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De Melo, Nigel (2020) Stem cell control through tailored ion release profiles from resorbing calcium phosphate materials. PhD thesis, University of Nottingham.

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Abstract

Control of stem cell fate has been traditionally enacted by applications of proteins, cytokines, and small molecules. Recent studies suggest that inorganic ions can similarly influence stem cell behaviour and fate. Phosphate based glasses (PBGs) demonstrate tailorable dissolution rates for the local release of these ions, demonstrating potential for both hard and soft tissue regeneration. PBG microspheres have been proposed as a promising biomaterial for orthopaedic applications due to their chemical similarity to the inorganic phase of bone and the potential for their ionic dissolution products to promote bone healing and repair. This project explored the effects of ionic dissolution products on human mesenchymal stem cells (hMSCs) in vitro and how they may be used to control stem cell fate.

This was investigated though the application of precursor compounds in isolation and in combination onto hMSCs to optimize metabolic and osteogenic effects of the core components of a PBG system. Solid and porous PBG microspheres of seven different formulations were produced and characterized for elemental composition, thermal properties and Qn species distribution before testing with hMSC culture. hMSC were cultured on PBGs directly to investigate the effects of PBG formulation on adhesion. PBGs were also indirectly tested through the application of ionic dissolution products into hMSC culture to identify effects on metabolic activity, alkaline phosphatase activity (ALP), matrix mineralization and gene expression changes though the use of TaqMan Low Density Arrays (TLDAs).

Compound and indirect PBG treatments demonstrated that phosphate content in culture exhibited a dose specific cytotoxic effect on hMSC (>1.25 mM) that could be reversed through the application of calcium (5 mM), magnesium (5 mM) or both in combination. Specific phosphate species such as pyrophosphate and polyphosphate likely contributed to the promotion of ALP activity while inhibiting matrix mineralization. This suggested that a balance between pyrophosphate/polyphosphate and orthophosphate may need to be targeted to promote ALP activity without inhibiting matrix mineralization due to oversaturation. Direct hMSC culture on PBG discs revealed significant increases in cell adhesion with decreasing PBG degradation rate. Indirect studies and gene expression analysis on hMSCs revealed no cytotoxicity from the dissolution products, and no upregulation of integrin gene expression with increasing calcium and magnesium content in the PBGs tested. These findings suggest that degradation of the glass surface itself may be responsible for the lowered number of adherent cells through surface destabilization or creation of high pH/osmolarity layer at the material-cell interface. This study also investigated the previously unexplored strategy of PBG microsphere mixing. Utilizing two different formulations of PBG microspheres allowed for the leveraging of their respective properties of faster degradation and higher calcium content from one formulation, while promoting nominal metabolic activity from the other. Finally, analysis of gene expression following PBG treatment revealed the promotion of β-catenin destabilization through Notch receptor and secreted frizzled related protein 1 (SFRP1) upregulation which may result in the disruption of RUNX2 and β-catenin activity, key osteoblastic promoter genes.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Sottile, Virginie Ahmed, Ifty
Keywords:	Biomaterials, Phosphate-based glasses, Cell culture, Degradation, Stem cells, Bone, Osteoblasts
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QT Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	61119
Depositing User:	De Melo, Nigel
Date Deposited:	11 Dec 2020 04:40
Last Modified:	11 Dec 2022 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/61119

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