Informing on mineral and matrix deposition in bone and primary cells in health and disease using Raman spectroscopy and label-free imaging

The therapeutic outcome for many musculoskeletal diseases often results in the replacement of the diseased joint with an artificial implant. New implant coatings and surfaces are being developed to improve their clinical outcome with much focus on the bone cell- implant interface. Osteoblast adherence and differentiation are key events in the early stages of implant osseointegration; however, limitations in molecular research techniques make it challenging to evaluate the response of osteoblasts to titanium and other opaque surfaces in vitro. Raman spectroscopy and label-free imaging are an attractive non- destructive alternative to conventional biomolecular techniques. The overall aim of this work was to investigate the potential of Raman spectroscopy and label-free imaging techniques to monitor osteogenic behaviour in primary cells and to further examine components of the mineralised matrix in bone tissue. Raman spectroscopy was used to evaluate and characterise the proliferation and differentiation of calvarial osteoblasts (COBS). Early changes (between 3 and 5 days of osteogenic culture) were detected by Raman spectroscopy before gene expression and standard tissue culture assays. Further Raman spectral analysis revealed fluctuations in phosphate species after 5 days of culture. Human osteoblasts (HOBS) isolated from trabecular and cortical bone cultured directly on titanium-coated disks were measured using Raman spectroscopy. Differences in matrix components of the Raman spectrum, relating to collagen structure, were detected between trabecular and cortical HOBS within patients. Analysis of SHG images obtained from trabecular and cortical bone chips revealed differences in collagen type I fibre width and length between trabecular and cortical bone samples. These results provide specific information about the matrix structure of individual patients. Label-free techniques could be applied to the assessment of individual patients, leading to the development of individualised medicine. For the first time, a combined approach of Raman spectroscopy, SHG and TPF imaging was employed to characterise the effects of the deletion of osteoblast specific VEGF in intracortical murine bone sections. Raman spectroscopic analysis revealed decreased hydroxyapatite deposition in VEGF deleted bone sections compared to wildtype (WT). Measurement of the full width at half the maximum height (FWHM) of the 959 cm-1 phosphate peak, revealed increased crystallinity in the posterior region of both VEGFKO and WT bone sections, although the mineral in the VEGFKO posterior region was more mature than the WT bones sections. Second harmonic generation (SHG) demonstrated an increase in collagen type I fibres in VEGF deleted bone sections suggesting an autocrine regulation of matrix mineralisation role for VEGF on osteoblasts. Overall, this thesis demonstrates the sensitivity of Raman spectroscopy together with non- linear label-free imaging techniques (SHG and TPF), and their potential for evaluating mineral and matrix composition at a tissue and cellular level in a non-destructive manner.