Although the effects of dehydration on the mechanical behavior of cortical bone are known, the underlying mechanisms for such effects are not clear. We hypothesize that the interactions of water with the collagen and mineral phases each have a unique influence on mechanical behavior. To study this, strength, toughness, and stiffness were measured with three-point bend specimens made from the mid-diaphysis of human cadaveric femurs and divided into six test groups: control (hydrated), drying in a vacuum oven at room temperature (21 °C) for 30 min and at 21, 50, 70, or 110 °C for 4 h. The experimental data indicated that water loss significantly increased with each increase in drying condition. Bone strength increased with a 5% loss of water by weight, which was caused by drying at 21 °C for 4 h. With water loss exceeding 9%, caused by higher drying temperatures (≥70 °C), strength actually decreased. Drying at 21 °C (irrespective of time in vacuum) significantly decreased bone toughness through a loss of plasticity. However, drying at 70 °C and above caused toughness to decrease through decreases in strength and fracture strain. Stiffness linearly increased with an increase in water loss. From an energy perspective, the water-mineral interaction is removed at higher temperatures than the water-collagen interaction. Therefore, we speculate that loss of water in the collagen phase decreases the toughness of bone, whereas loss of water associated with the mineral phase decreases both bone strength and toughness.
The objective of this study was first to prove the concept of a low field pulsed nuclear magnetic resonance (NMR) process for assessing the cortical porosity and pore size distribution of human bone in vitro, and then to apply the technique to detect age-related changes of bone in these parameters. The Carr-~Purcell--Meiboom-Gill NMR spin echo train method is used to determine the porosity, and an inversion NMR spin-spin relaxation (T2) spectrum is used to assess the pore size distribution in cortical bone. Using these techniques, cortical porosity and pore size distribution of 19 specimens of human cadaveric bone, ranging from 16 to 89 years of age, were assessed. The NMR results were compared with the histomorphometric data of the same bone samples to verify the efficacy of the NMR approach. Moreover, a coefficient (surface relaxivity) relating the pore size to the T2 relaxation time was determined empirically for the Haversian canals and the osteocytic lacunae. The results of this study demonstrate that the in vitro NMR approach using T2 relaxation techniques can directly assess the porosity and pore size distribution (Haversian canals and osteocytic lacunae) in human cortical bone. In addition, this study indicates that the age-related changes in cortical porosity relate predominantly to Haversian canals, whereas the porosity of osteocytic lacunae appears to be independent of age.
The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37-200°C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three-point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone. thereby supporting the hypothesis of this study.
A NMR spin–spin (T2) relaxation technique has been described for determining water distribution changes in human cortical bone tissue. The advantages of using NMR T2 relaxation techniques for bone water distribution are illustrated. The CPMG T2 relaxation data can be inverted to T2 relaxation distribution and this distribution then can be transformed to a pore size distribution with the longer relaxation times corresponding to larger pores. The FID T2 relaxation data can be inverted to T2 relaxation distribution and this distribution then can be transformed to bound- and mobile-water distribution with the longest relaxation time corresponding to mobile water and the middle relaxation time corresponding to bound water. The technique is applied to quantify apparent changes in porosity, bound and mobile water in cortical bone. Overall bone porosity is determined using the calibrated NMR fluid volume from the proton relaxation data divided by overall bone volume. The NMR bound and mobile water changes were determined from cortical bone specimens obtained from male and female donors of different ages. Differences in water distribution were found between specimens from male and female donors. Furthermore, the distribution of water within a single specimen was found to be non-homogeneous. Our results show that the ratio of the average bound to mobile water in bone from male donors is higher than in bone from female donors when the bone porosities are similar between male and female groups. We also show that the average bone porosity multiplied by the ratio of bound to mobile water is constant for both male and female bone groups. This parameter may be used as a measure of bone quality describing both porosity and water content, both of which may be important determinants of bone strength and fracture resistance.
The purpose of this study was to explore the effects of changes in Type I collagen on the viscoelasticity of bone. Bone coupons were heated at either 100 or 200 degrees C to induce the thermal denaturation of Type I collagen. Half of these specimens were rehydrated after heat treatment; the other half were tested in a dry condition. The degree of denatured collagen (DC%) was analyzed by a selective digestion technique with the use of alpha-chymotrypsin. Isothermal (37 degrees C) and variable temperature tests (scans from 35 to 200 degrees C) were performed with the use of a dynamic mechanical analyzer to evaluate changes in bone viscoelastic properties as a function of collagen damage, specifically, changes in the loss factor (tan delta) and storage modulus (E') were assessed. Significant collagen denaturation occurred only when bone was heated at 200 degrees C irrespective of the hydration condition. Also, DC% did not show a significant effect on tan delta. However, higher values of tan delta were observed in wet samples compared to dry specimens. The temperature-scan tests revealed that the hydration condition, but not DC%, significantly affected the behavior of tan delta. However, E' was not strongly influenced either by DC% or by water content. These results suggest that at a constant frequency the denaturation of collagen triple-helical molecules may have few effects on the viscoelasticity of bone, but moisture may play a prominent role in determining this property.
The purpose of this study was to examine the use of a dynamic mechanical analyzer (DMA) system to study the viscoelastic nature of bone. Cortical bone specimens from human femora were tested isothermally for 150 min at 37 degrees C and the loss factor (tan delta) and storage modulus (E') were measured. To explore the effects of test conditions on tan delta and E', different levels of applied stress, two specimen sizes, and two hydration conditions (wet and vacuum-dried) were evaluated. Finally, nonisothermal tests were performed, wherein specimens were heated up to 70 degrees C at different heating rates: 1 degrees C/min, 3 degrees C/min, and 5 degrees C/min. The results indicated that a threshold level of minimum applied stress was required to obtain repeatable and relatively constant values of tan delta. Specimen size did not significantly affect tan delta although it influenced E'. Moisture content had a significant effect on tan delta; vacuum-dried specimens exhibited a lower tan delta compared to wet specimens. Lastly, heating rates influenced tan delta values with lower rates producing more consistent results. The study demonstrated that DMA can be used as an effective tool to test bone.
Collagen crosslinks are important to the quality of bone and may be contributors to the age-related increase in bone fracture. This study was performed to investigate whether age and gender effects on collagen crosslinks are similar in osteonal and interstitial bone tissues. Forty human cadaveric femurs were collected and divided into two age groups: Middle aged (42-63 years of age) and Elderly (69-90 years of age) with ten males and ten females in each group (n = 10). Micro-cores of bone tissue from both secondary osteons (newly formed) and interstitial regions (biologically old) in the medial quadrant of the diaphysis were extracted using a custom-modified, computer numerical controlled machine. The bone specimens were then analyzed using high performance liquid chromatography to determine the effects of age and gender on the concentration of mature, enzymatic crosslinks (hydroxylysyl-pyridinoline -HP and lysylpyridinoline -LP) and a non-enzymatic crosslink (pentosidine -PE) at these two bony sites. The results indicate that age has a significant effect on the concentration of LP and PE, while gender has a significant effect on HP and LP. In addition, the concentration of the crosslinks in the secondary osteons is significantly different from that in the interstitial bone regions. These results suggest that the rate of non-enzymatic crosslinking may increase while the formation of maturate enzymatic crosslinks may decrease with age. Such changes could potentially reduce the inherent quality of the bone tissue in the elderly skeleton.
Advanced glycation end products (AGEs) have been observed to accumulate in bone with increasing age and may impose effects on bone resorption activities. However, the underlying mechanism of AGEs accumulation in bone is still poorly understood. In this study, human cortical bone specimens from young (31±6 years old), middle-aged (51±3 years old) and elderly (76±4 years old) groups were examined to determine the spatial-temporal distribution of AGEs in bone matrix and its effect on bone resorption activities by directly culturing osteoclastic cells on bone slices. The results of this study indicated that the fluorescence intensity (excitation wave length 360 nm and emission wave length 470±40 nm) could be used to estimate the relative distribution of AGEs in bone (pentosidine as its marker) under an epifluorescence microscope. Using the fluorescence intensity as the relative measure of AGEs concentration, it was found that the concentration of AGEs varied with biological tissue ages, showing the greatest amount in the interstitial tissue, followed by the old osteons, and the least amount in newly formed osteons. In addition, AGEs accumulation was found to be dependent on donor ages, suggesting that the younger the donor the less AGEs were accumulated in the tissue. Most interestingly, AGEs accumulation appeared to initiate from the region of cement lines, and spread diffusively to the other parts as the tissue aged. Finally, it was observed that the bone resorption activities of osteoclasts were positively correlated with the in situ concentration of AGEs and such an effect was enhanced with increasing donor age. These findings may help elucidate the mechanism of AGEs accumulation in bone and its association with bone remodeling process.
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