VDR-Null Mice Lack Mineralized Tethers Stabilizing Epiphyseal and Metaphyseal Bone

Objectives: Micro-CT of rat tibial growth plates showed the presence of X-ray opaque structures termed tethers linking the epiphysis and metaphysis and increasing in number with age as the growth plate (GP) becomes thinner. We hypothesized that tethers might provide mechanical stability during long bone growth and the altered chondro-osseous morphology typical of rickets might involve changes in tether number, distribution, or structure. Methods: GPs were isolated from micro-CT scans of right distal femurs from 15 VDR-/- and 15 VDR+/+ mice. Randomly selected GPs were processed for decalcified (8) and undecalcified (1) histomorphometry; serial sagittal sections were stained with H&E or silver nitrate, respectively. Tissue shrinkage following histology processing was calculated. GP width and area were measured (Image-Pro Plus) and GP volume, GP projection area, and tether number, volume and occupancy rate were determined by reconstruction of consecutive histological images. 3D micro-CT images were aligned with histologic images for analysis. Tether number was counted directly, GP and tether projection area were measured (Image-Pro Plus), tether volume was calculated from tether area and average GP thickness. Results: Shrinkage was great in VDR-/- GPs. Tethers occurred in all VDR+/+ GPs by all imaging methods. No tethers were seen in VDR-/- femurs by undecalcified histology; 30% VDR-/- GPs had tethers by micro-CT and these were only in the central region; all VDR-/- femurs had tethers by decalcified histology. Although VDR-/- GPs had greater area and thickness, tether number, total tether area, occupancy rate and volume fraction were reduced. Histology showed that individual tether volume was comparable in VDR-/- and VDR+/+ GPs. Conclusions: Rickets does not affect all regions of the GP equally. Failure of the GP to develop tethers properly has consequences for establishing the appropriate interface between two mechanically dissimilar tissues. Funded by Childrens Healthcare of Atlanta.