Development of regenerative extracellular vesicles for bone tissue engineering

Tsaroucha, Dimitra ORCID: 0000-0002-9621-9378 (2019). Development of regenerative extracellular vesicles for bone tissue engineering. University of Birmingham. M.Sc.

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Abstract

There are a number of clinical strategies to promote fracture healing and treat bone defects, such as autologous or allogenic bone grafting procedures and stem cell therapies. Stem cell therapies have shown great potential in the area of regenerative medicine and tissue engineering. However, numerous considerable limitations are associated with the presented techniques, including limited supply in donor tissue, often insurmountable regulatory hurdles, ethical and economic issues. Extracellular vesicles (EVs) have gained considerable attention in tissue engineering as a new potential therapy that enables bone and tissue healing. EVs are nano-sized particles that are released into the cellular environment from all types of cells contributing to cell-to-cell communication. Naturally EVs deliver important biological cargo including nucleic acids and osteoinductive proteins. An EV approach to tissue engineering could eliminate the significant issues with grafting procedures and provide an alternative therapy to the current biological therapies. However, the separation and identification of regenerative EV populations is currently problematic due to the variability that exists in vesicles and isolation protocols.

The vast part of this study concerns the isolation and characterisation of osteoblastic EVs. EVs were isolated by two different methods; sequential and differential ultracentrifugation. Their size distribution and morphologies wеrе determined via three different microscopy techniques such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The total protein content was analysed using a protein quantification assay (BCA) and the influences of these EVs on osteoblast metabolism were assessed using an Alamar Blue assay (AB).

Finally, formulation of an injectable biomaterial in an effort to facilitate localised EV delivery to a bone defect was explored. The biomaterial of interest in this study was a natural hydrogel called Gellan Gum (GG). Gellan Gum (GG) is commonly usеd in the food and pharmaceutical industries as a thickening agent. In this study, different cross-linker solutions were prepared and evaluated as a potential injectable system that may be used to controllably deliver therapeutic doses of isolated vesicles.

In order to produce an injectable gel, toothpaste was considered as a baseline material with desirable rheological characteristics. The rheological behavior of GG sample was tested and compared to that of a toothpaste.

EVs were successfully isolated using both protocols. The highest centrifugal forces; 75,000 x g and 120,000 x g gave the highest concentration of EVs that were capable to enhance cell proliferation in culture. Also, at these two centrifugal forces in both protocols, EVs with similar average sizes appeared. Future work would focus on the incorporation of EVs into optimised GG hydrogels and in-vitro testing of mineralisation.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):