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

Raman spectroscopy links differentiating osteoblast matrix signatures to pro-angiogenic potential
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 (HA) and is central in the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are often coated with poly-l-lysine (PLL) which facilitates cell attachment. This 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 HA. Deconvolution analysis of murine derived long bone OB Raman spectra revealed collagen species were 4.01-fold higher in OBs grown on PLL. Further, an increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) was coupled with a 9.32-fold reduction in mature mineral species (carbonated apatite) on PLL versus controls. These unique low mineral signatures identified in OBs were linked with reduced alkaline phosphatase enzymatic activity, reduced Alizarin Red staining and altered osteogenic gene expression. The promotion of immature mineral species and restriction of mature mineral species of OB grown on PLL were linked to increased cell viability and pro-angiogenic vascular endothelial growth factor (VEGF) production. These results demonstrate the utility of Raman spectroscopy to link distinct matrix signatures with OB maturation and VEGF release. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone during fracture repair or degenerative bone loss.
Sharma, Aikta
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Goring, Alice
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Staines, Katherine A.
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Emery, Roger J.
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Pitsillides, Andrew A.
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Oreffo, Richard
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Mahajan, Sumeet
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Clarkin, Claire
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Sharma, Aikta
de245946-e5d9-434a-9c08-07ab87ae7691
Goring, Alice
30fc2dd7-a73b-469d-ab7a-f33ad13a3cee
Staines, Katherine A.
34707083-f62f-40a3-8ccb-a949e476c055
Emery, Roger J.
f443dd99-20e8-465e-b03c-8923a514ef1a
Pitsillides, Andrew A.
0181cd36-d160-4955-8b4b-06bc96bba25d
Oreffo, Richard
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
Mahajan, Sumeet
b131f40a-479e-4432-b662-19d60d4069e9
Clarkin, Claire
05cd2a88-1127-41aa-a29b-7ac323b4f3c9

Sharma, Aikta, Goring, Alice, Staines, Katherine A., Emery, Roger J., Pitsillides, Andrew A., Oreffo, Richard, Mahajan, Sumeet and Clarkin, Claire (2019) Raman spectroscopy links differentiating osteoblast matrix signatures to pro-angiogenic potential. Matrix Biology Plus, [100018]. (doi:10.1016/j.mbplus.2019.100018).

Record type: Article

Abstract

Mineralization of bone is achieved by the sequential maturation of the immature amorphous calcium phase to mature hydroxyapatite (HA) and is central in the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are often coated with poly-l-lysine (PLL) which facilitates cell attachment. This 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 HA. Deconvolution analysis of murine derived long bone OB Raman spectra revealed collagen species were 4.01-fold higher in OBs grown on PLL. Further, an increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) was coupled with a 9.32-fold reduction in mature mineral species (carbonated apatite) on PLL versus controls. These unique low mineral signatures identified in OBs were linked with reduced alkaline phosphatase enzymatic activity, reduced Alizarin Red staining and altered osteogenic gene expression. The promotion of immature mineral species and restriction of mature mineral species of OB grown on PLL were linked to increased cell viability and pro-angiogenic vascular endothelial growth factor (VEGF) production. These results demonstrate the utility of Raman spectroscopy to link distinct matrix signatures with OB maturation and VEGF release. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone during fracture repair or degenerative bone loss.

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Accepted/In Press date: 9 October 2019
e-pub ahead of print date: 20 November 2019

Identifiers

Local EPrints ID: 436688
URI: http://eprints.soton.ac.uk/id/eprint/436688
PURE UUID: 79fa2eaa-ed29-41d2-bb13-4ca1c519a514
ORCID for Aikta Sharma: ORCID iD orcid.org/0000-0002-5449-358X
ORCID for Richard Oreffo: ORCID iD orcid.org/0000-0001-5995-6726
ORCID for Sumeet Mahajan: ORCID iD orcid.org/0000-0001-8923-6666

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Date deposited: 20 Dec 2019 18:31
Last modified: 18 Feb 2021 17:32

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