T. C. Lee

Detecting microdamage in bone

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.

Bone adaptation to load: microdamage as a stimulus for bone remodelling

Mechanical loading in the proximal radius was increased by ulnar osteotomy (Group O), altered by Steinmann pinning (Group P) or unaltered in sham operated controls (Group C) in skeletally mature female sheep, aged 2–4 years. A series of intravenous fluorochromes were given to label bone formation and fuchsin‐stained microdamage assessed at intervals of up to 24 weeks. Microcracks were present in all groups and were found in the original cortex near the periosteal surface. No microcracks were found in the new, fibrolamellar bone laid down at periosteal or endosteal surfaces. Mean microcrack length (49 µm, SD 10 µm) did not differ between groups or over time. Microcrack numerical and surface densities and resorption cavity density peaked in all groups at 6 weeks, consistent with a regional acceleratory phenomenon (RAP), but the peaks were significantly greater in Group O. The density of refilling or secondary osteons peaked at 10 weeks and the mean time required for the formation of an osteon was 7.51 ± 0.59 weeks. Fatigue‐induced microdamage is normally present in bone and is increased due to repetitive loading of the mechanically overloaded radius. The location and timing of microcracks, resorption cavities and secondary osteons are consistent with the activation‐resorption‐formation remodelling cycle and suggest that microdamage is a stimulus for bone remodelling.

Visualisation of three‐dimensional microcracks in compact bone

Microdamage in bone contributes to the loss of bone quality in osteoporosis and is thought to play a major role in both fragility and stress fractures (Schaffler et al. 1995). In this study, in vivo microcracks in human ribs were bulk‐stained in basic fuchsin and viewed in longitudinal section and in 3 dimensions using 2 different computer‐based methods of reconstruction: (1) serial sectioning of methylmethacrylate embedded sections using a sledge macrotome and identification of microcracks using UV epifluorescence followed by computerised reconstruction of microcracks using software and (2) laser scanning confocal microscopy of thick sections followed by reconstruction of microcracks into a 3‐D image. The size and shape of microcracks were found to be similar using both techniques. Both techniques of reconstruction showed microcracks to be approximately elliptical in shape. From the serial sectioning reconstructions (n = 9), microcracks were found to have a mean length of 404±145 μm (mean±S.D.) (in the longitudinal direction) and mean width of 97±38 μm (in the transverse direction). Using epifluorescence microscopy, 92 microcracks were identified; mean microcrack length was 349±100 μm in the longitudinal direction. This was consistent with other results (Burr & Martin, 1993) and with the theoretical prediction of an elliptical crack shape with aspect ratio (longitudinal∶transverse) of 5∶1 deduced from analysis of random 2‐D sections (Taylor & Lee, 1998). The results obtained provide new data on the nature of microcracks in bone and the method has the potential to become a useful tool in the calculation of stress intensity values which indicate the probability of an individual microcrack propagating to cause a stress or fragility fracture.

Fluorescence-aided detection of microdamage in compact bone

En bloc staining with basic fuchsin is an established method for demonstrating microdamage in bone. Using transmitted light microscopy, variations in light intensity, depth of focus and magnification are necessary to distinguish fully‐stained microcracks generated in vivo, from partially‐stained or unstained artefactual cracks due to cutting and machining. This process is both difficult and time‐consuming. In this study, 2 methods were used to examine fuchsin‐stained microcracks in human rib sections, transmitted light and epifluorescence microscopy. No differences were found in crack number, density or length between the 2 methods indicating comparable accuracy. Using green epifluorescence, only microcracks containing fuchsin fluoresced orange against the darkfield background, enabling unstained, artefactual cracks to be screened out. Under UV epifluorescence, microcracks stained through the full 100 μm depth of the section fluoresced purple. Partially‐stained artefactual cracks failed to fluoresce and were screened out. Epifluorescence is a simple, rapid and accurate screening method for differentiating fully‐stained from artefactual microcracks in bone.

Microdamage and mechanical behaviour: predicting failure and remodelling in compact bone.

This paper reports on the development of a theoretical model to simulate the growth and repair of microdamage in bone. Unlike previous theories, which use simplified descriptions of damage, this approach models each individual microcrack explicitly, and also models the basic multicellular units (BMUs) that repair cracks. A computer simulation has been developed that is capable of making a variety of predictions. Firstly, we can predict the mechanical behaviour of dead bone in laboratory experiments, including estimates of the number of cycles to failure and the number and length of microcracks during fatigue tests. Secondly, we can predict the results of bone histomorphometry, including such parameters as BMU activation rates and the changing ratio of primary to secondary bone during ageing. Thirdly, we can predict the occurrence of stress fractures in living bone: these occur when the severity of loading is so great that cracks grow faster than they can be repaired. Finally, we can predict the phenomenon of adaptation, in which bone is deposited to increase cortical thickness and thus prevent stress fractures. In all cases results compare favourably with experimental and clinical data.

K-Wire position in tension band wiring of the Olecranon — A comparison of two techniques

Tension band wiring is a recognised standard treatment for olecranon fractures. We studied the effect of K-wire position on backing out of the wire in a group of 80 patients with closed transverse olecranon fractures with a minimum follow-up time of 9 months. The rate of wires backing out as seen on X-ray was three times greater in patients who had K-wires passed down the long axis of the ulna rather than across the anterior cortex as recommended by the AO group. There was a corresponding higher rate of local complications in these patients. 42% of this group had to have the metal removed compared with 11.4% of the transcortical group. We compared the biomechanical properties of both K-wires positions in a human cadaveric model. The maximum pull-out strength for each configuration was recorded in 20 elbow joints. The average maximum pullout strength for the intramedullary wires was 56.3 N (range 27. 7-95.6 N) and 122.7 N for the transcortical wires (range 56.7-201.2). The results of both the clinical study and biomechanical data support the routine use of transcortical placement of K-wires in tension-band wiring of transverse olecranon fractures.

Osteonal crack barriers in ovine compact bone

Bone is an anisotropic structure which can be compared to a composite material. Discontinuities within its microstructure may provide stress concentration sites for crack initiation, but act as a barrier to its propagation. This study looks specifically at the relationship between crack length and propagation in compact bone. Beam‐shaped bone samples from sheep radii were prepared and stained with fluorochrome dyes and tested in cyclic fatigue under four‐point bending in an INSTRON 1341 servo‐hydraulic fatigue‐testing machine. Samples were tested at a frequency of 30 Hz and stress range of 100 MPa under load control. Specimens were sectioned transversely using a diamond saw, slides prepared and examined using epifluorescence microscopy. Cracks in transverse sections were classified in terms of their location relative to cement lines surrounding secondary osteons. Mean crack length, crack numerical density and crack surface density were examined. Short microcracks (100 µm or less) were stopped at the cement lines surrounding osteons, microcracks of intermediate length (100–300 µm) were deflected as they hit the cement line, and microcracks that were able to penetrate through cement lines were longer (> 400 µm). These data show that bone microstructure allows the initiation of microcracks but acts as a barrier to crack propagation.

Biomechanical properties across trabeculae from the proximal femur of normal and ovariectomised sheep.

The elastic behaviour of trabecular bone is a function not only of bone volume and architecture, but also of tissue material properties. Variation in tissue modulus can have a substantial effect on the biomechanical properties of trabecular bone. However, the nature of tissue property variation within a single trabecula is poorly understood. This study uses nanoindentation to determine the mechanical properties of bone tissue in individual trabeculae. Using an ovariectomised ovine model, the modulus and hardness distribution across trabeculae were measured. In both normal and ovariectomised bone, the modulus and hardness were found to increase towards the core of the trabeculae. Across the width of the trabeculae, the modulus was significantly less in the ovariectomised bone than in the control bone. However, in contrast to this hardness was found not to differ significantly between the two groups. This study provides valuable information on the variation of mechanical material properties in healthy and diseased trabecular bone tissue. The results of the current study will be useful in finite element modelling where more accurate values of trabecular bone modulus will enable the prediction of the macroscale behaviour of trabecular bone.

The fatigue strength of compact bone in torsion

We have conducted a series of fatigue tests on samples of bovine compact bone loaded in cyclic torsion. The fatigue strength (i.e. the range of stress needed to cause failure in a given number of cycles) was found to be lower than the fatigue strength of the same material in compression by more than a factor of two. We also tested intact chicken metatarsals and found a similar reduction in strength compared to compression testing of chicken tibiae. These results were predicted using a theoretical model in which fatigue failure was assumed to be dependent on the growth of microcracks, oriented approximately parallel to the bones longitudinal axis but having misorientation angles of up to 30 degrees. An effective stress range was derived which is a function of the normal and shear stresses, and thus of the Mode I and Mode II stress intensities experienced by the crack. These results may have important consequences for the understanding of fatigue in bone in vivo; relatively small amounts of longitudinal shear stress, which are often ignored in analysis, may contribute significantly to fatigue failures. This may shed light on the phenomenon of stress fractures and on the need for repair and adaptation in living bone.

Microcracks in compact bone: a three-dimensional view.

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 µm, respectively.