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Detection of early osteogenic commitment in primary cells using Raman spectroscopy

Detection of early osteogenic commitment in primary cells using Raman spectroscopy
Detection of early osteogenic commitment in primary cells using Raman spectroscopy
Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate υ3 (1030 cm-1) and B-type carbonate (1072 cm-1), DNA (782 cm-1) and collagen matrix (CH2 deformation at 1450 cm-1) and weaker phosphate bands (948 and 970 cm-1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945-952 cm-1) and carbonated apatite (957-962 cm-1) after only 3 days in culture and octacalcium phosphate (970 cm-1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells.
Raman spectroscopy, osteogenesis, bone cells, osteoblasts
0003-2654
1962-1973
Smith, Stephanie J.
b93e230c-cc90-42f5-ae47-19354008d2fa
Emery, Roger
bc4e1ca5-b6fd-498d-b78a-d26059f36c7a
Pitsillides, Andrew
e4ebf5b6-5e10-47dd-8bc9-f3baa8e29707
Clarkin, Claire E.
05cd2a88-1127-41aa-a29b-7ac323b4f3c9
Mahajan, Sumeet
b131f40a-479e-4432-b662-19d60d4069e9
Smith, Stephanie J.
b93e230c-cc90-42f5-ae47-19354008d2fa
Emery, Roger
bc4e1ca5-b6fd-498d-b78a-d26059f36c7a
Pitsillides, Andrew
e4ebf5b6-5e10-47dd-8bc9-f3baa8e29707
Clarkin, Claire E.
05cd2a88-1127-41aa-a29b-7ac323b4f3c9
Mahajan, Sumeet
b131f40a-479e-4432-b662-19d60d4069e9

Smith, Stephanie J., Emery, Roger, Pitsillides, Andrew, Clarkin, Claire E. and Mahajan, Sumeet (2017) Detection of early osteogenic commitment in primary cells using Raman spectroscopy. Analyst, 142 (11), 1962-1973. (doi:10.1039/C6AN02469F).

Record type: Article

Abstract

Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate υ3 (1030 cm-1) and B-type carbonate (1072 cm-1), DNA (782 cm-1) and collagen matrix (CH2 deformation at 1450 cm-1) and weaker phosphate bands (948 and 970 cm-1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945-952 cm-1) and carbonated apatite (957-962 cm-1) after only 3 days in culture and octacalcium phosphate (970 cm-1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells.

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Osteogenesis Raman accepted - Accepted Manuscript
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More information

Accepted/In Press date: 18 April 2017
e-pub ahead of print date: 3 May 2017
Published date: 7 June 2017
Keywords: Raman spectroscopy, osteogenesis, bone cells, osteoblasts
Organisations: Institute for Life Sciences, Biomedicine, Centre for Biological Sciences

Identifiers

Local EPrints ID: 408602
URI: http://eprints.soton.ac.uk/id/eprint/408602
ISSN: 0003-2654
PURE UUID: 951ad688-8a9d-456e-8d29-67bb18760254
ORCID for Sumeet Mahajan: ORCID iD orcid.org/0000-0001-8923-6666

Catalogue record

Date deposited: 25 May 2017 04:02
Last modified: 22 Nov 2021 05:22

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Contributors

Author: Stephanie J. Smith
Author: Roger Emery
Author: Andrew Pitsillides
Author: Sumeet Mahajan ORCID iD

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