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Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition
Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition
Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF) in bone forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we have used a murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Furthermore, we also deleted Vegf in vitro in OBs extracted from male and female mice in an attempt to link sex-specific matrix signatures to deviations in gene expression. Label-free and non-destructive polarisation-resolved second harmonic generation microscopy (p-SHG) revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied only in males by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-stand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure including Adamts2, Spp1, Mmp9 and Lama1. The current results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders which clinically manifest in both pathological nano and macro-level disorganisation.
Collagen, Extracellular matrix, Polarisation-resolved second-harmonic generation, Raman spectroscopy, Sexual dimorphism, VEGF
1754-8403
Sharma, Aikta
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Goring, Alice L
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Johnson, Peter
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Boyde, Alan
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Emery, R.J.
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Hesse, Eric
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Olsen, Bjorn
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Pitsillides, A.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 L
ac8955ad-5d55-478f-a370-447f6fa1798a
Johnson, Peter
862b092d-ac8b-4c9b-804f-10d42d8b0d21
Boyde, Alan
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Emery, R.J.
5e119a17-dbd0-45a6-82b4-e47b9051ac7c
Hesse, Eric
e70cc75d-270f-432a-9047-1e65dfc10627
Olsen, Bjorn
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Pitsillides, A.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, Goring, Alice L, Johnson, Peter, Boyde, Alan, Emery, R.J., Hesse, Eric, Olsen, Bjorn, Pitsillides, A.A., Oreffo, Richard, Mahajan, Sumeet and Clarkin, Claire (2021) Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition. Disease Models and Mechanisms, 14 (3), [dmm048116]. (doi:10.1242/dmm.048116). (In Press)

Record type: Article

Abstract

Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF) in bone forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we have used a murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Furthermore, we also deleted Vegf in vitro in OBs extracted from male and female mice in an attempt to link sex-specific matrix signatures to deviations in gene expression. Label-free and non-destructive polarisation-resolved second harmonic generation microscopy (p-SHG) revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied only in males by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-stand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure including Adamts2, Spp1, Mmp9 and Lama1. The current results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders which clinically manifest in both pathological nano and macro-level disorganisation.

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dmm.048116.full - Accepted Manuscript
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Accepted/In Press date: 15 January 2021
Keywords: Collagen, Extracellular matrix, Polarisation-resolved second-harmonic generation, Raman spectroscopy, Sexual dimorphism, VEGF

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Local EPrints ID: 446529
URI: http://eprints.soton.ac.uk/id/eprint/446529
ISSN: 1754-8403
PURE UUID: 09707a4c-ae6b-432b-961a-21dcf32a573e
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: 12 Feb 2021 17:31
Last modified: 29 Apr 2021 01:53

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Contributors

Author: Aikta Sharma ORCID iD
Author: Alice L Goring
Author: Peter Johnson
Author: Alan Boyde
Author: R.J. Emery
Author: Eric Hesse
Author: Bjorn Olsen
Author: A.A. Pitsillides
Author: Richard Oreffo ORCID iD
Author: Sumeet Mahajan ORCID iD
Author: Claire Clarkin

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