Microcracks in compact bone: a three-dimensional view

Abstract

Microcracks have been implicated in the loss of bone quality for osteoporosis. In order to detect and monitor their growth, and to use these data to predict fractures, it is essential to obtain quantitative data regarding their shape in three dimensions. Beam-shaped bone samples from sheep radii were prepared and stained with fluorochrome dyes and tested in cyclical fatigue under four-point bending in a servo-hydraulic fatigue-testing machine. Samples were tested at a frequency of 30 Hz under load control at a stress range of 100 MPa. Holes were drilled into bone samples and used as reference points for reconstructions. A series of thin longitudinal sections were cut using a sledge macrotome. A two-dimensional image of each section was examined using an epifluorescence microscope and images transferred to a PC via a CCD low-light colour video camera. A three-dimensional image of each microcrack was reconstructed using computer software, and its dimensions measured. Cracks were elliptical in shape, longer in the longitudinal direction and with a mean aspect ratio of 5.5 +/- 1.05. The mean (+/- SD) length and width of labelled microcracks were 488 +/- 151 and 88 +/- 21 microm, respectively.

Fig. 1

Beam-shaped specimen. Mean width (w) and height (h) was 7 × 2.6 mm and each specimen was 40 mm long.

Fig. 2

Longitudinal section of fluorochrome-labelled bone viewed with UV epifluorescence (λ = 365 nm). An artefactual microcrack is arrowed. Scale bar = 200 µm.

Fig. 3

Transverse section of fluorochrome-labelled bone viewed with UV epifluorescence (λ = 365 nm) to test the visibility of fluorochromes in the new embedding medium. Calcein was used to stain fatigued bone specimens and has penetrated the bone tissue and labelled canaliculi, lacunae, Haversian canal (H.C) and Volkmann's canals (V.C) connecting two Haversian canals. Alizarin (red arrow) was administered in vivo in an earlier study (Lee & Taylor, 2003). Scale bar = 250 µm.

Fig. 4

Beam-shaped bone specimens were stained with alizarin to label pre-existing microcracks. Alizarin fluoresces red under UV light (λ = 365 nm) and has labelled the pre-existing microcrack (arrow) on the edge of a sectioned Haversian canal. An osteon labelled with alizarin (inner red label) and calcein (outer green label) is also visible. Scale bar = 50 µm.

Fig. 5

Example of calcein-stained oblique microcracks shown (arrows) in a longitudinal section of bone. The drill hole (D), which is used as a reference point for aligning sections, is shown. Slide is viewed under UV light (λ = 365 nm). Scale bar = 100 µm.

Fig. 6

Initial reconstruction of a microcrack and hole (arrow) drilled into the bone specimen. (a) An example of a ‘skeletal’ reconstruction demonstrating alignment of individual sections. (b) Image of the same microcrack and drill hole shown with a ‘skin’ covering placed around it using Surfdiver™.

Fig. 7

(a–d) Stepwise reconstruction of a microcrack rotated at various angles (yellow). The drill hole is also reconstructed as a cylinder (h) and allows understanding of the orientation of the microcrack as it is rotated at various angles.