provides us with ideal access to a community of academics and clinicians.
The multidisciplinary approach adopted here will contribute directly to our knowledge economy since the PDRA will benefit from high level training integrated into this project across a broad range of techniques, equipping her with a robust skills set suitable for application in many areas of science.