The bone mineralisation process and its influence on bone strength remains poorly understood. While histomorphometric methods use fluorescent labels to measure bone mineralisation rates this fails to provide information on bone matrix maturation or composition. To overcome this, a spectroscopic sFTI-RM method was developed to analyse a brittle bone mouse model caused by ephrinB2 deletion in late osteoblasts/osteocytes (Dmp1Cre.EfnB2f/f).
The cortical midshaft was analysed in 3µm-thick tibial sections from 12-week old female Dmp1Cre.EfnB2f/f mice and controls (w/w) (n=13/group). 15μm2 regions were measured, commencing at the periosteal edge to the mature bone measuring bone matrix maturation in situ. Polarized FT-IR imaging was used to characterize collagen organization. Data was combined with 3-point bending results to determine relationships between bone strength and composition.
Region-based sFT-IRM showed gradual accrual of mineral into matrix in control samples, with a 12% higher mineral:matrix ratio at the periosteal edge in Dmp1Cre.EfnB2f/f bones compared to w/w controls; this was associated with greater carbonate incorporation. Regression analyses with 3-point bending data showed that the ultimate strength of Dmp1Cre.EfnB2f/f bones was determined by the carbonate:mineral ratio. A 13% reduction in the amide I:amide II ratio within the maturing bone suggests changes in collagen fiber alignment that underlie the high mineral and carbonate content. Sub-peak analysis of the amide I band revealed that the mature:immature crosslink ratio was significantly higher in Dmp1Cre.EfnB2f/f bones compared to w/w controls. Altered collagen organization was confirmed through polarization studies which demonstrated more heterogeneous distribution of amide I and II throughout the cortical bone in Dmp1Cre.EfnB2f/f bones.
These data indicate that the brittle bone phenotype presents higher mineral deposition and carbonate incorporation, but also an altered collagen organization. We also show that FT-IR microscopy allows investigating the changes in bone composition, thus complementing standard histological techniques.