Detecting microdamage in bone

Abstract

Fatigue-induced microdamage in bone contributes to stress and fragility fractures and acts as a stimulus for bone remodelling. Detecting such microdamage is difficult as pre-existing microdamage sustained in vivo must be differentiated from artefactual damage incurred during specimen preparation. This was addressed by bulk staining specimens in alcohol-soluble basic fuchsin dye, but cutting and grinding them in an aqueous medium. Nonetheless, some artefactual cracks are partially stained and careful observation under transmitted light, or epifluorescence microscopy, is required. Fuchsin lodges in cracks, but is not site-specific. Cracks are discontinuities in the calcium-rich bone matrix and chelating agents, which bind calcium, can selectively label them. Oxytetracycline, alizarin complexone, calcein, calcein blue and xylenol orange all selectively bind microcracks and, as they fluoresce at different wavelengths and colours, can be used in sequence to label microcrack growth. New agents that only fluoresce when involved in a chelate are currently being developed--fluorescent photoinduced electron transfer (PET) sensors. Such agents enable microdamage to be quantified and crack growth to be measured and are useful histological tools in providing data for modelling the material behaviour of bone. However, a non-invasive method is needed to measure microdamage in patients. Micro-CT is being studied and initial work with iodine dyes linked to a chelating group has shown some promise. In the long term, it is hoped that repeated measurements can be made at critical sites and microdamage accumulation monitored. Quantification of microdamage, together with bone mass measurements, will help in predicting and preventing bone fracture failure in patients with osteoporosis.

Fig. 1

Fuchsin-stained microcrack. Scale bar = 50 µm.

Fig. 2

Fuchsin-stained microcrack viewed using green epifluorescence (546 nm). Scale bar = 50 µm.

Fig. 3

Periosteal fibrolamellar bone formation sequentially labelled with chelating fluorochromes: oxytetracycline (yellow), alizarin complexone (red) and calcein (green). UV epifluorescence, 365 nm. Scale bar = 100 µm.

Fig. 4

Microcracks labelled with (a) calcein blue (UV epifluorescence, 365 nm), (b) alizarin complexone (green epifluorescence, 546 nm) and (c) oxytetracycline (UV epifluorescence, 365 nm). Scale bar = 50 µm.

Fig. 5

Fracture surface sequentially labelled with alizarin complexone (red), calcein (green) and oxytetracycline (yellow). UV epifluorescence, 365 nm. Scale bar = 100 µm.

Fig. 6

Three-iodinated contrast agent with one binding site.

Fig. 7

Micro-CT of bone specimen of cross-section 2 mm × 2 mm. Scratch labelled with iodinated contrast agent shown in Fig. 6.