Micro-Brillouin scattering measurements in mature and newly formed bone tissue surrounding an implant

The evolution of implant stability in bone tissue remains difficult to assess because remodeling phenomena at the bone-implant interface are still poorly understood. The characterization of the biomechanical properties of newly formed bone tissue in the vicinity of implants at the microscopic scale is of importance in order to better understand the osseointegration process. The objective of this study is to investigate the potentiality of micro-Brillouin scattering techniques to differentiate mature and newly formed bone elastic properties following a multimodality approach using histological analysis. Coin-shaped Ti-6Al-4V implants were placed in vivo at a distance of 200?μm from rabbit tibia leveled cortical bone surface, leading to an initially empty cavity of 200?μm×4.4?mm. After 7 weeks of implantation, the bone samples were removed, fixed, dehydrated, embedded in methyl methacrylate, and sliced into 190?μm thick sections. Ultrasonic velocity measurements were performed using a micro-Brillouin scattering device within regions of interest (ROIs) of 10?μm diameter. The ROIs were located in newly formed bone tissue (within the 200?μm gap) and in mature bone tissue (in the cortical layer of the bone sample). The same section was then stained for histological analysis of the mineral content of the bone sample. The mean values of the ultrasonic velocities were equal to 4.97×10(-3)?m/s in newly formed bone tissue and 5.31×10(-3)?m/s in mature bone. Analysis of variance (p=2.42×10(-4)) tests revealed significant differences between the two groups of measurements. The standard deviation of the velocities was significantly higher in newly formed bone than in mature bone. Histological observations allow to confirm the accurate locations of the velocity measurements and showed a lower degree of mineralization in newly formed bone than in the mature cortical bone. The higher ultrasonic velocity measured in newly formed bone tissue compared with mature bone might be explained by the higher mineral content in mature bone, which was confirmed by histology. The heterogeneity of biomechanical properties of newly formed bone at the micrometer scale may explain the higher standard deviation of velocity measurements in newly formed bone compared with mature bone. The results demonstrate the feasibility of micro-Brillouin scattering technique to investigate the elastic properties of newly formed bone tissue.     

The use of ultrasound in vivo to determine acute change in the mechanical properties of bone following intense physical activity

The velocity of ultrasound was measured transcutaneously across the patella and tibia in 98 volunteers both before and after running the 26 mile Boston Marathon. Absolute sound velocities were 2.9% higher in those runners finishing before 3 h when compared to runners finishing after 3 h. Tibial velocities in males were 8.8% higher than in female runners. The mean velocity across the patella of three wheelchair racers was 28% lower than the mean combined patella velocity measured in all runners. These data suggest that 'faster' velocities are associated with bone that is better suited for high functional demands. Surprisingly, when pre- and post-race velocities were compared in each runner, there was a 1.6% increase in ultrasonic velocity across the tibia, and a 3.5% increase across the patella. An increase in ultrasonic velocity following extreme physical activity suggests that adaptive mechanisms exist in healthy bone to withstand, or possibly avoid, the microdamage which might be caused by repetitive cyclic loading.     

Noninvasive measurement of bone mineral mass in the unanesthetized rabbit.

Direct photon absorptiometry was used for the in vivo measurement of bone mineral mass in the rabbit tibia. Bone mineral mass in 124 male and female rabbits was correlated (p less than 0.001) with age to 26 weeks and weight to 5.5 kg. Examining reproducibility and repositioning error, three rabbits were measured 12 times each. Bone mineral mass in the three rabbits was: 0.432 +/- 0.020 g/cm2, 0.414 +/- 0.010 g/cm2 and 0.487 +/- 0.037 g/cm2. The greatest coefficients of variance observed for measurements without repositioning and measurements with repositioning were 4.8% and 7.6%, respectively. The technique provided sensitive, reproducible and noninvasive measurement of bone mineral mass without anesthesia or undue restraint.     

Theory of longitudinal plasma waves with allowance for ion motion

A study is made of the propagation of steady-state large-amplitude longitudinal plasma waves in a cold collisionless plasma with allowance for both electron and ion motion. Conditions for the existence of periodic potential waves are determined. The electric field, potential, frequency, and wavelength are obtained as functions of the wave phase velocity and ion-to-electron mass ratio. Taking into account the ion motion results in the nonmonotonic dependence of the frequency of the waves with the maximum possible amplitudes on the wave phase velocity. Specifically, at low phase velocities, the frequency is equal to the electron plasma frequency for linear waves. As the phase velocity increases, the frequency first decreases insignificantly, reaches its minimum value, and then increases. As the phase velocity increases further, the frequency continues to increase and, at relativistic phase velocities, again becomes equal to the plasma frequency. Finally, as the phase velocity approaches the speed of light, the frequency increases without bound.     

Direct in vivo strain measurements in human bone-a systematic literature review

Bone strain is the governing stimuli for the remodeling process necessary in the maintenance of bone's structure and mechanical strength. Strain gages are the gold standard and workhorses of human bone experimental strain analysis in vivo. The objective of this systematic literature review is to provide an overview for direct in vivo human bone strain measurement studies and place the strain results within context of current theories of bone remodeling (i.e. mechanostat theory). We employed a standardized search strategy without imposing any time restriction to find English language studies indexed in PubMed and Web of Science databases that measured human bone strain in vivo. Twenty-four studies met our final inclusion criteria. Seven human bones were subjected to strain measurements in vivo including medial tibia, second metatarsal, calcaneus, proximal femur, distal radius, lamina of vertebra and dental alveolar. Peak strain magnitude recorded was 9096 με on the medial tibia during basketball rebounding and the peak strain rate magnitude was -85,500 με/s recorded at the distal radius during forward fall from standing, landing on extended hands. The tibia was the most exposed site for in vivo strain measurements due to accessibility and being a common pathologic site of stress fracture in the lower extremity. This systematic review revealed that most of the strains measured in vivo in different bones were generally within the physiological loading zone defined by the mechanostat theory, which implies stimulation of functional adaptation necessary to maintain bone mechanical integrity.     

A full body musculoskeletal model based on flexible multibody simulation approach utilised in bone strain analysis during human locomotion

Load-induced strains applied to bone can stimulate its development and adaptation. In order to quantify the incident strains within the skeleton, in vivo implementation of strain gauges on the surfaces of bone is typically used. However, in vivo strain measurements require invasive methodology that is challenging and limited to certain regions of superficial bones only such as the anterior surface of the tibia. Based on our previous study [Al Nazer et al. (2008) J Biomech. 41:1036-1043], an alternative numerical approach to analyse in vivo strains based on the flexible multibody simulation approach was proposed. The purpose of this study was to extend the idea of using the flexible multibody approach in the analysis of bone strains during physical activity through integrating the magnetic resonance imaging (MRI) technique within the framework. In order to investigate the reliability and validity of the proposed approach, a three-dimensional full body musculoskeletal model with a flexible tibia was used as a demonstration example. The model was used in a forward dynamics simulation in order to predict the tibial strains during walking on a level exercise. The flexible tibial model was developed using the actual geometry of human tibia, which was obtained from three-dimensional reconstruction of MRI. Motion capture data obtained from walking at constant velocity were used to drive the model during the inverse dynamics simulation in order to teach the muscles to reproduce the motion in the forward dynamics simulation. Based on the agreement between the literature-based in vivo strain measurements and the simulated strain results, it can be concluded that the flexible multibody approach enables reasonable predictions of bone strain in response to dynamic loading. The information obtained from the present approach can be useful in clinical applications including devising exercises to prevent bone fragility or to accelerate fracture healing.     

Fat Mass Measured by DXA Varies with Scan Velocity

Objective: To study the influence of scan velocities of DXA on the measured size of fat mass, lean body mass, bone mineral content and density, and total body weight. Research Methods and Procedures: The subjects were 71 healthy white adults, 38 women and 33 men. The mean age was 41.7 ± 13.5 years and body mass index was 28.6 ± 5.6 kg/m². The subjects were scanned consecutively in slow, medium, and fast scan mode by a Lunar DPX-IQ DXA scanner. Results: Throughout the body mass index and sagittal height ranges, scanned lean body mass significantly decreased with higher scan velocity and lean body mass was 2.7% lower in fast than in medium mode (p < 0.0001). In contrast, fat mass, percentage of body fat, and bone mineral contents were higher with increasing scan velocity. Areas not analyzed by the scanner, so called blue spots, increased with scan velocity and sagittal height, and their presence significantly enhanced the error. Body weight estimated by DXA in slow mode was −0.8% lower than scale weight in the women (p < 0.001) and −0.2% in men (not significant), and the difference was greater with increasing scan velocity. Discussion: Scan velocity significantly influences the measured fat mass size, lean body mass, bone mineral content, and body weight. To obtain the most accurate results, slow mode is preferable and fast scans should be avoided. Future studies should report and take scan velocity into consideration.     

Flexural wave propagation in discontinuous model and in vitro tibiae

The change in the characteristics of propagating flexural waves due to the introduction of a discontinuity in an aluminum beam representing a zero-order model of a fractured human tibia and of an in vitro tibia was investigated. It was found that amplitudes of the transmitted waves decreased linearly, while reflected waves increased with the depth of cuts simulating a partial fracture for both types of specimen above a certain threshold. Completely severed segments reglued by silicone rubber transmitted only small-amplitude and low-frequency flexural waves. The experimental data for the beam tests generally agreed with the results of a corresponding finite element analysis based on the Euler-Bernoulli equation, although some of the important details were absent in the prediction.

Transverse impact tests were also performed on in vitro tibiae in an intact state, in a fractured state, and in a condition having a surgical compressional plate mounted across the fracture site. The resultant wave forms were different from those of the beam because of dissimilar geometrical configurations and material properties. These tests provided some basic data that might find application in the possible use of flexural waves as a diagnostic tool for the assessment of the state of healing of fractured long bones.     

Relationships of the group velocity of the time-reversed Lamb wave with bone properties in cortical bone in vitro

The present study aims to investigate the feasibility of using the time-reversed Lamb wave as a new method for noninvasive characterization of long cortical bones. The group velocity of the time-reversed Lamb wave launched by using the modified time reversal method was measured in 15 bovine tibiae, and their correlations with the bone properties of the tibia were examined. The group velocity of the time-reversed Lamb wave showed significant positive correlations with the bone properties (r = 0.55–0.81). The best univariate predictor of the group velocity of the time-reversed Lamb wave was the cortical thickness, yielding an adjusted squared correlation coefficient (r²) of 0.64. These results imply that the group velocity of the time-reversed Lamb wave, in addition to the velocities of the first arriving signal and the slow guided wave, could potentially be used as a discriminator for osteoporosis.     

Fatigue evaluation of long cortical bone using ultrasonic guided waves

Bone fatigue fracture is a progressive disease due to stress concentration. This study aims to evaluate the long bone fatigue damage using the ultrasonic guided waves. Two-dimensional finite-difference time-domain method was employed to simulate the ultrasonic guided wave propagation in the long bone under different elastic modulus. The experiment was conducted on a 3.8 mm-thick bovine bone plate. The phase velocities of two fundamental guided modes, A1 and S1, were measured by using the axial transmission technique. Simulation shows that the phase velocities of guided modes A1 and S1 decrease with the increasing of the fatigue damage. After 20,000 cycles of fatigue loading on the bone plate, the average phase velocities of A1 and S1 modes were 6.6% and 5.3% respectively, lower than those of the intact bone. The study suggests that ultrasonic guided waves can be potentially used to evaluate the fatigue damage in long bones.     

Piezoelectricity in tendon and bone

We confirm that the electrical signals produced by samples of dried bone and tendon bent in the cantilever mode are d.c. square waves in response to step loading. The signals are similar to those produced by piezoelectric mineral crystals also tested, but with an important exception: the sign of the voltage reverses on end-for-end rotation of the mineral samples but does not change for bone and tendon. We deduce a theoretical expression for the expected signal using the standard theory of piezoelectricity and the contiuum theory of elasticity. The derived expression correctly describes the experimental results for the mineral samples but fails for bone and tendon.

We then extend the standard theory of piezoelectricity by including a term relating the polarization to the gradient of the stress via a tensor of rank four. This extension leads to agreement with experiment.     

Hindlimb unloading has a greater effect on cortical compared with cancellous bone in mature female rats.

This study was designed to determine the effects of 28 days of hindlimb unloading (HU) on the mature female rat skeleton. In vivo proximal tibia bone mineral density and geometry of HU and cage control (CC) rats were measured with peripheral quantitative computed tomography (pQCT) on days 0 and 28. Postmortem pQCT, histomorphometry, and mechanical testing were performed on tibiae and femora. After 28 days, HU animals had significantly higher daily food consumption (+39%) and lower serum estradiol levels (-49%, P = 0.079) compared with CC. Proximal tibia bone mineral content and cortical bone area significantly declined over 28 days in HU animals (-4.0 and 4.8%, respectively), whereas total and cancellous bone mineral densities were unchanged. HU animals had lower cortical bone formation rates and mineralizing surface at tibial midshaft, whereas differences in similar properties were not detected in cancellous bone of the distal femur. These results suggest that cortical bone, rather than cancellous bone, is more prominently affected by unloading in skeletally mature retired breeder female rats.     

A method for measurement of joint kinematics in vivo by registration of 3-D geometric models with cine phase contrast magnetic resonance imaging data

A new method is presented for measuring joint kinematics by optimally matching modeled trajectories of geometric surface models of bones with cine phase contrast (cine-PC) magnetic resonance imaging data. The incorporation of the geometric bone models (GBMs) allows computation of kinematics based on coordinate systems placed relative to full 3-D anatomy, as well as quantification of changes in articular contact locations and relative velocities during dynamic motion. These capabilities are additional to those of cine-PC based techniques that have been used previously to measure joint kinematics during activity. Cine-PC magnitude and velocity data are collected on a fixed image plane prescribed through a repetitively moved skeletal joint. The intersection of each GBM with a simulated image plane is calculated as the model moves along a computed trajectory, and cine-PC velocity data are sampled from the regions of the velocity images within the area of this intersection. From the sampled velocity data, the instantaneous linear and angular velocities of a coordinate system fixed to the GBM are estimated, and integration of the linear and angular velocities is used to predict updated trajectories. A moving validation phantom that produces motions and velocity data similar to those observed in an experiment on human knee kinematics was designed. This phantom was used to assess cine-PC rigid body tracking performance by comparing the kinematics of the phantom measured by this method to similar measurements made using a magnetic tracking system. Average differences between the two methods were measured as 2.82 mm rms for anterior/posterior tibial position, and 2.63 deg rms for axial rotation. An intertrial repeatability study of human knee kinematics using the new method produced rms differences in anterior/posterior tibial position and axial rotation of 1.44 mm and 2.35 deg. The performance of the method is concluded to be sufficient for the effective study of kinematic changes caused to knees by soft tissue injuries.     

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.     

Noninvasive determination of ulnar stiffness from mechanical response--in vivo comparison of stiffness and bone mineral content in humans

An approach referred to as Mechanical Response Tissue Analysis (MRTA) has been developed for the noninvasive determination of mechanical properties of the constituents of the intact limb. Of specific interest in the present study is the bending stiffness of the ulna. The point mechanical impedance properties in the low frequency regime, between 60 and 1,600 Hz are used. The procedure requires a proper design of the probe for good contact of the skin at midshaft and proper support of the proximal and distal ends of the forearm to obtain an approximation to "simple support" of the ulna. A seven-parameter model for the mechanical response is then valid, which includes the first mode of anterior-posterior beam bending of the ulna, the damping and spring effect of the soft tissue between probe and bone, and the damping of musculature. A dynamic analyzer (HP3562A) provides in seconds the impedance curve and the pole-zero curve fit. The physical parameters are obtained from a closed-form solution in terms of the curve-fit parameters. The procedure is automated and is robust and analytically reliable at about the five percent level. Some 80 human subjects have been evaluated by this mechanical response system and by the Norland single photon absorptiometer, providing for the first time in vivo, a comparison of elastic bending stiffness (ulna) and bone mineral content (radius). Three functional parameters of potential clinical value are the cross-sectional bending stiffness EI, the axial load capability Pcr (Euler buckling load) and the bone "sufficiency" S, defined as the ratio of Pcr to body weight. The correlation between EI and bone mineral (r = 0.81) is only slightly less than previous in vitro results with both measurements on the same bone (r = 0.89). When sufficiency is taken into consideration, the correlation of Pcr and bone mineral content is improved (r = 0.89). An implication is that "quality" of bone is a factor which is not indicated by bone mineral content but which is indicated by stiffness. Bone mineral is necessary for proper stiffness but not sufficient. Therefore mechanical measurement should provide a new dimension to be used toward a better understanding of the factors related to bone health and disease.     

Effects of dietary calcium and phosphorus deficiency and subsequent recovery on broiler chicken growth performance and bone characteristics

The ability of birds to modify dietary phosphorus utilisation when fed with low-phosphorus and calcium (Ca) diets was studied using different sequences of dietary phosphorus and Ca restriction (depletion) and recovery (repletion) during the grower and the finisher phases. A total of 3600 Ross 708 broilers were randomly divided into 10 replicate pens per treatment (60 per pen, six pens per block). Chicks were fed a common starter diet from days 0 to 10, then a grower control diet (C: 0.90% Ca, 0.39% non-phytate phosphorus, nPP), mid-level diet (M: 0.71% Ca, 0.35% nPP) or low Ca and nPP diet (L: 0.60% Ca, 0.30% nPP) from days 11 to 21, followed by a finisher diet C, M or L containing, respectively, 0.85%, 0.57% or 0.48% Ca and 0.35%, 0.29% or 0.24% nPP from days 22 to 37. Six treatment sequences were tested: CC, MM, LL, ML, LC and LM. Bone mineral content by dual-energy X-ray, tibia ash, toe ash weight and tibia breaking strength were measured on days 21 and 37. No significant effect was observed on growth performance throughout the experiment. Diet L reduced bone mineral content, breaking strength, tibia and toe ash by 9%, 13%, 11% and 10%, respectively, on day 21 (compared with diet C, for linear effect, P<0.05). On day 37, bone mineral content, breaking strength, tibia and toe ash remained lower compared with control values (CC v. MM v. LL, P<0.05 for linear and quadratic effects). Mineral depletion duration (ML v. LL) did not affect bone mineral status. Replenishing with the C diet during the finisher phase (LC) restored bone mineral content, tibia ash and toe ash weight better than the M diet did, but not to control levels (CC v. LC v. LM, for linear effect, P<0.05). These results confirm that dietary Ca and nPP may be reduced in the grower phase without affecting final growth performance or breaking strength as long as the finisher diet contains sufficient Ca and nPP. The practical applications of this strategy require further study in order to optimise the depletion and repletion steps.     

Disproportional skeletal growth and markedly decreased bone mineral content in growth hormone receptor -/- mice

Growth hormone (GH) is important for skeletal growth as well as for a normal bone metabolism in adults. The skeletal growth and adult bone metabolism was studied in mice with an inactivated growth hormone receptor (GHR) gene. The lengths of femur, tibia, and crown-rump were, as expected, decreased in GHR-/- mice. Unexpectedly, GHR-/- mice displayed disproportional skeletal growth reflected by decreased femur/crown-rump and femur/tibia ratios. GHR-/- mice demonstrated decreased width of the growth plates in the long bones and disturbed ossification of the proximal tibial epiphysis. Furthermore, the area bone mineral density (BMD) as well as the bone mineral content (BMC)/body weight were markedly decreased in GHR-/- mice. The decrease in BMC in GHR-/- mice was not due to decreased trabecular volumetric BMD but to a decreased cross-sectional cortical bone area In conclusion, GHR-/- mice demonstrate disproportional skeletal growth and markedly decreased bone mineral content.     

Attenuation of bone mass and increase of osteoclast formation in decoy receptor 3 transgenic mice

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT, and TL1A, induces osteoclast formation from monocyte, macrophage, and bone stromal marrow cells. However, the function of DcR3 on bone formation remains largely unknown. To understand the function of DcR3 in bone formation in vivo, transgenic mice overexpressing DcR3 were generated. Bone mineral density (BMD) and bone mineral content (BMC) of total body were significantly lower in DcR3 transgenic mice as compared with wild-type controls. The difference in BMD and BMC between DcR3 transgenic and control mice was confirmed by histomorphometric analysis, which showed a 35.7% decrease in trabecular bone volume in DcR3 transgenic mice in comparison with wild-type controls. The number of osteoclasts increased in DcR3 transgenic mice. In addition, local administration of DcR3 (30 microg/ml, 10 microl, once/day) into the metaphysis of the tibia via the implantation of a needle cannula significantly decreased the BMD, BMC, and bone volume of secondary spongiosa in tibia. Local injection of DcR3 also increased osteoclast numbers around trabecular bone in tibia. Furthermore, coadminstration of soluble tumor necrosis factor receptor inhibitor/Fc chimera (TNFRSF1A) but not osteoprotegerin inhibited the action of DcR3. In addition, in an assay of osteoclast activity on substrate plates, DcR3 significantly increased the resorption activity of mature osteoclasts. Treatment with higher concentrations of DcR3 slightly increased nodule formation and alkaline phosphatase activity of primary cultured osteoblasts. These results indicate that DcR3 may play an important role in osteoporosis or other bone diseases.     

A comparison of bone strain measurements at anatomically relevant sites using surface gauges versus strain gauged bone staples

Instrumented bone staples were first introduced as an alternative to surface-mounted strain gauges for use in human in vivo bone strain measurements because their fixation to bone is secure and requires not only minimally invasive surgery. Bench-top bone bending models have shown that the output from strain gauged bone staples compares favorably to that of traditional mounted gauges. However their within- and across-subject performance at sites typically instrumented in vivo has never been examined. This study used seven human cadaver lower extremities with an age range of 23-81 years old and a dynamic gait simulator to examine and compare axial strains in the mid tibial diaphysis and on the dorsal surface of the second metatarsal as measured simultaneously with strain gauged bone staples and with traditional surface-mounted gauges. Rosette configurations were used at the tibial site for deriving principal compression and tension, and shear strains. Axial outputs from the two gauge types demonstrated strong linear relationships for the tibia (r(2)=0.78-0.94) and the second metatarsal (r(2)=0.96-0.99), but coefficients (slopes) for the relationship were variable (range 7-20), across subjects and across sites. The apparent low reliability of strain gauged staples may be explained by the fact that both strain gauged staples and surface strain gauges are inexact to some degree, do not measure strains from exactly the same areas and strain gauged staples reflect surface strains as well as deformations within the cortex. There were no relationships for the principal tibia compression, tension or shear strain measurements derived from the two rosette gauge types, reflecting the very different anatomical areas measured by each of the constructs in this study. Strain gauged bone staples may be most useful in comparing relative axial intra-subject differences between activities, but inter-subject variability may require larger sample sizes to detect differences between populations.     

In vivo fatigue microcracks in human bone: material properties of the surrounding bone matrix

Human bones sustain fatigue damage in the form of in vivo microcracks as a result of the normal everyday loading activities. These microcracks appear to preferentially accumulate in certain regions of bone and most notably in interstitial bone matrix areas. These are remnants of old bone tissue left unremodelled, which show a higher than average mineral content and consequently the occurrence of microcracks has been attributed to the possible brittleness brought about by such hypermineralisation. There is a need, therefore, for information on the in situ bone matrix properties in the vicinity of such in vivo microcracks to elucidate the possible causes of their appearance. The present study examined the elastic, strain rate (viscous) and plastic properties of bone matrix in selectively targeted areas by nanoindentation and in both quasistatic and dynamic mode. The results showed that in vivo crack areas are not as stiff as some well-known extremely mineralised and brittle bone examples (bulla, rostrum); the strain rate effects of crack regions were identical to those of other regions of human bone and agreed well with values collected for human bone in the past at the macroscale; while the plasticity index of the crack regions was also not statistically different from most bone examples (including human at random, bovine, bulla and rostrum) except antler, which showed lower plasticity and thus a greater fraction of elastic recovery in indentation energy. It is difficult, therefore, to explain the susceptibility of these interstitial regions to crack in terms of the mineral content and its after-effects on elasticity, viscosity and plasticity alone, but one need to attribute the cracks to the cumulative loading history of these areas, or raise the suggestion that these areas of bone matrix are in some measure 'aged' or material/quality defective.