Correlation of quantitative computed tomographic subchondral bone density and ash density in horses.

The purpose of this study was to compare subchondral bone density obtained using quantitative computed tomography with ash density values from intact equine joints, and to determine if there are measurable anatomic variations in mean subchondral bone density. Five adult equine metacarpophalangeal joints were scanned with computed tomography (CT), disarticulated, and four 1-cm(3) regions of interest (ROI) cut from the distal third metacarpal bone. Bone cubes were ashed, and percent mineralization and ash density were recorded. Three-dimensional models were created of the distal third metacarpal bone from CT images. Four ROIs were measured on the distal aspect of the third metacarpal bone at axial and abaxial sites of the medial and lateral condyles for correlation with ash samples. Overall correlations of mean quantitative CT (QCT) density with ash density (r=0.82) and percent mineralization (r=0.93) were strong. There were significant differences between abaxial and axial ROIs for mean QCT density, percent bone mineralization and ash density (p<0.05). QCT appears to be a good measure of bone density in equine subchondral bone. Additionally, differences existed between axial and abaxial subchondral bone density in the equine distal third metacarpal bone.

Effect of fixation and embedding on Raman spectroscopic analysis of bone tissue.

Raman spectroscopy provides valuable information on the physicochemical properties of hard tissues. While the technique can analyze tissues in their native state, analysis of fixed, embedded, and sectioned specimens may be necessary on certain occasions. The information on the effects of fixatives and embedding media on Raman spectral properties is limited. We examined the effect of ethanol and glycerol as fixatives and a variety of embedding media (Araldite, Eponate, Technovit, glycol methacrylate, polymethyl methacrylate, and LR white) on Raman spectral properties (mineralization, crystallinity, and carbonation) measured from the cortical bone of mouse humeri. Humeri were fixed in ethanol or glycerol, followed by embedding in one of the media. Nonfixed, freeze-dried, and fixed but not embedded sections were also examined. Periosteal, endosteal, and midosteal regions of the intracortical envelope were analyzed. Raman spectra of fixative solutions and embedding media were also recorded separately in order to examine the specifics of overlap between spectra. We found significant effects of fixation, embedding, and anatomical location on Raman spectral properties. The interference of ethanol with tissue seemed to be relatively less pronounced than that of glycerol. However, there was no single combination of fixation and embedding that left Raman spectral parameters unaltered. We conclude that careful selection of a fixation and embedding combination should be made based on the parameter of interest and the type of tissue. It may be necessary to process multiple samples from the tissue, each using a combination appropriate for the Raman parameter in question.

Long-term ovariectomy decreases ovine compact bone viscoelasticity.

Changes in bone mineral density associated with estrogen depletion in humans do not account for all of the associated change in fracture risk, and it is possible that some of this variation may lie in changes of other aspects of bone quality. The purpose of this study was to investigate changes in viscoelastic behavior of compact bone that may be associated with estrogen depletion. Changes in compact bone viscoelastic properties associated with three years of ovariectomy were investigated with dynamic mechanical analysis (low-amplitude 3-point bending at frequencies of 1-20 Hz) using beams milled from the diaphysis of the ovine radius. The viscoelastic storage modulus was significantly (5.2%) lower at the higher frequencies for the ovariectomized animals. The general anatomic variation in storage modulus, in which cranial sectors had higher values than caudal sectors, did not change with ovariectomy. The loss tangent (tandelta, a measure of damping) was also greatly decreased (up to 83%) at high frequencies in the ovariectomized animals. Anatomic variation in tandelta at low (6-12 Hz) frequencies (cranial and caudal sectors having higher values than lateral or medial sectors) was enhanced with ovariectomy. Changes in viscoelastic properties associated with long-term estrogen depletion could be responsible for a significant reduction in the toughness or strength of a bone without concomitant changes in screening modalities used to evaluate bone quality (e.g., DXA, QCT, QUA).

Matrix concentration of insulin-like growth factor I (IGF-I) is negatively associated with biomechanical properties of human tibial cancellous bone within individual subjects.

Insulin-like growth factor-I (IGF-I), abundant in bone matrix, is believed to play an important role during bone development and remodeling. To our knowledge, however, few studies have addressed the relationship between the concentration of IGF-I in bone matrix and the biomechanical properties of bone tissue. In this study, forty-five cylindrical specimens of cancellous bone were harvested from six human tibiae and scanned using micro-computed tomography (microCT). The bone volume fraction (BV/TV) was calculated from three-dimensional (3D) microCT images. Mechanical tests were then performed on a servohydraulic testing system to determine the strength and stiffness of cancellous bone. Following mechanical testing, the concentration of IGF-I in bone matrix was measured by using an enzyme-linked immunoabsorbent assay (ELISA). Within each subject, the concentration of IGF-I in bone matrix had significant (P<0.01) negative correlations with the bone volume fraction, strength, and stiffness of cancellous bone. In particular, the anterior quadrant of the proximal tibia was significantly (P<0.02) greater in IGF-I matrix concentration and marginally significantly lower in strength (P=0.053) and stiffness (P=0.059) than the posterior quadrant. The negative correlations between the cancellous bone matrix concentration of IGF-I and cancellous bone biomechanical properties within subjects found in this study may help us understand the variation of the biomechanical properties of cancellous bone in proximal human tibiae.

Dietary-induced metabolic acidosis decreases bone mineral density in mature ovariectomized ewes.

Dietary-induced metabolic acidosis (DIMA) may be a significant confounder in the development of osteoporosis. Diets that are acidifying are typically rich in proteins and grains and relatively poor in fruits and vegetables. Previous studies have not examined whether an interaction between estrogen depletion and DIMA have a compounded affect on bone mineral density loss. Sheep have been used successfully in previous studies to examine the interaction of bone turnover and ovariectomy. Therefore, the goal of this pilot study was to determine if bone mineral density (BMD) loss could be induced using DIMA in skeletally mature ovariectomized (OVX) ewes.

Determinants of ovine compact bone viscoelastic properties: effects of architecture, mineralization, and remodeling.

Significant decreases in ovine compact bone viscoelastic properties (specifically, stress-rate sensitivity, and damping efficiency) are associated with three years of ovariectomy and are particularly evident at higher frequencies [Proc. Orthop. Res. Soc. 27 (2002) 89]. It is unclear what materials or architectural features of bone are responsible for either the viscoelastic properties themselves, or for the changes in those properties that were observed with estrogen depletion. In this study, we examined the relationship between these viscoelastic mechanical properties and features involving bone architecture (BV/TV), materials parameters (ash density, %mineralization), and histologic evidence of remodeling (%remodeled, cement line interface). The extent of mineralization was inversely proportional to the material's efficiency in damping stress oscillations. The damping characteristics of bone material from ovariectomized animals were significantly more sensitive to variation in mineralization than was bone from control animals. At low frequencies (6 Hz or less), increased histologic evidence of remodeling was positively correlated with increased damping efficiency. However, the dramatic decreases in stress-rate sensitivity that accompanied 3-year ovariectomy were seen throughout the bone structure and occurred even in areas with little or no secondary Haversian remodeling as well as in areas of complete remodeling. Taken together, these data suggest that, while the mineral component may modify the viscoelastic behavior of bone, the basic mechanism underlying bone viscoelastic behavior, and of the changes in that behavior with estrogen depletion, reside in a non-mineral component of the bone that can be significantly altered in the absence of secondary remodeling.

Age-related morphometry of equine calcified cartilage.

Although there are many studies in the equine literature focused on articular diseases and the aetiology of osteoarthritis, few have concentrated on normal articular structures and how they change with age. The objective of this investigation was to study the thickness and morphology of the calcified cartilage layer of the distal metacarpus over a range of ages. A parasagittal slab of bone was sectioned from the region of sesamoid contact on the medial condyle of the metacarpi from 34 horses. The slab of bone was preserved, dehydrated and embedded, undecalcified, in methylmethacrylate and then stained with toluidine blue. Six repeatable fields of interest from the distal aspect of each metacarpus were digitised and examined to determine the morphology of the calcified cartilage layer. The thickness of the calcified cartilage, range 88-426 microm, was estimated using a method of integration. The results indicate an age-related influence on the thickness of the calcified cartilage layer, generally increased in older horses. While this finding is significant, perhaps more importantly a positional relationship was also identified, indicating that pressures endured by different regions within a joint may dictate morphological development of the tissues. This study has begun to lay the groundwork to determine whether the calcified layer of the hyaline cartilage could be involved in the development of osteoarthritis.

Stiff and strong compressive properties are associated with brittle post-yield behavior in equine compact bone material.

Our hypothesis was that post-yield mechanical behavior of compact bone material in compression, defined as the stress, strain, or energy absorbed between 0.2% strain-offset and the point of maximum stress, is correlated with material density, modulus, strength, histomorphometric evidence of remodeling, and post-failure gross specimen morphology. Post-yield behavior of compact bone material from the third metacarpal bone of 10 horses, ages 5 months to 20 years, was investigated using single-load compression-to-failure. The post-yield stress, strain, and absorbed energy were compared with the compressive elastic modulus, yield stress, ash density. post-failure macroscopic appearance of the specimen, and histologic evidence of remodeling. High values of elastic modulus, yield stress, and ash density were associated with low values of post-yield mechanical properties (stress, strain, and absorbed energy). Macroscopic post-failure morphology was associated with post-yield mechanical behavior, in that specimens displaying fractures were associated with lower post-yield mechanical properties, and that those without evidence of frank fracture were associated with higher post-yield mechanical properties. Microscopic evidence of remodeling activity was associated with high post-yield mechanical properties, but not with gross post-failure morphology. There was an abrupt change from relatively high values to extremely low values of post-yield mechanical properties at intermediate levels of ash density. This feature may serve as a functional tipper limit to the maximization of bone material stiffness and strength.

The X-ray attenuation characteristics and density of human calcaneal marrow do not change significantly during adulthood.

Changes in the material characteristics of bone marrow with aging can be a significant source of error in measurements of bone density when using X-ray and ultrasound imaging modalities. In the context of computed tomography, dual-energy computed techniques have been used to correct for changes in marrow composition. However, dual-energy quantitative computed tomography (DE-QCT) protocols, while increasing the accuracy of the measurement, reduce the precision and increase the radiation dose to the patient in comparison to single-energy quantitative computed tomography (SE-QCT) protocols. If the attenuation properties of the marrow for a particular bone can be shown to be relatively constant with age, it should be possible to use single-energy techniques without experiencing errors caused by unknown marrow composition. Marrow was extracted by centrifugation from 10 mm thick frontal sections of 34 adult cadaver calcanei (28 males, 6 females, ages 17-65 years). The density and energy-dependent linear X-ray attenuation coefficient of each marrow sample were determined. For purposes of comparing our results, we then computed an effective CT number at two GE CT/i scan voltages (80 and 120 kVp) for each specimen. The coefficients of variation for the density, CT number at 80 kVp and CT number at 120 kVp were each less than 1%, and the parameters did not change significantly with age (p > 0.2, r2 < 0.02, power > 0.8 where the minimum acceptable r2 = 0.216). We could demonstrate no significant gender-associated differences in these relationships. These data suggest that calcaneal bone marrow X-ray attenuation properties and marrow density are essentially constant from the third through sixth decades of life.

Prediction of fracture location in the proximal femur using finite element models.

Finite element (FE) models of the proximal femur are often used to study hip fracture. To interpret the results of these models, it is important to know whether the models accurately predict fracture location and/or type. This study evaluated the ability of automatically generated, CT scan-based linear FE models of the proximal femur to predict fracture location and fracture type. Fracture location was defined as the specific location of the fracture. Fracture type was a categorical variable defined as either a cervical or a trochanteric fracture. FE modeling and mechanical testing of 18 pairs of human femora were performed under two loading conditions, one similar to joint loading during single-limb stance and one simulating impact from a fall. For the stance condition, the predicted and actual fracture locations agreed in 13 of the 18 cases (72% agreement). For the fall condition, the predicted and actual fracture locations agreed in 10 of the 15 cases where the actual fractures could be identified (67% agreement). The FE models correctly predicted that only cervical fractures occurred in the stance configuration. For the fall configuration, FE-predicted and actual fracture types agreed in 11 of the 14 cases that could be compared (9 trochanteric, 2 cervical; 79% agreement). These results provide evidence that CT scan-based FE models of the proximal femur can predict fracture location and fracture type with moderate accuracy.

Effect of disk fenestration on sagittal kinematics of the canine C5-C6 intervertebral space.

OBJECTIVE: To determine the effect that ventral intervertebral disk fenestration has on the sagittal range of motion about the C5-C6 intervertebral space. STUDY DESIGN: A repeated measures in vitro mechanical study of spinal range of motion under controlled loading conditions before and after fenestration. SAMPLE POPULATION: A total of 10 canine cervical vertebral specimens (C4-C7) collected from clinically normal animals within 12 hours of euthanasia. METHODS: Specimens were loaded as cantilever beams fixed at C7. Weights (1 to 5 kg) were progressively applied to C4 to produce flexion or extension in the sagittal plane. Radiographs were taken at each load, 3 times before and 3 times after fenestration of the C5-C6 disk. The positions of radiodense markers embedded in the vertebrae were used to calculate flexion and extension angles and range of motion. RESULTS: Range of motion (difference between flexion and extension) and flexion and extension angles (individually) significantly increased after fenestration (P < .0001). CONCLUSION: Ventral fenestration produces sagittal instability of the C5-C6 disk space. CLINICAL RELEVANCE: In surgical fusion of caudal cervical intervertebral spaces in dogs, radiographically normal disks adjacent to the affected space are often fenestrated to facilitate distraction before surgical stabilization. This study shows that ventral fenestration produces instability of a caudal cervical intervertebral space in the model used. Such instability may contribute to the development of the secondary instability ("domino lesions") seen in some surgically treated dogs.

Ex vivo simulation of in vivo strain distributions in the equine metacarpus.

The objective of this study was to examine several simple ex vivo loading conditions for the equine metacarpus, and to evaluate their ability to reproduce the mid-diaphyseal bone surface strain distributions previously reported in vivo at the walk and trot. Distributed axial compressive loads, and 9 different axial compressive point loads at -7.5 kN and -15 kN were applied to metacarpal-distal carpal bone preparations from 6 Thoroughbred horses, aged 1-5 years. The resulting dorsal, medial, palmar, and lateral mid-diaphyseal bone surface axial and shear strains were compared with previously reported in vivo surface strain distributions using a root mean square error (RMSE) protocol. The effects of loading condition and load magnitude on RMSE were assessed with a mixed-model analysis of variance. There were significant differences between loading conditions, and, in most cases, between load magnitudes, in the fit of the ex vivo to the in vivo strain distributions. In vivo mid-diaphyseal bone surface strain distributions at the walk can be well approximated ex vivo by a distributed axial compressive load, or by a point load positioned 0.5 cm medial to the sagittal midline, at -7.5 kN loads. In vivo mid-diaphyseal bone surface strain distributions at the trot can be well approximated by the -15 kN loads applied to the same locations. These simplified loading conditions can be used in designing biologically relevant loading protocols for ex vivo mechanical testing studies, as well as in developing boundary conditions for finite element analysis. As such, these loading conditions may be considered as tools to be used as a means of replicating in vivo loading conditions during the initial design and testing stages in the development of fracture fixation devices, as well as in the theoretical mechanical analysis of the metacarpal structure.

Development and validation of a series of three-dimensional finite element models of the equine metacarpus.

Three-dimensional finite element (FE) models of the left metacarpi of five adult horses were developed from quantitative computed tomography data, using the algorithms of Keyak et al. (1990, J Biomed. Engng 12, 389-397). The metacarpi were then equipped with 12 rosette strain gauges and loaded non-destructively in a mechanical testing machine. The bones and the models were loaded in axial compression, with the load evenly distributed across the distal row of carpal bones, and with a point load placed mediad to the sagittal midline, to a load equivalent to three times body weight (-15 kN); and in sagittal four-point bending to -2 kN. Maximum and minimum principal strains from the models were compared with those at the strain gauge rosettes. There were significant (p < 0.001) and strong (0.69 < r < 0.90) correlations between predicted and observed surface principal strains, most often resolving as second- or third-order polynomial relationships. In most cases, particularly at extreme strain magnitudes, the models tended to overestimate the observed strain magnitudes. These data suggest that the models are robust and accurate predictors of surface strains. Validation of these models lends further support for the use of this method of automated three-dimensional FE modeling, with its emphasis on accurate, individualized portrayal of structural geometry and material distribution, in research applications, and specifically for the use of these models in the study of the biology and mechanics of the equine metacarpus.

The distribution of material properties in the equine third metacarpal bone serves to enhance sagittal bending.

The distribution of material properties within the equine third metacarpal bone (MC3), and its possible effect on the mechanics of the structure, was quantitatively evaluated using single-load-to-failure compressive materials testing of specimens from ten horses. Bone samples from six regions within five proximodistal levels of MC3 were milled into right cylinders and compressed at a strain rate of 0.01 s-1. Diaphyseal MC3 bone material was stiffer, stronger, deformed less to yield and failure, and absorbed more energy to yield, than metaphyseal cortical bone material. Lateral and medial MC3 cortical bone material was stiffer and deformed less to yield and failure, than dorsal and palmar material. This distribution of material properties appears to increase the structural compliance in the sagittal plane, and may serve to enhance the predictability of the strain distribution during normal locomotion, as is provided in other bones by a sagittal curvature.

Necropsy survey of metacarpal fusion in the horse.

Paired metacarpi obtained at necropsy from 100 horses ranging in age from term fetus to 35 years were examined to estimate the prevalence and sites of metacarpal fusion. Metacarpal fusion was seen in 192 of 200 metacarpi, and 78% of all horses 2 years or older had 2 or more fusions. Fusion of the second metacarpal bone to the third metacarpal bone was significantly (P < 0.001) more common than was fusion of the fourth to the third metacarpal bone. Fusions appeared for the most part in pairs and were bilaterally symmetric. Rooney-Prickett type-A carpometacarpal joint configurations (in which there is no measurable articulation between the third carpal and second metacarpal bones) were rare in this population, and Rooney-Prickett type-B configurations (in which there is a measurable articulation between the third carpal and second metacarpal bones) were observed in 98.5% of metacarpi. Medial metacarpal fusion was positively correlated with age, occupation, and proportion of the proximal projection of the carpometacarpal distal joint surface that was taken by the second metacarpal bone. Lateral metacarpal fusion was positively correlated with age and the proportion of the proximal projection of the carpometacarpal distal joint surface taken by the fourth metacarpal bone. Horses in performance careers (racing, race training, or show ring occupations) had an earlier development of the first 2 fusions than did horses in other or unknown occupations; development of the third and fourth fusions were not significantly different between occupation groups. The rate of metacarpal fusion per horse-year appeared to be at least 10 times higher than a clinically evident rate. A variety of gross morphologic features was observed in the fusions from this sample, some of which were small, subtle, and possibly difficult to detect in vivo. It is hypothesized that many instances of metacarpal fusion may be a result of functional adaptation of the metacarpus to increased or changed loading conditions, rather than a response to isolated traumatic events.

Estimation of material properties in the equine metacarpus with use of quantitative computed tomography.

The purpose of this study was to investigate the relationships between data obtained from quantitative computed tomography and mechanical properties in the equine metacarpus, as measured in vitro in bone specimens. Three hundred and fifty-five bone specimens from the metacarpi of 10 horses were machined into right cylinders aligned with the long axis of the bone. A computed tomographic scan of the specimens, along with a Cann-Genant K2HPO4 calibration standard, was obtained. The specimens then were compressed to failure, and the elastic modulus, yield stress, yield strain, strain energy density at yield, ultimate stress, ultimate strain, and strain energy density at ultimate failure were calculated. The specimens were dried and ashed. Quantitative computed tomography-derived K2HPO4 equivalent density proved to be an excellent estimator (r2 > 0.9) of elastic modulus, yield stress, ultimate stress, wet density, dry density, and ash density; a moderately good estimator (0.4 < r2 < 0.9) of strain energy density at yield and at ultimate failure; and a poor estimator (r2 < 0.2) of yield strain and ultimate strain. It was concluded that the relationships between quantitative computed tomography data and mechanical properties of the equine metacarpus were strong enough to justify the use of these data in automated finite element modeling.

An in vitro biomechanical investigation of an interlocking nail for fixation of diaphyseal tibial fractures in adult horses.

The compressive, bending and torsional mechanical properties of osteotomized adult equine tibiae stabilized with an interlocking intramedullary nail (nail-tibia composite) were compared with those of intact tibiae to determine the clinical applicability of the the nail for repair of tibial fractures in adult horses. The mean yield load, failure load, and stiffness for the nail-tibia composites were significantly less (P < .05) than those for the intact tibiae in all loading configurations. The mean compressive yield load for the nail-tibia composites was greater than the compressive load calculated from previously reported in vivo data for walking and trotting, and was equal to the load calculated for recovery from anesthesia. The mean yield bending moment for the nail-tibia composites was greater than the bending moment previously calculated for standing, walking, and recovery from anesthesia. The mean torsional yield load for the nail-tibia composites was less than the torsional load determined for the walk from another in vivo study. The design of the interlocking nail evaluated in the present study should be modified to increase torsional and compressive yield strengths and torsional stiffness before reasonable success could be expected for the treatment of adult equine tibial fractures.

Epidemiologic study of toxoplasmosis on a sheep ranch.

An epidemiologic study was done on a ranch in northern California on a flock of ewes that had a history of abortions, mummified fetuses, weak or stillborn lambs, and failure to conceive. Of 56 ewes tested, 33 (59%) had serum agglutinating antibodies against Toxoplasma gondii, with an unusually high proportion of high titers. Over a 2-year-period, reproductive problems were higher (39% to 42%) among the seropositive ewes than among the seronegative ewes (9% to 33%). Of 89 sera received from persons and 7 species of animals on the ranch, 44 (49%) were found to be seropositive to T gondii, including seropositive members of a family of 6. The rancher's wife and teenage daughter, both of whom were involved with lambing, had serum titers exceeding 4,096. Other members of the family not involved with lambing were seronegative by the indirect hemagglutination test. The 2 infected persons are known to have come in contact with placentas, birth fluids, fetuses, and colostrums from these infected ewes.