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.

Comparison of the linear finite element prediction of deformation and strain of human cancellous bone to 3D digital volume correlation measurements.

The mechanical properties of cancellous bone and the biological response of the tissue to mechanical loading are related to deformation and strain in the trabeculae during function. Due to the small size of trabeculae, their motion is difficult to measure. To avoid the need to measure trabecular motions during loading the finite element method has been used to estimate trabecular level mechanical deformation. This analytical approach has been empirically successful in that the analytical models are solvable and their results correlate with the macroscopically measured stiffness and strength of bones. The present work is a direct comparison of finite element predictions to measurements of the deformation and strain at near trabecular level. Using the method of digital volume correlation, we measured the deformation and calculated the strain at a resolution approaching the trabecular level for cancellous bone specimens loaded in uniaxial compression. Smoothed results from linearly elastic finite element models of the same mechanical tests were correlated to the empirical three-dimensional (3D) deformation in the direction of loading with a coefficient of determination as high as 97% and a slope of the prediction near one. However, real deformations in the directions perpendicular to the loading direction were not as well predicted by the analytical models. Our results show, that the finite element modeling of the internal deformation and strain in cancellous bone can be accurate in one direction but that this does not ensure accuracy for all deformations and strains.

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.

Fatigue damage-fracture mechanics interaction in cortical bone.

Fatigue loading causes accumulation of damage that may lead to the initiation of a macrocrack and result in a catastrophic failure of bone. The objective of this study was to examine the influence of fatigue damage on crack growth parameters in bovine cortical bone. Nineteen rectangular beam specimens (4 x 4 x 48 mm) were machined from bovine tibiae. The long axis of the beams was aligned with the long axis of bones. Using a four-point bending fatigue setup, ten specimens were fatigue-damaged to different levels as indicated by stiffness loss. A through-thickness notch was machined at the center of each damaged and undamaged beam. The notched specimens were then monotonically loaded beyond failure using a three-point bending protocol. Critical stress intensity factor, K(I), and work to critical load, W(Q), were significantly lower in the damaged group than in the undamaged group (p < 0.03). When the undamaged specimens were assigned a percent stiffness loss of zero and pooled with the damaged group, significant negative correlations of percent stiffness loss with K(I) (R = 0.58, p < 0.01), W(Q) (R = 0.54, p < 0.02), maximum load, P(max) (R = 0.59, p < 0.008), deflection at maximum load, Delta(max) (R = 0.48, p < 0.04), structural stiffness, S(max) (R = 0.53, p < 0.02), W(max) (R = 0.55, p < 0.02), and load at 1.4 mm deflection (a value beyond failure but without complete fracture), P(1.4) (R = 0.47, p < 0.05), were found. Post hoc analysis revealed that the average load-deflection curve from the damaged group was transformable into that from the undamaged group through a special shift on the load-deflection plane. Fatigue damage reduces bone stiffness and resistance to crack initiation, maximum load-carrying capacity, and deflection before and after failure in cortical bone. The data suggest there is a single rule that governs the overall effect of fatigue damage on the fracture behavior of cortical bone.

Finite element calculated uniaxial apparent stiffness is a consistent predictor of uniaxial apparent strength in human vertebral cancellous bone tested with different boundary conditions.

Strong correspondence between the uniaxial apparent strength and stiffness of cancellous bone allows the use of stiffness as a predictor of bone strength. Measured values of mechanical properties in cancellous bone can be different between experiments due to different experimental conditions. In the current study, bone volume fraction, experimentally determined and finite element (FE) predicted stiffness were examined as predictors of cancellous bone ultimate strength in two different groups each of which was tested using a different end constraint. It is demonstrated that, although always significant, the relationships of strength with bone volume fraction and experimentally determined stiffness are different between test groups. Apparent stiffness, estimated by FE modeling, predicts the ultimate strength of human cancellous bone consistently for all examined experimental protocols.

Trabecular shear stress in human vertebral cancellous bone: intra- and inter-individual variations.

Correlation of the mean and standard deviation of trabecular stresses has been proposed as a mechanism by which a strong relationship between the apparent strength and stiffness of cancellous bone can be achieved. The current study examined whether the relationship between the mean and standard deviation of trabecular von Mises stresses can be generalized for any group of cancellous bone. Cylindrical human vertebral cancellous bone specimens were cut in the infero-superior direction from T12 of 23 individuals (inter-individual group). Thirty nine additional specimens were prepared similarly from the T4-T12 and L2-L5 vertebrae of a 63 year old male (intra-individual group). The specimens were scanned by micro-computed tomography (microCT) and trabecular von Mises stresses were calculated using finite element modeling. The expected value, standard deviation and coefficient of variation of the von Mises stress were calculated form a three-parameter Weibull function fitted to von Mises stress data from each specimen. It was found that the average and standard deviation of trabecular von Mises shear stress were: (i) correlated with each other, supporting the idea that high correlation between the apparent strength and stiffness of cancellous bone can be achieved through controlling the trabecular level shear stress variations, (ii) dependent on anatomical site and sample group, suggesting that the variation of stresses are correlated to the mean stress to different degrees between vertebrae and individuals, and (iii) dependent on bone volume fraction, consistent with the idea that shear stress is less well controlled in bones with low BV/TV. The conversion of infero-superior loading into trabecular von Mises stresses was maximum for the tissue at the junction of the thoracic and lumbar spine (T12-L1) consistent with this junction being a common site of vertebral fracture.

Estimation of bone matrix apparent stiffness variation caused by osteocyte lacunar size and density.

The role of osteocyte lacunar size and density on the apparent stiffness of bone matrix was predicted using a mechanical model from the literature. Lacunar size and lacunar density for different bones from different gender and age groups were used to predict the range of matrix apparent stiffness values for human cortical and cancellous tissue. The results suggest that bone matrix apparent stiffness depends on tissue type (cortical versus cancellous), age, and gender, the magnitudes of the effects being significant but small in all cases. Males had a higher predicted matrix apparent stiffness than females for vertebral cancellous bone (p< I0(-7)) and the difference increased with age (p =0.0007). In contrast, matrix apparent stiffness was not different between males and females forfemoral cortical bone and increased with age in both males (p < 0.0001) and females (p < 0.0364). Osteocyte lacunar density and size may cause significant gender and age-related variations in bone matrix apparent stiffness. The magnitude of variations in matrix apparent stiffness was small within the physiological range of lacunar size and density for healthy bone, whereas the variations can be profound in certain pathological cases. It was proposed that the mechanical effects of osteocyte density be uncoupled from their biological effects by controlling lacunar size in normal bone.

Effect of in ovo administration of insulin-like growth factor-I on composition and mechanical properties of chicken bone.

The influence of in ovo administration of insulin-like growth factor-I (IGF-I) on long bone growth (tibiae and femora) of 42-d-old broiler chickens was investigated. Eggs were divided into three groups: uninjected control, vehicle-injected control, and recombinant human (rh) IGF-I. Eggs were injected once with 100 microL vehicle (10 mM acetic acid and 0.1% BSA) per embryo or vehicle containing 100 ng rh IGF-I/100 microL per embryo (n = 555 eggs total) on Days 1, 2, 3, or 4 of embryonic development. Males had greater bone length and moment of inertia than did females for the tibia and the femur (P < or = 0.01 for all). Although fracture load was significantly affected by gender (P < or = 0.02 and P < or = 0.006 for the femur and tibia, respectively), there was no treatment effect on these variables. However, when the fracture load was normalized with body weight of the animal, treatment and gender effects were found for femora (P < or = 0.04). Hydroxyproline concentrations of bones from male broilers were increased by the treatment (P < or = 0.02), whereas it had no effect on female broilers. There was no treatment effect on ash content, stiffness, yield load, yield deflection, and ultimate deflection and elastic, plastic, and total work for the femur or the tibia. We suggest that the effect of in ovo administration of IGF-I on bone mechanical properties was site-specific, and treated femora tended to have a lower fracture load relative to increased body weight.

Calculation of porosity and osteonal cement line effects on the effective fracture toughness of cortical bone in longitudinal crack growth.

Based on the microscopic analyses of cracks and correlational studies demonstrating evidence for a relationship between fracture toughness and microstructure of cortical bone, an equation was derived for bone fracture toughness in longitudinal crack growth, using debonding at osteonal cement lines and weakening effect of pores as main crack mechanisms. The correlation between the measured and predicted values of fracture toughness was highly significant but weak for a single optimal value of matrix to cement line fracture toughness ratio. Using fracture toughness values and histomorphometrical parameters from an available data set, matrix to cement line fracture toughness ratio was calculated for human femoral bone. Based on these calculations it is suggested that the effect of an osteon on fracture toughness will depend on the cement line's ability to compensate for the pore in an osteon. Matrix to cement line fracture toughness ratio significantly increased with increasing age, suggesting that the effectiveness of osteons in energy absorption may be reduced in the elderly due to a change in cement line properties.

Fracture toughness of human femoral neck: effect of microstructure, composition, and age.

The effects of porosity and pore size; osteonal area, size, and density; mineral content; water content; wet and dry apparent densities; and age on mode I (tensile) and mode II (shear) strain energy release rate were investigated for femoral neck cortical bone from human cadavers aged >/=50 years. The results suggest that porosity- and density-based parameters that are related to bone quantity are more consistently determinant for femoral neck fracture toughness than morphology-based parameters that are related to microstructural organization. Bone features examined here were more explanatory for shear than tension fracture toughness. Tension and shear fracture toughness did not change with age, unlike in previous reports investigating the femoral and tibial shaft. It was concluded that the femoral neck is different from the femoral and tibial shaft in terms of its microstructure and composition and in its relationship of fracture toughness to its constituents and age.

In vivo diffuse damage in human vertebral trabecular bone.

Accumulation of microdamage in vivo may lead to loss of bone quality. Until recently, linear microcracks were the only known form of in vivo microdamage, but through the use of confocal microscopy an additional level of damage (diffuse damage) has been identified. In this study, in vivo diffuse damage was characterized and quantified in human vertebral trabecular bone as a function of tissue morphology, age, race, gender, and previously quantified in vivo linear microcracks. Presence of diffuse damage in human vertebral tissue was confirmed and validated by simultaneous use of polarized, ultraviolet, and laser confocal microscopy. Diffuse damage was found to occur preferentially within trabecular packets rather than in interstitial bone (p < 0.05). It was consistently higher in men compared with women (p < 0.05), but was not different by race or age group. Diffuse damage did not correlate with linear microcracks, but both exhibited the same probability distribution in which the percentage of individuals having a particular amount of damage decreased exponentially as damage content increased. These findings suggest that diffuse damage accumulation and repair are governed by the same biological phenomena as microcracks, but diffuse damage contributes independently to the microdamage content of bone.

Fracture toughness is dependent on bone location--a study of the femoral neck, femoral shaft, and the tibial shaft.

The fracture toughness of the right femoral neck, femoral shaft, and tibial shaft of matched cadaveric bones, ages 50 to 90 years, was compared. Results of this study indicate that tensile (G(Ic)) and shear (G(IIc)) fracture toughness vary depending on bone location. The femoral neck has the greatest resistance to crack initiation for both tension and shear loading while the femoral shaft has the least. The relationship between age and the fracture toughness of the femoral neck and shaft was investigated. G(c) of the femoral shaft significantly decreased with age for mode I and was nearly significant for mode II. Fracture toughness of the femoral neck did not change with age for the later decades of life. Implications of these findings are discussed.

Postnatal growth of broilers in response to in ovo administration of chicken growth hormone.

The effect of in ovo administration of chicken growth hormone (cGH) on growth rate and efficiency of gain, organ, and long bone growth of 42-d-old broiler chickens was investigated. Eggs were injected once with 100 microL vehicle (0.03 M NaHCO3, 0.15 M NaCl, pH 8.3) per embryo or vehicle containing 100 ng cGH/100 microL per embryo (n = 630 eggs total) on one of the following Days: 1, 4, or 7 through 18 of embryogenesis. There was no significant difference in hatchability between control and cGH treatment groups on any given injection day. Cumulative feed conversion of all treatment groups was improved relative to their respective control groups (P < 0.05). In ovo administration of cGH on Day 15 or 16 of incubation increased body weights (P < 0.01) of female broilers. On the other hand, body weights of male broilers were significantly increased by treatment on Day 1 (P < 0.04). Breast weights of female broilers from treatment groups Day 15 or 16 were increased (P < 0.01, P < 0.05, respectively). Liver weights of female broilers from treatment groups Day 1 and 15 were increased (P < 0.05, P < 0.01, respectively). In contrast, in ovo administration of cGH on Day 11 of incubation increased liver weights of male broilers (P < 0.03). There was no significant difference between control and treatment groups, in terms of heart or leg weights, or in Warner-Bratzler shear force of Pectoralis profundus muscle. Hydroxyproline concentration and cross-sectional area of female broiler tibias from treatment groups Day 11 or Day 16 were increased (P < 0.05), and ultimate breaking strength (stress) of tibias from the same groups was reduced (P < 0.05). In ovo administration of cGH altered growth and tissue development of broiler chickens in a time by sex dependent fashion.

Influence of microdamage on fracture toughness of the human femur and tibia.

The relationship between microdamage accumulation and bone fragility is not well understood. Previous work has demonstrated a positive relationship between microdamage and age in human cortical bone. Prior investigations have also demonstrated that fracture toughness decreases with age in the same bone. Based on these findings, the objective of this study was to test the hypothesis that a decrease in fracture toughness can be attributed to an increase in microdamage density. Microdamage parameters (density, surface density, and average crack length) were measured from bone taken from the shaft of the human femur and tibia and correlated with results from fracture toughness tests of the same bone. Results indicated that there was a weak but significant inverse relationship between fracture toughness and microdamage parameters for tension loading of the femur. These findings suggest that in vivo microdamage observable at the transmitted light level (100x) plays a secondary role to other contributory factors to decreased fracture toughness with age. Results also indicate that this relationship depends on bone ductility that apparently differs between the femur and the tibia. This study, in addition to prior investigations, suggests that crack size (microscopic vs. submicroscopic) and crack origin or type (in vivo vs. stress induced de novo) may influence the relationship between microdamage and bone toughness.

Influence of bone composition and apparent density on fracture toughness of the human femur and tibia.

The influence of wet and dry apparent density, apparent and real percentage of mineral, organic and water contents on the tension and shear fracture toughness, i.e., the mode I and mode II strain energy release rate (GIc and GIIc), respectively, was investigated for the human femur and the tibia. The results suggest that the water content, apparent density, and age were the best explanatory parameters for GIc and GIIc. Both GIc and GIIc significantly increase with increasing wet or dry apparent density. They also decrease with increasing water content; the decrease is nonsignificant for GIIc in the femur only. Mineral and organic percentage did not change in the bone with age, while the apparent percentages did change. Compositional parameters altogether can explain 35%-59% of the variation in fracture toughness. We conclude that bone composition and density have an important influence on fracture resistance of the cortical bone.

The influence of bone morphology on fracture toughness of the human femur and tibia.

The influence of porosity, osteon density, osteonal area, osteonal lamellar area, osteon size, and haversian canal size on the tension and shear fracture toughness, that is, the mode I and mode II strain energy release rate (GIc and GIIc), respectively, were investigated for the human femur and the tibia. The results suggest that porosity and osteon density were the best explanatory morphological parameters for GIc and GIIc. Both GIc and GIIc significantly decrease with increasing porosity. They also increase with increasing osteon density, the increase being significant for the femur only. Morphological parameters, altogether, can explain 49%-68% of the variation in fracture toughness. We concluded that, although there must be other factors such as biochemical components and microdamage, osteon morphology has an important influence on fracture resistance of the cortical bone.

Effect of medication on biomechanical properties of rabbit bones: heparin induced osteoporosis.

The aim of this controlled study is to investigate the effect of heparin on osteoporosis initiation and of calcitonin and tamoxifen on the progress of osteoporosis induced by heparin through biomechanical means and, thus, to assist in clinical usage of these drugs. 32 four-month-old female New Zealand white rabbits were divided into four different experimental groups. The animals in group A were administered heparin (Liquemine) intraperitoneally at the dosage of 1000 IU/kg/day. The animals in group B were injected the same amount of heparin as those of group A, and in addition, were given calcitonin at the dosage of 100 IU/kg/day. The animals in group C were medicated the same way as group B but 2 mg/kg/day tamoxifen (Nolvadex) was orally added into their intestine via cannula, one side connected to the injector. The animals in group D were the control. The experiment lasted 8 weeks. The animals in all experimental groups showed the same growth pattern as that of the control group. Whole-bone femur, humerus and tibia specimens were subjected to 3-point bending tests while sections from the proximal ends of the same specimens were subjected to compression tests. The data, recorded as load vs deflection, were converted into stress vs strain using the strength of materials formulae. The data obtained from the bending and the compression experiments were treated separately. The stiffness of the bones of the medicated groups were compared with those of the control groups. Our data indicated that the tamoxifen treated humera, femora and tibiae attained the largest bending stiffness in all cases investigated. However, this was not the case for compression. None of the drug administered groups attained the stiffness of the control group except for the case of tamoxifen treated femora which attained stiffness close to that of the controls. The results show that heparin altered the mechanical properties of bones indicating osteoporosis, tamoxifen was effective in reducing the effect of heparin while calcitonin yielded no conclusive result.