Plenary Poster 30th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2020

Molecular imaging at the multi-scale discloses novel regulators of matrix organisation during bone pathology (#22)

Aikta Sharma 1 , Alice Goring 1 , Peter B Johnson 2 , Roger JH Emery 3 , Eric Hesse 4 , Alan Boyde 5 , Bjorn R Olsen 6 , Andrew A Pitsillides 7 , Richard OC Oreffo 8 , Sumeet Mahajan 2 , Claire E Clarkin 1
  1. School of Biological Sciences, University of Southampton, Southampton, Hampshire, United Kingdom
  2. School of Chemistry, University of Southampton, Southampton, Hampshire, United Kingdom
  3. Faculty of Medicine, Imperial College London, London, United Kingdom
  4. Institute of Molecular Musculoskeletal Research, LMU Munich, Hamburg, Germany
  5. Dental Physical Sciences, Queen Mary University of London, London, United Kingdom
  6. Department of Developmental Biology, Harvard School of Dental Medicine, Boston, Massachusetts, United States of America
  7. Department of Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom
  8. Institute of Developmental Sciences, University of Southampton, Southampton, Hampshire, United Kingdom

Objectives: Deletion of vascular endothelial growth factor (VEGF) in osteoblasts (OBs) induces pathological and sex-specific alterations to the conformation and mineralisation of the bone matrix in addition to vascular organisation in males. Whether the sexual dimorphism of the bone vasculature is due to the divergent control of matrix architecture is unknown, and the focus of this study.

Methods: Raman spectroscopy (RS) and polarised-second harmonic generation (p-SHG) microscopy were performed to compare the composition and organisation of the bone matrix in male and female osteocalcin-specific Vegf knockout (OcnVEGFKO) long bones. Vegf expression was deleted in vitro in OBs from male and female Vegffl/fl mice using a Cre-recombinase adenovirus prior to screening of mRNA transcripts of extracellular matrix (ECM) components.

Results: p-SHG revealed a sex-specific macro-level disorganisation of bone matrix in male OcnVEGFKO versus females, attributed by; regionalised alterations to collagen fibril number (-1.16-fold) extensive osteoid and enhanced cortical porosity at the tibiofibular junction. Polarisation anisotropy revealed reductions in fibrillar anisotropy around the endosteal regions in female OcnVEGFKOs (-4.19-fold) and periosteal and perivascular regions (-1.97-fold and -2.64-fold, respectively) in male OcnVEGFKO. RS detected nano-molecular sexual dimorphism in the levels of carbonate following OcnVEGFKO, with reductions detected in females (-2.79-fold) and elevations in males (1.21-fold) versus WTs. Collagen intra-strand stability was exclusively reduced in OcnVEGFKO males (-3.06-fold) versus WT controls. OcnVEGFKO also induced reductions in hydroxyapatite mineralisation in both males and females versus WTs (-1.07-fold and -1.26-fold). Further divergence in genes encoding ECM proteins and pro-angiogenic factors in male and female Vegf deficient cells were detected in vitro (Spp1, Mmp13 and Thbs1).

Conclusions: We demonstrate the utility of label-free and non-destructive approaches for the detection of multi-scale sex-specific variations in the bone matrix following VEGF deletion. Identification of sex-specific genetic regulators of the ECM could be targeted in bone disorders that present nano and macro-level matrix disorganisation.