From ageing to space travel: Developing an organotypic model of skeletal tissue disuse for understanding degeneration in altered environments

The role of bone tissue in maintaining calcium balance and organ function is essential. Calcium acts as a universal currency and is essential for most physiological processes, including cellular communication, muscle contraction, blood clotting and nerve function. A substantial loss of skeletal tissue takes place in several clinical contexts, including disuse osteoporosis, ageing, spinal cord injury, immobilisation and weightlessness in microgravity, all characterised by rapid and significant loss in bone mass in the load-bearing regions, including lower limbs, spine and hip. This increases the risk in fractures and impairs the healing process, placing a significant burden on the healthcare system and the costs associated with interventions.
While bone mass is known to decrease proportionally with reduced loading, the cellular processes governing it require further understanding. It is widely accepted that bone resorption is increased and bone deposition is decreased, however, there is no robust way of studying the imbalance in bone remodelling.
There are well-established animal models for studying musculoskeletal disuse and bone loss, which cause a reduction in bone mass in animals either through surgical removal of glands involved in bone metabolism, immobilisation using toxins, surgical resection of nerves, tendons or the spinal cord, or a tail suspension method facilitating hindlimb unloading. Some of these processes are very detrimental for the animals and others interfere with the biochemistry of skeletal homeostasis. Moreover, the results are not entirely representative of the human conditions, as differences exist in the bone remodelling process between the two species and between strains of the same laboratory animal.
The aim of this work is to produce a model that can refine and reduce the number of animals used for understanding musculoskeletal degeneration and to provide a method to study bone loss in a dish. The work will generate a physiologically-relevant model, in which specialised bone cells of human origin as well as combinations of these cell types will be cultured inside human-derived biological scaffolds. Cells will be provided with mechanical unloading using several rotary culture bioreactors that can keep cells in a constant suspension using constantly rotating vessels, thus simulating a weightless state. This platform will be used in combination with a range of active matrices derived from human tissue, such as fibrin (blood clot-like), which are degradable and pathologically representative. These will be morphologically adapted into spheroids for suspended culture and can provide a degree of support while allowing bone cells to replace them with collagenous matrix, heavily mineralise this template and bury themselves inside it, as shown by foundation work.
This model will help in studying early bone loss processes which are essential for understanding disuse pathology, can provide a first-stage elimination step of cytotoxic, genotoxic and incompatible compounds leading to less harmful agents being progressed for in vivo testing, and will allow the testing of numerous promising drugs and potential therapeutics. It will also help researchers developing treatments in a wide range of skeletal conditions, not only relevant to disuse osteoporosis, but also in excessive bone research, bone cancers, inflammatory degradation and multi-systemic research.

A substantial loss of skeletal tissue takes place in several clinical contexts, including disuse osteoporosis, ageing and weightlessness in microgravity, all characterised by rapid and significant loss in bone mass in the load-bearing regions. This increases the risk in fractures and places a significant burden on the healthcare system and the costs associated with interventions. While bone mass is known to decrease proportionally with reduced loading, the cellular processes governing it require further understanding. There are relevant animal models available which involve surgical excision of glands, immobilisation using toxins, surgical resection, or a tail suspension method facilitating hindlimb unloading. Some of these processes are very detrimental for the animals and others interfere with systemic signalling paths. Moreover, the translation of findings to human applications is difficult, due to differences in bone remodelling between species and between strains of the same species. The aim of this work is to produce a physiologically-relevant model that can reduce the number of animals used for understanding skeletal degeneration. Specialised human skeletal cells will be cultured inside spheroidal human-derived fibrin scaffolds within a low-shear and load culture system. Cells will be provided with mechanical unloading using rotary culture bioreactors that can keep cells in a constantly suspended state. Foundation work showed that these templates can provide a degree of support, while allowing bone cells to replace them with collagenous matrix, heavily mineralise and construct lacunar structures inside it. This model will help in studying early bone loss processes which are essential for understanding disuse pathology, can provide a first-stage elimination step of toxic and incompatible compounds before being progressed to in vivo testing, and will allow the testing of numerous promising drug formulations that can identify potential therapeutics.

This research has the potential to make improvements across all of the 3R areas. The work is aimed at developing pathologically-relevant microstructural features as found in atrophied human tissue, which could deliver a refinement step in optimising different conditions and parameters before they are used for animal experiments. It would help in reducing the number of parameters to a range of better-defined conditions, and could act as a first-stage screening platform for identifying cytotoxic drugs and compounds, meaning that animals used for this purpose would be subjected to less severe conditions. To achieve this, I am planning to optimise the model to a point of sufficient complexity that will allow comparable results to ex vivo human tissue from the micron to the centimetre scale.
Such a model would allow the recreation of molecular signalling and cellular interactions and might serve as a predictive tool for quantifying the rate of bone loss that other technologies are not able to achieve. A better understanding of the cellular behaviour in these stages can lead to the identification of novel drug targets and within the healthcare system, with the design of preventative care and better-timed interventions in patients with osteoporosis, osteopenia or prolonged bed rest atrophy.
The model has the potential to become a standard of practice for studying different aspects of degenerative bone loss. I believe the advanced organotypic features that can be generated would drive its adoption and wider use and would enable the faster and cheaper translation of promising interventions and compounds to the clinic. From an industrial perspective, the model could potentially provide a more cost effective and lower maintenance tool for selecting promising drug formulations and for repurposing 'shelved' drugs, using a personalised human-derived system.
The model could contribute towards an envisaged 20% decrease in the annual reduction coefficients based on 2016-2017 figures, that could lead to almost 1000 animals being replaced nationally in basic research (0.77%), 116 reduced for translational research (0.24%), and 89 reduced in refinement work (0.38%).
To achieve these objectives, I am planning to publish methodological information regarding the model in reputable, international open-access type journals in order to drive the wider use of this model with the ambition of it enabling faster and cheaper translation of promising healthcare products to the clinic.
Publication of protocol-type papers would be another important step, as it would capture know-how that would enable other users to modify the model using alternative conditions and to further develop it into a tool that may facilitate clinical translation of their own technologies.
These steps are essential for driving adoption of this model across research laboratories, in industry and potentially towards acceptance by regulatory bodies for healthcare products. Companies awaiting such technologies unanimously state the lack of both multi-centre validation of promising models and censuses over appropriate outcome measures/methods as barriers to wider adoption. An open access protocol forum may provide an enabler for multi-centre validation.
I am planning to organise key events and a set up a consortium of investigators involving former and new contacts to generate an exchange forum for ideas and collaborations in this domain.
I am planning to solidify my collaborations with my network of contacts working in ageing, bone pathology, biomaterials and microgravity research to maximise the research outputs and hence the scientific impact. I am planning to use these opportunities to establish a point of intensive research in this domain that will be unique not only in Birmingham but also in the UK, running collaboratively with similar facilities in Germany, France and the US.