Towards Osteogenesis—Utilizing the Power of Cell-Free Protein Synthesis for Regenerative Medicine

Bone healing complications resulting from non-unions and delayed non-unions are continuing to burden the clinical sector, therefore, there is a growing demand for advancing bone regeneration research. During one of the many, complex, steps in wound healing, inflammatory cells present at the site release a plethora of signalling factors that recruit nearby mesenchymal stem cells (MSCs). Upon binding of these factors to transmembrane receptors on MSCs, cell signalling cascades are engaged, resulting in the initiation of processes such as differentiation. MSCs also have the capacity to secrete paracrine factors in the instance of tissue injury, contributing to the repair response. Due to these important attributes, MSCs and growth factors are extensively researched in tissue engineering to reconstruct or repair mesenchymal tissues. A particular growth factor, namely Bone Morphogenetic Protein 2 (BMP2) is critical for efficient bone formation and mineralisation, as it has the ability to induce osteoblast differentiation in MSCs. So far, the administration of growth factors, such as BMP2, as therapeutics, has primarily focused on bolus or systemic intravenous injections. However, these proteins have a short half-life and tend to diffuse away from their target tissues. To tackle this, we have explored a way in which the growth factor could be produced on-site to minimise the problematic administration of BMP2. It involves the embedment of a Cell-Free Protein Synthesis (CFPS) system into 3D hydrogel networks, giving rise to biologically active, protein-producing bioink.<br/><br/>Firstly, a collection of growth factor-reporter fusion constructs was designed and selected for the highest yielding by <i>E.coli</i> CFPS. The efficiency of protein synthesis was further optimised by varying the input DNA concentrations, temperature and CFPS buffer additives. Secondly, an investigation was launched into the interactions and effect on MSCs with CFPS-made fusion protein and native BMP2. The phosphorylation of downstream BMP2 signalling molecules detected by Western Blotting was a good indicator that the CFPS-made fusion and the native BMP2 were comparable in their receptor-binding activity. Flow cytometry was also conducted to confirm the interaction of the fusion protein with MSCs. Furthermore, an upregulation of osteogenic markers, calcium deposition and enhanced alkaline phosphatase activity were observed, indicating that the fusion protein is capable of MSCs differentiation towards the osteogenic lineage. Thirdly, CFPS was incorporated into a series of hydrogel networks, where the increasing fluorescence of the fusion protein produced in-gel was successfully detected. Eventually, we aim to develop this CFPS system into an injectable bioink capable of spatio-temporal osteogenesis stimulation, which would pose as a powerful technique in tissue engineering research.