Effect of bone mineral content on the tensile properties of cortical bone: experiments and theory

The effect of mineral volume fraction on the tensile mechanical properties of cortical bone tissue is investigated by theoretical and experimental means. The mineral content of plexiform, bovine bone was lowered by 18% and 29% by immersion in fluoride solutions for 3 days and 12 days, respectively. The elastic modulus, yield strength and ultimate strength of bone tissue decreased, while the ultimate strain increased with a decrease in mineral content. The mechanical behavior of bone tissue was modeled by using a micromechanical shear lag theory consisting of overlapped mineral platelets reinforcing the organic matrix. The decrease in yield stress, by the 0.002 offset method, of the fluoride treated bones were matched in the theoretical curves by lowering the shear yield stress of the organic matrix. The failure criterion used was based on failure stresses determined from a failure envelope (Mohr's circle), which was constructed using experimental data. It was found that the model predictions of elastic modulus got worse with a decrease in mineral content (being 7.9%, 17.2% and 33.0% higher for the control, 3-day and 12-day fluoride-treated bones). As a result, the developed theory could not fully predict the yield strain of bones with lowered mineral content, being 12.9% and 21.7% lower than the experimental values. The predicted ultimate stresses of the bone tissues with lower mineral contents were within +/- 10% of the experimental values while the ultimate strains were 12.7% and 26.3% lower than the experimental values. Although the model developed in this study did not take into account the presence of hierarchical structures, voids, orientation of collagen molecules and micro cracks, it still indicated that the mechanical properties of the organic matrix depend on bone mineral content.     

Combined Effects of Fluoride and Cadmium on Liver and Kidney Function in Male Rats

Although cadmium (Cd) and fluoride may both have adverse effects on bone, most studies focus on a single agent. In this study, we investigated the effects of cadmium and fluoride on bone at a relative low level. Sprague–Dawley male rats were assigned randomly into four groups which were given sodium chloride, cadmium (50mg/L), and fluoride (20mg/L) alone, or in combination via drinking water. At the 12th week, urine, blood, and bone tissues were collected for biomarker assay, biomechanical assay, and histological assay. Cadmium had significantly adverse effects on bone mineral density, bone biomechanical property, and bone microstructure. Fluoride slightly increased vertebral bone mineral density but negatively affected bone biomechanical property and bone microstructure. Fluoride could reverse the decrease of vertebral bone mineral density caused by cadmium but could not improve the damage of bone biomechanical property and microstructure caused by cadmium. Tartrate-resistant acid phosphatase 5b levels in rats treated with cadmium and fluoride or in combination were 1–2.5 folds higher than the control. Our data suggest that low level of fluoride could reverse the decrease of vertebral bone mineral density caused by cadmium exposure but has no influence on appendicular skeleton damage caused by cadmium.     

Fluoride reduces the rate of dissolution of bone

Recently, fluoride has been used in the treatment of osteoporosis in an attempt to rebuild bone lost due to this disease. Fluoride has been shown to have a profound effect on osteoblasts and bone formation. Studies have shown that synthetic fluorapatite is more stable and less soluble than hydroxyapatite. This study was designed to determine the dissolution properties of bones from rats fed a normal diet versus rats fed a high fluoride diet. Intact and deproteinized bones were dissolved in buffered solutions, containing physiological amounts of inorganic Ca and P, at pH 3.4, 4.4 and 5.4 The data demonstrate that fluoridated bones dissolve slower than normal bones, deproteinized bones dissolve faster than intact bones and that the initial rate of dissolution of fluoridated bone is always significantly lower than that of normal bone. This may help to explain the reduced rate of osteoclastic resorption in patients undergoing fluoride therapy.     

Serum Fluoride and Sialic Acid Levels in Osteosarcoma

Osteosarcoma is a rare malignant bone tumor most commonly occurring in children and young adults presenting with painful swelling. Various etiological factors for osteosarcoma are ionizing radiation, family history of bone disorders and cancer, chemicals (fluoride, beryllium, and vinyl chloride), and viruses. Status of fluoride levels in serum of osteosarcoma is still not clear. Recent reports have indicated that there is a link between fluoride exposure and osteosarcoma. Glycoproteins and glycosaminoglycans are an integral part of bone and prolonged exposure to fluoride for long duration has been shown to cause degradation of collagen and ground substance in bones. The present study was planned to analyze serum fluoride, sialic acid, calcium, phosphorus, and alkaline phosphatase levels in 25 patients of osteosarcoma and age- and sex-matched subjects with bone-forming tumours other than osteosarcoma and musculo-skeletal pain (controls, 25 each). Fluoride levels were analyzed by ISE and sialic acid was analyzed by Warren’s method. Mean serum fluoride concentration was found to be significantly higher in patients with osteosarcoma as compared to the other two groups. The mean value of flouride in patients with other bone-forming tumors was approximately 50% of the group of osteosarcoma; however, it was significantly higher when compared with patients of group I. Serum sialic acid concentration was found to be significantly raised in patients with osteosarcoma as well as in the group with other bone-forming tumors as compared to the group of controls. There was, however, no significant difference in the group of patients of osteosarcoma when compared with group of patients with other bone-forming tumors. These results showing higher level of fluoride with osteosarcoma compared to others suggesting a role of fluoride in the disease.     

Changing the structurally effective mineral content of bone with in vitro fluoride treatment

Bovine femur cortical bone specimens were tested in tension after being treated in vitro for 3 days with sodium fluoride solutions of different molarity (0.145, 0.5, and 2.0M). The treatments alter the mechanical properties of the bone samples with different degrees as compared to control samples (untreated). The mechanical properties of the treated samples have lower elastic modulus, yield and ultimate stress, acoustic impedance and hardness, and higher ultimate strain and toughness as compared to control samples. The observed effects were intensified with the increasing molarity of the treatment solutions. This study shows that the fluoride treatment can be used to investigate the composite behavior of bone tissue by altering the structurally important bone mineral content in a controlled manner.     

Remnant Woven Bone and Calcified Cartilage in Mouse Bone: Differences between Ages/Sex and Effects on Bone Strength

IntroductionMouse models are used frequently to study effects of bone diseases and genetic determinates of bone strength. Murine bones have an intracortical band of woven bone that is not present in human bones. This band is not obvious under brightfield imaging and not typically analyzed. Due to the band’s morphology and location it has been theorized to be remnant bone from early in life. Furthermore, lamellar and woven bone are well known to have differing mechanical strengths. The purpose of this study was to determine (i) if the band is from early life and (ii) if the woven bone or calcified cartilage contained within the band affect whole bone strength.

Woven Bone Origin Studies

In twelve to fourteen week old mice, doxycycline was used to label bone formed prior to 3 weeks old. Doxycycline labeling and woven bone patterns on contralateral femora matched well and encompassed an almost identical cross-sectional area. Also, we highlight for the first time in mice the presence of calcified cartilage exclusively within the band. However, calcified cartilage could not be identified on high resolution cone-beam microCT scans when examined visually or by thresholding methods.

Mechanical Strength Studies

Subsequently, three-point bending was used to analyze the effects of woven bone and calcified cartilage on whole bone mechanics in a cohort of male and female six and 13 week old Balb/C mice. Three-point bending outcomes were correlated with structural and compositional measures using multivariate linear regression. Woven bone composed a higher percent of young bones than older bones. However, calcified cartilage in older bones was twice that of younger bones, which was similar when normalized by area. Area and/or tissue mineral density accounted for >75% of variation for most strength outcomes. Percent calcified cartilage added significant predictive power to maximal force and bending stress. Calcified cartilage and woven bone could have more influence in genetic models where calcified cartilage percent is double our highest value.     

Effects of differences in mineralization on the mechanical properties of bone

There is a considerable variation in the mineralization of bone; normal, non-pathological compact bone has ash masses ranging from 45 to 85% by mass. This range of mineralization results in an even greater range of mechanical properties. The Young modulus of elasticity can range from 4 to 32 GPa, bending strength from 50 to 300 MPa, and the work of fracture from 200 to 7000 Jm-2. It is not possible for any one type of bone to have high values for all three properties. Very high values of mineralization produce high values of Young modulus but low values of work of fracture (which is a measure of fracture toughness). Rather low values of mineralization are associated with high values of work of fracture but low values of Young modulus and intermediate values of bending strength. The reason for the high value for the Young modulus associated with high mineralization is intuitively obvious, but has not yet been rigorously modelled. The low fracture toughness associated with high mineralization may be caused by the failure of various crack-stopping mechanisms that can act when the mineral crystals in bone have not coalesced, but which become ineffective when the volume fraction of mineral becomes too high. The adoption of different degrees of mineralization by different bones, leading to different sets of mechanical properties, is shown to be adaptive in most cases studied, but some puzzles still remain.     

A comparison of primate, carnivoran and rodent limb bone cross-sectional properties: are primates really unique?

The cross-sectional properties of mammalian limb bones provide an important source of information about their loading history and locomotor adaptations. It has been suggested, for instance, that the cross-sectional strength of primate limb bones differs from that of other mammals as a consequence of living in a complex arboreal environment (Kimura, 1991, 1995). In order to test this hypothesis more rigorously, we have investigated cross-sectional properties in samples of humeri and femora of 71 primate species, 30 carnivorans and 59 rodents. Primates differ from carnivorans and rodents in having limb bones with greater cross-sectional strength than mammals of similar mass. This might imply that primates have stronger bones than carnivorans and rodents. However, primates also have longer proximal limb bones than other mammals. When cross-sectional dimensions are regressed against bone length, primates appear to have more gracile bones than other mammals. These two seemingly contradictory findings can be reconciled by recognizing that most limb bones experience bending as a predominant loading regime. After regressing cross-sectional strength against the product of body mass and bone length, a product which should be proportional to the bending moments applied to the limb, primates are found to overlap considerably with carnivorans and rodents. Consequently, primate humeri and femora are similar to those of nonprimates in their resistance to bending. Comparisons between arboreal and terrestrial species within the orders show that the bones of arboreal carnivorans have greater cross-sectional properties than those of terrestrial carnivorans, thus supporting Kimura's general notion. However, no differences were found between arboreal and terrestrial rodents. Among primates, the only significant difference was in humeral bending rigidity, which is higher in the terrestrial species. In summary, arboreal and terrestrial species do not show consistent differences in long bone reinforcement, and Kimura's conclusions must be modified to take into account the interaction of bone length and cross-sectional geometry.     

Mammalian collagenase predisposes bone surfaces to osteoclastic resorption

Summary The cell-free endocranial surface of young adult rat parietal bones was used as a substrate for bone cell-derived mammalian collagenase. Incubation of parietal bones in a concentration of enzyme comparable to that secreted by osteoblastic cells in vitro caused destruction of surface osteoid, and resulted in exposure of mineral onto the bone surface. Bones so pre-treated were considerably more susceptible to osteoclastic resorption than bones preincubated in the absence of collagenase. These results are consistent with the view that the osteoid layer which covers bone surfaces acts as a barrier to osteoclastic contact with underlying, resorption — stimulating bone mineral; and that cells of the osteoblastic lineage induce osteoclastic resorption through collagenase secretion which, by digestion of the surface osteoid, exposes bone mineral to osteoclastic contact.     

Determination of mechanical stiffness of bone by pQCT measurements: correlation with non-destructive mechanical four-point bending test data

Mechanical tests of bone provide valuable information about material and structural properties important for understanding bone pathology in both clinical and research settings, but no previous studies have produced applicable non-invasive, quantitative estimates of bending stiffness. The goal of this study was to evaluate the effectiveness of using peripheral quantitative computed tomography (pQCT) data to accurately compute the bending stiffness of bone. Normal rabbit humeri (N=8) were scanned at their mid-diaphyses using pQCT. The average bone mineral densities and the cross-sectional moments of inertia were computed from the pQCT cross-sections. Bending stiffness was determined as a function of the elastic modulus of compact bone (based on the local bone mineral density), cross-sectional moment of inertia, and simulated quasistatic strain rate. The actual bending stiffness of the bones was determined using four-point bending tests. Comparison of the bending stiffness estimated from the pQCT data and the mechanical bending stiffness revealed excellent correlation (R2=0.96). The bending stiffness from the pQCT data was on average 103% of that obtained from the four-point bending tests. The results indicate that pQCT data can be used to accurately determine the bending stiffness of normal bone. Possible applications include temporal quantification of fracture healing and risk management of osteoporosis or other bone pathologies.     

Nanoindentation and whole-bone bending estimates of material properties in bones from the senescence accelerated mouse SAMP6

The senescence accelerated mouse, strain P6 (SAMP6) has been described as a model of senile osteoporosis. Recent results from whole-bone bending tests indicate that, despite having increased moments of inertia, SAMP6 long bones are weak and brittle compared to SAMR1 controls. In the current study we determined material properties of cortical bone from SAMP6 and SAMR1 femora and tibiae by two methods-nanoindentation and whole-bone bending tests combined with simple beam theory. We hypothesized that: (1) SAMP6 mice have reduced cortical bone material properties compared to SAMR1 controls; and (2) modulus estimated from whole-bone bending tests correlates well with modulus determined by nanoindentation. Results from nanoindentation indicated that modulus and hardness are approximately 10% higher in SAMP6 mice compared to SAMR1 controls (p<0.001), a finding consistent with slightly higher mineralization in SAMP6 bones. Despite their superior elastic and hardness properties, the bending failure properties of SAMP6 bones were markedly inferior--ultimate stress and toughness were reduced by 40% and 60%, respectively (p<0.001). Comparisons between the two testing methods for determining modulus showed poor agreement. Modulus estimated from whole-bone bending tests was not correlated with modulus determined by nanoindentation (p=0.054; r2=0.03) and the absolute values differed by a factor of five between the two methods (bending [wet], 6GPa; nanoindentation [dry], 31GPa). Moreover, relative differences between groups were inconsistent between the two methods. We conclude: (1) cortical bone from the SAMP6 mouse has increased modulus and hardness but poor material strength and toughness, which underscores the relevance of the SAMP6 mouse for studies of skeletal fragility, and (2) values of elastic modulus of bone tissue estimated using simple beam theory and bending tests of mouse femora and tibiae are inaccurate and should be interpreted with caution.     

Bone mineral density and bone structure parameters as predictors of bone strength: an analysis using computerized microtomography and gastrectomy-induced osteopenia in the rat

In this study the relationships of bone mineral density (BMD) and bone structure parameters calculated from 2D microtomography images to bone strength were investigated. Femurs from 21 male Sprague Dawley rats were subjected to dual-energy X-ray absorptiometry, computerized microtomography (CmicroT) and either three-point cantilever bending (femoral shaft) or two-point bending compression (femoral neck). Gastrectomy was performed on 12 animals and 9 were sham operated. From the tomograms bone structure analysis was performed using a software routine based on grey level run-length method. Correlations of BMD and bone structure parameters to mechanical parameters were investigated as were differences between the gastrectomized and the control samples. The reductions of BMD between the groups were 21 and 27% in the femoral neck and shaft, respectively. For the shaft, the correlations of BMD to all mechanical parameters were significant and BMD was a consistent predictor of bone strength for cortical bone. However, in the femoral neck where cancellous bone predominates, BMD was weakly correlated only to deflection. A significant correlation between trabecular thickness and neck bone strength was found. Hence, compared to trabecular thickness, BMD was of limited value in predicting bone strength in the femoral neck.     

Incidence and mechanical significance of pneumatization in the long bones of birds

The incidence of pneumatization in avian long bones was studied, by direct observation, in a large sample of species. Only proximal bones (humerus and femur) presented pneumatization in the sample studied. The incidence obtained was related to the variation of the maximum cortical thickness and mechanical properties, such as bending strength and flexural Young's modulus. Cortical thickness, bending strength and flexural Young's modulus were significantly lower in pneumatized bones than in marrow-filled bones. Furthermore, some congruence was found between pneumatization and systematic groups when compared. In this sense, Charadriformes was the only order studied with total absence of long bone pneumatization. Results on cortical thickness appear to be in agreement with modelling predictions previously made and with results obtained on other groups of flying vertebrates. The possible selective advantage of reduction in cortical thickness in relation to flying is suggested.     

Fluoride and environmental health: a review

The relationship between environmental fluoride and human health has been studied for over 100 years by researchers from a wide variety of disciplines. Most scientists believe that small amounts of fluoride in the diet can help prevent dental caries and strengthen bones, but there are a number of adverse affects that chronic ingestion at high doses can have on human health, including dental fluorosis, skeletal fluorosis, increased rates of bone fractures, decreased birth rates, increased rates of urolithiasis (kidney stones), impaired thyroid function, and lower intelligence in children. Chronic occupational exposure to fluoride dust and gas is associated with higher rates of bladder cancer and variety of respiratory ailments. Acute fluoride toxicity and even death from the ingestion of sodium fluoride pesticides and dental products have also been reported. The distribution of fluoride in the natural environment is very uneven, largely a result of the geochemical behavior of this element. Fluorine is preferentially enriched in highly evolved magmas and hydrothermal solutions, which explains why high concentrations are often found in syenites, granitoid plutonic rocks, alkaline volcanic, and hydrothermal deposits. Fluoride can also occur in sedimentary formations that contain fluoride-bearing minerals derived from the parent rock, fluoride-rich clays, or fluorapatite. Dissolved fluoride levels are usually controlled by the solubility of fluorite (CaF₂); thus, high concentrations are often associated with soft, alkaline, and calcium-deficient waters. Although much is known about the occurrence and health effects of fluoride, problems persist in Third World countries, where populations have little choice in the source of their drinking water and food. However, even in developed nations, fluoride ingestion can exceed the recommended dose when sources other than drinking water are ignored.     

Compressive axial mechanical properties of rat bone as functions of bone volume fraction,apparent density and micro-ct based mineral density

Mechanical testing has been regarded as the gold standard to investigate the effects of pathologies on the structure–function properties of the skeleton. With recent advances in computing power of personal computers, virtual alternatives to mechanical testing are gaining acceptance and use. We have previously introduced such a technique called structural rigidity analysis to assess mechanical strength of skeletal tissue with defects. The application of this technique is predicated upon the use of relationships defining the strength of bone as a function of its density for a given loading mode. We are to apply this technique in rat models to assess their compressive skeletal response subjected to a host of biological and pharmaceutical stimulations. Therefore, the aim of this study is to derive a relationship expressing axial compressive mechanical properties of rat cortical and cancellous bone as a function of equivalent bone mineral density, bone volume fraction or apparent density over a range of normal and pathologic bones.We used bones from normal, ovariectomized and partially nephrectomized animals. All specimens underwent micro-computed tomographic imaging to assess bone morphometric and densitometric indices and uniaxial compression to failure.We obtained univariate relationships describing 71–78% of the mechanical properties of rat cortical and cancellous bone based on equivalent mineral density, bone volume fraction or apparent density over a wide range of density and common skeletal pathologies. The relationships reported in this study can be used in the structural rigidity analysis introduced by the authors to provide a non-invasive method to assess the compressive strength of bones affected by pathology and/or treatment options.     

Computed-tomography-based finite-element models of long bones can accurately capture strain response to bending and torsion

Finite element (FE) models of long bones constructed from computed-tomography (CT) data are emerging as an invaluable tool in the field of bone biomechanics. However, the performance of such FE models is highly dependent on the accurate capture of geometry and appropriate assignment of material properties. In this study, a combined numerical-experimental study is performed comparing FE-predicted surface strains with strain-gauge measurements. Thirty-six major, cadaveric, long bones (humerus, radius, femur and tibia), which cover a wide range of bone sizes, were tested under three-point bending and torsion. The FE models were constructed from trans-axial volumetric CT scans, and the segmented bone images were corrected for partial-volume effects. The material properties (Young's modulus for cortex, density-modulus relationship for trabecular bone and Poisson's ratio) were calibrated by minimizing the error between experiments and simulations among all bones. The R(2) values of the measured strains versus load under three-point bending and torsion were 0.96-0.99 and 0.61-0.99, respectively, for all bones in our dataset. The errors of the calculated FE strains in comparison to those measured using strain gauges in the mechanical tests ranged from -6% to 7% under bending and from -37% to 19% under torsion. The observation of comparatively low errors and high correlations between the FE-predicted strains and the experimental strains, across the various types of bones and loading conditions (bending and torsion), validates our approach to bone segmentation and our choice of material properties.     

Type I collagen mutation alters the strength and fatigue behavior of Mov13 cortical tissue

Despite advances in understanding the molecular basis of Osteogenesis Imperfecta, the mechanisms by which type I collagen mutations compromise whole bone function are not well understood. Previously, we have shown that a heterozygous type I collagen mutation is associated with increased brittleness of long bones from Mov13 transgenic mice, a model of the mild form of Osteogenesis Imperfecta. In the current study, we investigated tissue-level damage processes by testing the hypothesis that the fatigue properties of Mov13 tissue were significantly compromised relative to littermate controls. We also quantified tissue structure and mineral content to explain variations in the fatigue behavior. Micro-beam specimens were machined from the anterior and posterior quadrants of Mov13 and control femurs and subjected to cyclic bending at one of four stress levels. Mov13 tissue exhibited a 22–25% reduction in tissue bending strength and a similar reductions in fatigue life and the stress level at which damage was apparent. These results provided tissue-level evidence that damage accumulation mechanisms were significantly compromised in Mov13 cortical tissue. Given that significant alterations in tissue structure were observed in Mov13 femurs, the results of this study support the idea that Mov13 femurs were brittle because alterations in tissue structure associated with the mutation interfered with normal damage processes. These results provide new insight into the pathogenesis of Osteogenesis Imperfecta and are consistent with bone behaving as a damaging composite material, where damage accumulation is central to bone fracture.     

Sexual dimorphism in human lower limb bone structure: relationship to subsistence strategy and sexual division of labor

Cross-sectional geometric properties of the human femur and tibia are compared in males and females in a number of recent and archaeological population samples extending back to the Middle Paleolithic. There is a consistent decline in sexual dimorphism from hunting-gathering to agricultural to industrial subsistence strategy levels in properties which measure relative anteroposterior bending strength of the femur and tibia in the region about the knee. This trend parallels and is indicative of reductions in the sexual division of labor, in particular differences in the relative mobility of males and females. Sexual dimorphism in mediolateral bending strength near the hip shows no consistent temporal trend, probably reflecting relatively constant sex differences in pelvic structure related to the requirements of childbirth. Upper and Middle Paleolithic samples are indistinguishable in terms of sexual dimorphism from modern huntergatherers, suggesting a similar sexual division of labor. The results illustrate the utility of cross-sectional geometric parameters of long bone diaphyses in reconstructing behavioral differences within and between past populations. Some variations in the accuracy of sexing techniques based on diaphyseal measurements of the lower limb long bones may also be explained by these behavioral and structural factors.     

Mechanical properties of cranial sutures

Many bones in mammalian skulls are linked together by cranial sutures, connective tissue joints that are morphologically variable and show different levels of interdigitation among and within species. The goal of this investigation was to determine whether sections of skull with cranial sutures have different mechanical properties than adjacent sections without sutures, and if these properties are enhanced with increased interdigitation. To test these hypotheses, bending strength and impact energy absorption were measured for samples of goat (Capra hircus) cranial bone without sutures and with sutures of different degrees of interdigitation. Bending strength was measured under both dynamic (9.7 mm displacement s-1) and relatively static (0.8 mm s-1) conditions, and at either speed, increased sutural interdigitation provided increased strength during three-point bending. However, except for very highly interdigitated sutures loaded slowly, sutures were not as strong in bending as bone. In contrast, sutures absorbed from 16% to 100% more energy per unit volume during impact loading than did bone. This five-fold increase in energy absorption by the sutures was significantly correlated with increased sutural interdigitation.     

Effects of fluoride ingestion on white-tailed deer (Odocoileus virginianus)

The effects of the addition of 25 or 50 ppm fluoride (F), as sodium fluoride (NaF), to the rations of 5-mo-old male white-tailed deer were similar to those observed in domestic cattle fed similar amounts of fluoride. The ingestion of 50 ppm F for 2 yr resulted in the accumulation of over 7,000 ppm F in bone ash. Accumulation of fluoride in antlers was extensive and occurred more rapidly than in skeletal tissue. Fluoride ingestion resulted in lesions on the developing incisors that were similar, but not identical to those seen in other species. Increased molar wear in the deer fed 50 ppm F was minimal, and no gross pathology of the mandible was observed. Only mild hyperostosis of the long bones was evident.