Professor Liam Grover was awarded funding to develop an in vitro model for studying osteogenesis.
Principal Investigator: Professor Liam Grover
Organisation: University of Birmingham
Award type: PhD studentship
Start date: 2014
Duration: 3 years
Osseous tissue forms in various physiological circumstances ranging from normal bone development and fracture repair, to pathological heterotopic bone formation in extraskeletal tissues following muscle trauma, traumatic brain or spinal cord injury, or surgical procedures of the hip and knee. Animals such as rodents, rabbits and sheep are used in research on normal and heterotopic ossification to understand the dynamic role of osteoclasts, osteoblasts and osteocytes. Many studies focus on manipulating the expression of bone morphogenetic proteins in transgenic mice or involve subcutaneous or intramuscular implantation of bone marrowderived mesenchymal stem cells. However, for trauma models it can be necessary to cause physical damage by crushing a muscle or fracturing a bone.
The animal models are often associated with severe suffering and while they have been useful in understanding some aspects of ossification, they are not always representative of pathological states and often are too complex to investigate early phase bone formation. There are few alternative models available due to difficulties differentiating and maintaining osteocytes in vitro and although immortalised cell lines exist, these show reduced levels of key markers such as sclerostin, the secreted protein which inhibits osteoblast differentiation.
3Rs benefits (actual and potential)
Alexandra Iordachescu, the PhD student, has developed a self-structuring in vitro model for the development of mature bone, which has replaced the use of animals for screening potential demineralisation agents and studies of early bone formation in the Grover laboratory.
The model consists of a fibrin gel cast between two ceramic anchors into which osteoblastic cells, from the rat femoral periosteum, are seeded. The culture is maintained with a continuous source of calcium phosphate, supplemented with osteogenic factors. Using various imaging modalities, Alexandra has shown that the periosteal cells deposit an ordered matrix that closely resembles mature bone in terms of chemistry (e.g. the collagen/mineral ratio) and cellular composition (e.g. osteoblasts and osteocytes are present). The model remains viable in culture for over a year, recapitulating the successive phases of ossification from initiation of bone formation through to the differentiation of osteocytes from osteoblasts. Importantly, the model includes the presence of canalicular networks, essential for intracellular communication, which have not been previously observed in in vitro culture. Details of the model development and validation were published in Advanced Biosystems in 2017.
Alexandra and Liam are working with groups at the University of Oxford and Imperial College London to transfer the model into their laboratories, helping to further replace the use of animals.
Scientific and technological benefits
Alexandra demonstrated in a pilot study that it is possible to test compounds that inhibit ossification in the in vitro model. The study was conducted with two compounds, one which is used to treat acquired and congenital heterotopic ossification and the other which has been shown to reduce heterotopic ossification in transgenic murine models of Fibrodysplasia ossificans progressiva. Both compounds led to a decrease in matrix and mineral formation. Since then, the model has been used by the Grover laboratory to identify new compounds that block ossification. The model also has other benefits as it is possible to harvest matrix vesicles from it. These exosomes are thought to have a central role in controlling bone mineralisation but are poorly understood due to difficulties extracting them. Alexandra has developed a new protocol for harvesting the vesicles using immunoprecipitation, which should allow more detailed study of the process and regulation of mineralisation. The protocol was published in RSC Advances in 2018.
Alexandra has presented the in vitro model at international conferences including the World Biomaterials Congress (WBC) in Montreal, where she was the recipient of a WBC Trainee Award. In 2019, Alexandra was awarded an NC3Rs training fellowship to further develop the in vitro model by including mechanical unloading. This will allow the model to be used to study the loss of bone in ageing and during osteoporosis, helping to replace procedures such as the hindlimb unloading model in which the rat is suspended from its tail for two to three weeks so that only the forelimbs touch the cage floor.
Liam also secured further funding from the NC3Rs in 2018 for a PhD studentship in collaboration with Dr Amy Naylor at the University of Birmingham. This also aims to further advance the model developed by Alexandra, by including human osteoblasts and osteoclasts (primary cells and mesenchymal stem cells) so that the process of bone re-modelling can be studied.
This case study was published in our 2019 Research Review.