Microcracking in bone acts as a stimulus for bone remodelling, contributes to the loss of bone quality in osteoporosis and is thought to play a major role in both fragility and stress fractures. This study seeks to refine a method of crack detection using fluorescent chelating agents and to label microcrack initiation, growth and coalescence during fatigue testing in compression. The three dimensional shape of microcracks and their interaction with the microstructure of bone will also be investigated.

Five fluorescent stains have been identified which chelate calcium ions lining crack walls. However, when applied in sequence, agents displaced each other by chemical substitution, preventing accurate measurement of crack growth. The calcium affinity of each agent was measured using ion chromatography and the agents ranked in descending order:​ alizarin (A), xylenol orange (X), calcein blue (CB), calcein (C) and oxytetracycline (O). This sequence was then tested on polished bovine bone specimens on which surface scratches had been made to mimic microcracks. The quality, clarity and degree of substitution of each agent were measured and concentration and tissue penetration time were varied. The optimal sequence was A - X - C - CB, with a penetration time of 30 minutes and the A, X and C at 0.0005M concentration and CB at 0.0001 M. The chemical behaviour of O was variable and so it was excluded from the study.

Specimens from fresh bovine tibiae were machined into typical, waisted type of circular cross section and fatigue tested in cyclic compression at 3 Hz. They were initially stained with alizarin, to label pre-existing damage, and the other agents applied in sequence during the test. Specimens were then sectioned and examined using UV epifluorescence microscopy. Microcracks accumulated early in a specimen’s life but the rate then levelled off before a second increase in the period prior to failure. Bone micro structure greatly influenced microcrack growth with the 75 - 95% of microcracks found in interstitial bone between Haversian systems. Microcracks grew in length primarily in the longitudinal direction, parallel to Haversian systems and the longitudinal axis of the bone. These results support the concept of a microstructural barrier effect in bone.

Human ribs were stained with basic fuchsin to label in vivo microcracks and viewed in the longitudinal direction and in three dimensions using two different computer-based methods of reconstruction:​ (i) laser scanning confocal microscopy of sections stained in basic fuchsin followed by reconstruction of microcracks into a 3D image and (ii) serial sectioning of methy 1-methacrylate embedded sections with identification of microcracks using UV epifluorescence followed by computerised reconstrucfion individual cracks. Both LSCM and serial sectioning showed similar results regarding the shape and size of microcracks in bone. Microcracks were elliptical in shape with a ratio of longitudinal length:​ transverse width of 4.6:​ 1, consistent with theoretical predictions.