Nanobioengineering strategies for bone repair: osteoblast-derived extracellular vesicles and their mimetics

Brunet, Mathieu, Yves (2024). Nanobioengineering strategies for bone repair: osteoblast-derived extracellular vesicles and their mimetics. University of Birmingham. Ph.D.

Brunet2024PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (7MB)

Abstract

Introduction: Nanobioengineering strategies combine recent advances in nanotechnologies with bioengineering methods to accelerate the development of nanotherapeutics. In the last decade, extracellular vesicles (EVs) have been intensively researched as an innovative acellular approach to overcome the current limitations of cell-based therapeutic solutions. For bone repair, EVs derived from osteoblasts have exhibited a strong osteogenic potency highlighting their potential as the next generation of bone regenerative therapy. Nevertheless, the clinical translation of EVs remains hindered by several barriers including scale-up and purity. Aims: The aim of this project was to advance the development of nanotherapeutics for bone repair by harnessing the potential of osteoblast-derived EVs. Comprehensively characterised, the osteoinductive capacity of mineralising-osteoblast-derived EVs (MO-EVs) was evaluated to determine their therapeutic relevance with the development of an innovative functional assay. Based on these results, synthetic MO-EVs were then developed and produced as bioinspired mimetics overcoming translational hurdles. Finally, the formulation of a hydrogel 3D system was investigated to develop a new delivery strategy for EVs suited to bone repair applications.
Methods: MO-EVs were separated using differential ultracentrifugation after comparing isolation methods. MO-EVs were fully characterised to determine their physico-chemical properties, the validation of the presence of EV biomarkers as well as their storage conditions. The composition of MO-EVs was also determined with a particular focus on protein content via a proteomic analysis and on mineral content. The effects of MO-EVs on osteoblast migration and proliferation were assessed as well as their osteogenic potency on osteoblast cultures. To that end, an innovative biomineralisation in vitro assessment method was also developed harnessing the potential of μ-X-ray fluorescence spectroscopy. Using both top-down and bottom-up approaches, synthetic MO-EVs were formulated, and both were compared to the biological activity of MO-EVs. Cell-derived nanovesicles were generated from mineralising osteoblasts (MO-NVs) via serial extrusion, whereas proteoliposomes were formulated using the thin film hydration method. An injectable alginate/collagen hydrogel system was developed harnessing the potential of competitive ligand exchange cross-linking gelation.
Results: MO-EVs were successfully isolated via ultracentrifugation and their extensive characterisation revealed the presence of key proteins such as annexins as well as minerals which can be mechanistically linked to their pro osteogenic potency which was demonstrated in vitro on osteoblasts. Moreover, MO-EVs were found to modulate mineral production in a time- and dose- dependent manner as demonstrated by μ-XRF. Both synthetic EVs were successfully formulated presenting with MO-EVs features and the incorporation of both alkaline phosphatase (ALP) and annexin VI was validated. Nevertheless, neither of these synthetic EVs matched the activity of the MO-EVs. Finally, alginate-collagen composite hydrogels were successfully produced, and the MO-EV release kinetics was found dependent on the collagen concentration.
Conclusion: In this thesis, novel insights were provided supporting the therapeutic use of MO- EVs for bone repair. Through the use of bioengineering strategies, the foundations of the development of synthetic MO-EVs were laid. Associated with the successful development of an injectable hydrogel delivery system for these nanovesicles, the work presented has supported the development of MO-EV based therapies for bone repair.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):