Remnant Woven Bone and Calcified Cartilage in Mouse Bone: Differences between Ages/Sex and Effects on Bone Strength.

INTRODUCTION:Mouse models are used frequently to study effects of bone diseases and genetic determinates of bone strength. Murine bones have an intracortical band of woven bone that is not present in human bones. This band is not obvious under brightfield imaging and not typically analyzed. Due to the band's morphology and location it has been theorized to be remnant bone from early in life. Furthermore, lamellar and woven bone are well known to have differing mechanical strengths. The purpose of this study was to determine (i) if the band is from early life and (ii) if the woven bone or calcified cartilage contained within the band affect whole bone strength. WOVEN BONE ORIGIN STUDIES:In twelve to fourteen week old mice, doxycycline was used to label bone formed prior to 3 weeks old. Doxycycline labeling and woven bone patterns on contralateral femora matched well and encompassed an almost identical cross-sectional area. Also, we highlight for the first time in mice the presence of calcified cartilage exclusively within the band. However, calcified cartilage could not be identified on high resolution cone-beam microCT scans when examined visually or by thresholding methods. MECHANICAL STRENGTH STUDIES:Subsequently, three-point bending was used to analyze the effects of woven bone and calcified cartilage on whole bone mechanics in a cohort of male and female six and 13 week old Balb/C mice. Three-point bending outcomes were correlated with structural and compositional measures using multivariate linear regression. Woven bone composed a higher percent of young bones than older bones. However, calcified cartilage in older bones was twice that of younger bones, which was similar when normalized by area. Area and/or tissue mineral density accounted for >75% of variation for most strength outcomes. Percent calcified cartilage added significant predictive power to maximal force and bending stress. Calcified cartilage and woven bone could have more influence in genetic models where calcified cartilage percent is double our highest value

Calcified cartilage in 6 and 13 week old male and female Balb/C mice.

<p>Calcified cartilage at the femur mid-shaft was significantly higher in 13 week old animals than 6week old animals and higher in males than females. However, when normalized by cortical area the percent calcified cartilage was similar between sexes of one age but remained higher in 13week animals.</p

Three point bending correlative results.

<p>Three point bending correlative results.</p

Comparison of early bone formation and ICWB in 12-14wk old mice.

<p>Bone formed up to 3 wks old (green signal, A) was similar in pattern and amount as woven bone (B) in the contralateral limb (C, n = 5).</p

Comparison of ICWB visualization with standard histological stains and brightfield imaging versus polarized imaging.

<p>Two serial sections from the mid-shaft of a 13-week-old male mouse were stained with either H&E (A) or picrosirius red and alcian blue (B & C). (A) Under brightfield imaging neither the ICWB nor calcified cartilage is easily differentiated from surrounding lamellar bone using an H&E stain. (B) Alcian blue staining allows visualization of calcified cartilage, but the ICWB is still not obvious. (C) Only under polarized light imaging can the ICWB (white dashed lines) be differentiated.</p

Example of DOX bone labeling in 12-14wk old mice with different administration times.

<p>Doxycycline (DOX) is a fluorescent tetracycline that can be administered to animals via drinking water and incorporates into actively mineralizing tissues. (A) Animals never exposed to DOX show no fluorescence even when imaged with a lengthy exposure (1000ms, no gain). (B) Animals exposed to DOX <i>in utero</i> until 3 weeks old have an intracortical band of labeled bone (exposure 500ms, no gain). (C) If DOX was administered until 8 weeks old, almost the entirety of the bone is labeled (exposure 500ms, no gain).</p

Example mouse femur histological cross section demonstrates calcified cartilage presence.

<p>(A-C) An intracortical band of woven bone (ICWB) apposed by lamellar bone was present in all samples of both studies. (A’-C’) Calcified cartilage (blue tissue, black arrow heads) were present in all samples. (D) Calcified cartilage resided exclusively within ICWB. None could be found in lamellar bone.</p

Example mouse femur histological cross section compared to high resolution microCT.

<p>(A) Calcified cartilage, which is reportedly hyper mineralized compared to surrounding bone, is easily visible in histological sections stained with alcian blue and picrosirius red (blue tissue). (B-F) However, even with high-resolution cone-beam microCT (3.5um voxel), calcified cartilage cannot be distinguished from surrounding bone via mineral density thresholding. Thus, cone-beam microCT at 3.5um resolution cannot be used to quantify calcified cartilage.</p

Woven Bone and Calcified Cartilage Outcomes at the Femur mid-shaft in Balb/C Mice.

<p>(A) Absolute WB.A was the same between age groups of each sex with males tending to be higher than females (ANOVA, p = 0.042). (B) However, when normalized by Ct.A, percent WB was significantly higher in the 6 week old mice than 13 week olds and there were no differences attributable to sex. (C) In contrast, the absolute amount of CC differed between ages and sex with older having twice that of younger and males having more than female. (D) When normalized by Ct.A it remained elevated in older animals but was not significant (ANOVA, p = 0.0581). (E) When normalized by WB.A, percent CC.A was significantly higher in 13week old mice than 6 week old mice.</p