Towards osteogenesis
: utilising the power of cell-free protein synthesis for regenerative medicine

  • Agata K Jakimowicz

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

The annual incidence of bone disorders and breakages is on the rise due to an ageing population. Numerous drawbacks and constraints of the current clinical treatments for bone repair have been observed, with current demand for bone grafts outstripping supply from donors. Recently, interest in bone regeneration has shifted to the field of tissue engineering, which uses techniques to generate new tissue in vitro, such as engineered grafts for implementation, or in vivo, by prompting the tissue to self-repair through the use of biomolecules such as growth factors. Here, the latter approach of tissue engineering is combined with synthetic biology to provide the foundations for generating a growth-factor-producing gel capable of stimulating osteogenic differentiation of mesenchymal stems cells (MSCs). The component that enables programmable production of the growth factor, specifically bone morphogenetic protein 2 (BMP2), is created by a cell-free expression system (CFES), an in vitro protein synthesis approach. In this project, a collection of growth factor-reporter fusion constructs were designed and selected for the highest yielding by E.coli CFES. Secondly, the effect of the fusion interactions with MSCs was visualised, confirming bioactivity of the novel protein. Phosphorylation of downstream BMP2 signalling molecules was detected from fusion samples, indicating significant deGFP-BMP2 receptor-binding activity. Furthermore, an upregulation of osteogenic markers, calcium deposition and enhanced alkaline phosphatase activity were observed, and in same cases to a greater effect than a positive control, rhBMP2, indicating that the fusion protein is capable of MSCs differentiation towards the osteogenic lineage. Lastly, CFES was incorporated into a series of hydrogel networks, where the increasing fluorescence signalled the successful in-gel production of the fusion protein. Looking ahead, this CFPS system could be developed into an injectable gel capable of spatio-temporal osteogenic stimulation, which would be a powerful technique in tissue engineering. This thesis serves as an integral step towards this goal.
Date of Award6 Dec 2022
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorAdam W Perriman (Supervisor) & J L R Anderson (Supervisor)

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