Deciphering the Role of Osteoblast-Derived VEGF in Osteogenesis Using Raman Spectroscopy and Multimodal Imaging.

Osteoblast-derived vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and regulator of angiogenic and osteogenic coupling during skeletal development and postnatal skeletal homeostasis. Raman spectroscopy, a label-free vibrational technique, poses as an alternative platform for the biomolecular interrogation of osteoblast activities. The main aim of this PhD was therefore to utilise Raman spectroscopy to characterise the effects of VEGF during osteogenic differentiation, and consequently the angiogenic effects of osteoblasts on vascular endothelial cell function, in order to test the hypothesis that spectral signatures of osteoblasts and the bone matrix can determine angiogenic and osteogenic potential. This thesis describes three main objectives; i) Characterisation of the spectroscopic signatures of differentiated osteoblasts in vitro to determine the osteogenic phases associated with pro-angiogenic VEGF-A (VEGF) production. Univariate spectral deconvolution analysis indicated that spectral features associated with type I collagen and the earliest mineral precursor of hydroxyapatite (amorphous calcium phosphate, ACP), dominated in osteogenic signatures of immature, pre-mineralising primary murine long bone osteoblasts (LOBs) where VEGF release was pronounced. Mature LOBs, conversely, released less VEGF and were shown to engage in matrix mineralisation characterised by spectral signatures that were dominated by carbonated apatite (CAP), the last precursor to hydroxyapatite. Osteogenic assays such as gene expression analysis, viability assays and histochemical staining, performed in parallel, validated the association of spectroscopic osteoblast signatures with differentiation status. ii) Examination of the direct effects of osteoblast-derived VEGF on matrix formation and mineralisation following the in vitro and in vivo Vegf deletion. Osteoblast-derived VEGF was deleted in mature osteoblasts (OcnVEGFKO) and the long bone cortex of adult mice were interrogated ex vivo using both multimodal polarisation-resolved second-harmonic generation microscopy (p-SHG) and Raman spectroscopy. p-SHG revealed regionalised organisational deficits in collagen fibre arrangement, fibre number and orientation following OcnVEGFKO, which was additionally observed to exert a degree of sex-specificity in males versus wildtype littermates (WTs). Raman spectroscopy confirmed conformational and organisational deficits were directly associated with sex-specific alterations in matrix composition, with complementary sex-differences evident in the Raman bands of type I collagen, B-type carbonate and hydroxyapatite. Additional sexual dimorphisms were identified in the expression of genes associated with matrix formation and mineralisation, matrix remodelling and angiogenesis between primary murine male and female WT and Vegf-deficient (OBVEGFKO) LOBs in vitro, including Adamts2, Spp1, Mmp9 and Lama1. iii) Investigation of the functional, angiogenic effect of osteoblasts on vascular endothelial cell activity. As osteoblast-derived VEGF was shown to differentially influence the matrix formation and mineralisation capacity of male and female LOBs, the consequential sex-differences in LOB activities on murine bone marrow endothelial cell (BMEC) function were examined in a direct-contact co-culture model. BMEC survival was supported in male LOB:BMEC cultures with the collagen content of the LOB matrix being lower than female LOBs, confirmed by Sirius Red staining, respectively. Raman spectroscopy identified that such divergence in BMEC survival was directly associated with sex-differences in LOB functionality with respect to mineral and matrix synthesis capacity. Spectral signatures of female LOBs were characterised by type I collagen associated bands, indicative of matrix deposition whereas those of male LOBs were dominated by bands corresponding to ACP, CAP and the intermediary mineral species, octacalcium phosphate (OCP), by univariate spectral analysis. Collectively my findings indicate that the identification of repertoires associated with the osteoblast activity and the bone matrix could therefore be used to directly assess osteogenic and angiogenic status, thus endorse the use of label-free technologies within an explorative and diagnostic capacity for skeletal research