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NC3Rs: National Centre for the Replacement Refinement & Reduction of Animals in Research
PhD Studentship

Bone healing and regeneration in an ex vivo bioreactor – evaluation of angiogenesis and reparation for clinical application

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At a glance

Award date
September 2012 - September 2016
Grant amount
Principal investigator
Professor Richard Oreffo


University of Southampton


  • Replacement
Read the abstract
View the grant profile on GtR



This research aims to develop an accurate model of bone repair and regeneration, using ex vivo human fetal femur tissue, to test the efficacy of novel bone regenerative therapies. Human-derived bone tissue will be studied using a natural bioincubator, the chorioallantoic membrane (CAM) of the chicken egg. This human ex vivo model could serve as an alternative to existing mouse models which involve invasive surgical procedures.


Skeletal tissue loss as a result of injury or disease causes a significantly reduced quality of life at a high socioeconomic cost. A variety of natural and synthetic materials are currently under investigation as potential scaffolds for bone regenerative therapies, including alginate, collagen and synthetic polymers. At present these scaffolds are tested using invasive surgical procedures in mice which are likely to cause pain.

This research aims to substitute the use of the mouse with models using human-derived bone tissue. An important component of any bone development strategy is the ability to support and actively promote the invasion of the host vasculature, and this cannot currently be accurately modelled in vitro. Initially chick, followed by human-derived tissue will be grown using the chicken egg as a surrogate blood supply and natural bioincubator.

Research details and methods

A novel, self-assembling, synthetic, smectite clay hydrogel known as Laponite will be used to develop the scaffold for bone regeneration. Chick and human fetal femur cells will be used for regeneration as these have already demonstrated unique biological properties, including markedly increased proliferative and multi-lineage potential compared with adult skeletal cell sources, reflecting their earlier developmental origin. The cellular construct will be placed upon the CAM of a ten day old chick embryo and will become infiltrated and nourished by CAM derived vessels. A range of growth factors and clay concentrations will be investigated to determine which conditions best support the infiltration of new blood vessels into new bone tissue.

Defects will then be created in chick and human femur bone samples using a 2mm drill bit and the bone pieces cultured on the CAM will be placed in the defects, along with either control, growth factor or cell-containing gels. Vascularisation and osteogenesis will be measured at various time points over a nine day time course to assess the quality and efficacy of bone repair. Bone regeneration will be analysed by a range of techniques, including immunochemistry, computer x-ray tomography and targeted microarrays.

Application abstract

Skeletal tissue loss as a result of injury or disease causes a significantly reduced quality of life at a high socioeconomic cost. We are investigating tissue engineered cellular constructs, consisting of a novel smectite hydrogel and a highly potent source of skeletal stem cells derived from human fetal femurs as a potential tissue replacement. To test the efficacy of such constructs in promoting tissue healing and regeneration, we propose to develop a new ex vivo model to study bone formation that accurately model bone repair and regeneration without the need for often highly invasive surgical procedures in murine hosts. Our primary aim is to refine animal use in our research by substituting these surgical procedures in sentient mouse models with realistic and novel models of bone regeneration in a natural painless bioincubator – the chorioallantoic membrane (CAM) of the chicken egg.



Moreno-Jiménez I et al. (2018). Remodelling of human bone on the chorioallantoic membrane of the chicken egg: De novo bone formation and resorption. Journal of Tissue Engineering and Regenerative Medicine 12(8):1877-1890. doi: 10.1002/term.2711

Moreno-Jiménez I et al. (2017). The Chorioallantoic Membrane Assay for Biomaterial Testing in Tissue Engineering: A Short-Term In Vivo Preclinical Model. Tissue engineering. Part C, Methods 23(12):938-52. doi: 10.1089/ten.TEC.2017.0186

Gibbs DM et al. (2016). A review of hydrogel use in fracture healing and bone regeneration. Journal of Tissue Engineering and Regenerative Medicine 10(3):187-98. doi: 10.1002/term.1968

Moreno-Jiménez I et al. (2016). The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering. Scientific Reports 6:32168. doi: 10.1038/srep32168