Raman spectroscopy links differentiating osteoblast matrix signatures to pro-angiogenic potential

Mineralization of bone is achieved by the sequential maturation of the immature amorphous calcium phase to mature hydroxyapatite and is central to the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are typically coated with poly-L-lysine (PLL) to facilitate cell attachment. The current study has used Raman spectroscopy to investigate the effect of PLL coating on osteoblast (OB) matrix composition during differentiation, with a focus on collagen specific proline and hydroxyproline and precursors of hydroxyapatite. Deconvolution analysis of murine derived long bone OB Raman spectra demonstrated collagen species were 4.01-fold higher in OBs grown on PLL. An increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) and a reduction of 9.32-fold in mature mineral species (carbonated apatite) on PLL were detected. The unique low mineral signatures driven by PLL were associated with reduced alkaline phosphatase enzymatic activity, Alizarin Red staining, Alpl and Phospho-1 mRNA. The enhancement of immature mineral species and restriction of mature mineral species of OBs present on PLL were associated with increased cell viability and pro-angiogenic VEGF release. These results demonstrate the utility of Raman spectroscopy to characterize matrix signatures and their association with VEGF of differentiating OBs. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone in aged patients during fracture repair or in cases of pathological mineralization