Effect of microgravity on collagenase deproteinization and EDTA decalcification of bone fragments

Undecalcified (n = 140) and decalcified (n = 11) bone fragments were treated with either collagenase (to remove collagen portion; undecalcified n = 64, decalcified n = 11) or EDTA (to remove mineral portion; n = 76) under the reduced gravity environment on US Space Shuttle mission STS-57. The fragments were initially stored in Dulbecco's phosphate buffer solution. After orbit had been established, fragments were exposed to either a neutral buffered collagenase or EDTA solution. Reactions were terminated (neutral buffered formalin for collagenase, 21% CuSO4 5H2O for EDTA) before reentry to earth's atmosphere. Differences in bone samples mass from before flight to after flight were measured. EDTA-treated sample mass was corrected for CuSO4 content. Flight and matched ground (gravitational control) sample showed similar EDTA-induced loss of mineral mass. Collagenase treatments, however, appeared to be more effective in flight samples compared to ground control samples. The flight-exposed, collagenase-treated samples showed significantly more loss of mass than did ground samples. The microgravity environment appeared to promote proteolytic reactions in bone more than the EDTA decalcification reaction.     

Kinetic characterization of the deproteinization of trabecular and cortical bovine femur bones

The present study proposes an interpretation of the mechanism of bone deproteinization. Cortical and trabecular bovine femur bones were deproteinized using 6% NaOCl (37, 50, 60 °C). The kinetic parameters (rate constant and activation energy) were calculated, and the surface area of each type of bone was considered. A statistical analysis of the rate constants shows that cortical bone deproteinizes at a lower rate than trabecular. The activation energy is higher for trabecular than cortical bone, and no significant differences are found in the protein concentration values for both bones. Therefore, although trabecular bone deproteinizes at a higher rate than cortical, trabecular bone requires more energy for the deproteinization reaction to take place. Considering that both types of bones are constituted by mineral, protein, and water; the present work shows that the individual inner matrix architecture of trabecular and cortical bones, along with characteristics such as the mineral concentration and its bonding with collagen fibers, may be the responsible factors that control protein depletion.     

Multiscale modeling of elastic properties of cortical bone

We model cortical bone as a composite material with hierarchical structure. At a nanostructural level, bone is composed of cross-linked collagen molecules, containing water and non-collagenous proteins in their gaps, reinforced with hydroxyapatite-like nanocrystals. Such a nanocomposite structure represents a mineralized collagen fibril, which serves as a primary building block of bone. At a sub-microstructural level (few microns), the mineralized collagen fibrils are embedded in an extrafibrillar hydroxyapatite matrix to form a single lamella, which also contains the lacunar cavities. At a microstructural level (hundreds of microns) one can distinguish two lamellar structures in the mature cortical bone: osteons, made of concentric layers of lamellae surrounding long hollow Haversian canals, and interstitial lamellae made of remnants of old osteons. At a mesostructural level (several millimeters), the cortical bone is represented by a random collection of osteons and resorption cavities in the interstitial lamellae. A macrostructural level is the whole bone level containing both the cortical (compact) and trabecular (spongy) bone types. In this paper, we predict analytically the effective elastic constants of cortical bone by modeling its elastic response at these different scales, spanning from the nanostructural to mesostructural levels, using micromechanics methods and composite materials laminate theories. The results obtained at a lower scale serve as inputs for the modeling at a higher scale. The predictions are in good agreement with the experimental data reported in literature.     

Effects of fatigue induced damage on the longitudinal fracture resistance of cortical bone

As a composite material, cortical bone accumulates fatigue microdamage through the repetitive loading of everyday activity (e.g. walking). The accumulation of fatigue microdamage is thought to contribute to the occurrence of fragility fractures in older people. Therefore it is beneficial to understand the relationship between microcrack accumulation and the fracture resistance of cortical bone. Twenty longitudinally orientated compact tension fracture specimens were machined from a single bovine femur, ten specimens were assigned to both the control and fatigue damaged groups. The damaged group underwent a fatigue loading protocol to induce microdamage which was assessed via fluorescent microscopy. Following fatigue loading, non-linear fracture resistance tests were undertaken on both the control and damaged groups using the J-integral method. The interaction of the crack path with the fatigue induced damage and inherent toughening mechanisms were then observed using fluorescent microscopy. The results of this study show that fatigue induced damage reduces the initiation toughness of cortical bone and the growth toughness within the damage zone by three distinct mechanisms of fatigue–fracture interaction. Further analysis of the J-integral fracture resistance showed both the elastic and plastic component were reduced in the damaged group. For the elastic component this was attributed to a decreased number of ligament bridges in the crack wake while for the plastic component this was attributed to the presence of pre-existing fatigue microcracks preventing energy absorption by the formation of new microcracks.     

Effect of mineral content on the nanoindentation properties and nanoscale deformation mechanisms of bovine tibial cortical bone

In this paper, a multitechnique experimental and numerical modeling methodology was used to show that mineral content had a significant effect on both nanomechanical properties and ultrastructural deformation mechanisms of samples derived from adult bovine tibial bone. Partial and complete demineralization was carried out using phosphoric and ethylenediamine tetraacetic acid treatments to produce samples with mineral contents that varied between 37 and 0 weight percent (wt%). The undemineralized samples were found to have a mineral content of ~58 wt%. Nanoindentation experiments (maximum loads ~1000 μN and indentation depths ~500 nm) perpendicular to the osteonal axis for the ~58 wt% samples were found to have an estimated elastic modulus of ~7–12 GPa, which was 4–6× greater than that obtained for the ~0 wt% samples. The yield strength of the ~58 wt% samples was found to be ~0.24 GPa; 3.4× greater than that of the ~0 wt% sample. These results are discussed in the context of in situ and post-mortem atomic force microscopy imaging studies which show clear residual deformation after indentation for all samples studied. The partially demineralized samples underwent collagen fibril deformation and kinking without loss of the characteristic banding structure at low maximum loads (~300 μN). At higher maximum loads (~700 μN) mechanical denaturation of collagen fibrils was observed within the indent region, as well as disruption of interfibril interfaces and slicing through the thickness of individual fibrils leading to microcracks along the tip apex lines and outside the indent regions. A finite element elastic-plastic continuum mechanical model was able to predict the nanomechanical behavior of all samples on loading and unloading.     

Effects of ovariectomy on rat mandibular cortical bone: a study using Raman spectroscopy and multivariate analysis

To investigate the effects of ovariectomy (OVX) on rat mandibular bone, the physicochemical compositions of mandibular cortical bone of ovariectomy and sham operated rats 2, 4, and 8 months after surgery were compared using Raman spectroscopy. With principal component analysis and linear discriminant analysis based on the Raman spectra, the mandibular cortical bone of the OVX group was clearly distinguished from that of the sham-operated group 8 months after surgery with no overlap. Specifically, significant reductions in the mineral-to-matrix ratio and full width at half-maximum as well as a significant increase in the carbonate-to-phosphate ratio were observed in the mandibular cortical bone of the OVX group. Results support the hypothesis that Raman spectroscopy is sensitive enough to distinguish between OVX and sham-operated mandibles with multivariate analysis by detecting the chemical composition of the mandibular cortical bone. The parameters mineral-to-matrix ratio, carbonate-to-phosphate ratio, and full width at half-maximum can appropriately characterize changes in the chemical composition of the mandibular cortical bone after OVX.     

The effects of bovine trabecular bone matrix particulates on cortical bone repair

This paper reports the effects of a synthetic bone substitute and bone allograft on cortical bone repair in an experimental model. To test the hypothesis that bovine trabecular bone matrix, BBM, can enhance the repair rate of cortical bone, osteotomies were created in the rabbit fibula and filled with either allograft or BBM particulates or left empty as controls. At five weeks post-surgery, mechanical tests and histological evaluations were performed. No significant differences were observed in the mechanical properties of the healing bone in the three animal groups (n=6). Histologically, the medullary cavity was obstructed and the cross-sectional area ratio of the osteotomies to intact bone was approximately 3 : 1. Highly significant area differences were observed between the intact bone group and both the BBM and the allograft groups . At the junction between the original bone and the newly formed bone, both woven and lamellar bone microstructures were prevalent. However, in the BBM filled defects, the woven bone microstructure was not ostentatious. It is concluded that failure to demonstrate significantly differences between the treatments were due to the small sample sizes and or the efficacy of the tensile analysis.     

Effect of microstructure on micromechanical performance of dry cortical bone tissues

The mechanical properties of bone depend on composition and structure. Previous studies have focused on macroscopic fracture behavior of bone. In the present study, we performed microindentation studies to understand the deformation properties and microcrack–microstructure interactions of dry cortical bone. Dry cortical bone tissues from lamb femurs were tested using Vickers indentation with loads of 0.245–9.8 N. We examined the effect of bone microstructure on deformation and crack propagation using scanning electron microscopy (SEM). The results showed the significant effect of cortical bone microstructure on indentation deformation and microcrack propagation. The indentation deformation of the dry cortical bone was basically plastic at any applied load with a pronounced viscoelastic recovery, in particular at lower loads. More microcracks up to a length of approximately 20 μm occurred when the applied load was increased. At loads of 4.9 N and higher, most microcracks were found to develop from the boundaries of haversian canals, osteocyte lacunae and canaliculi. Some microcracks propagated from the parallel direction of the longitudinal interstitial lamellae. At loads 0.45 N and lower, no visible microcracks were observed.     

Effect of cryo-induced microcracks on microindentation of hydrated cortical bone tissue

Microcracks accumulate in cortical bone tissue as a consequence of everyday cyclic loading. However, it remains unclear to what extent microdamage accumulation contributes to an increase in fracture risk. A cryo-preparation technique was applied to induce microcracks in cortical bone tissue. Microcracks with lengths up to approximately 20 μm, which were initiated mainly on the boundaries of haversian canals, were observed with cryo-scanning electron microscopy. A microindentation technique was applied to study the mechanical loading effect on the microcracked hydrated bone tissue. The microindentation patterns were section-scanned using confocal laser scanning microscopy to understand the deformation and bone damage mechanisms made by mechanical loading. The results show that there was no significant difference with respect to microhardness between the original and microcracked hydrated cortical bone tissues (ANOVA, p > 0.05). The cryo-induced microcracks in the bone tissue were not propagated further under the mechanical loads applied. The deformation mechanism of the microcracked cortical bone tissue was plastic deformation, not brittle fracture.     

Hierarchical analysis and multi-scale modelling of rat cortical and trabecular bone

The aim of this study was to explore the hierarchical arrangement of structural properties in cortical and trabecular bone and to determine a mathematical model that accurately predicts the tissue''s mechanical properties as a function of these indices. By using a variety of analytical techniques, we were able to characterize the structural and compositional properties of cortical and trabecular bones, as well as to determine the suitable mathematical model to predict the tissue''s mechanical properties using a continuum micromechanics approach. Our hierarchical analysis demonstrated that the differences between cortical and trabecular bone reside mainly at the micro- and ultrastructural levels. By gaining a better appreciation of the similarities and differences between the two bone types, we would be able to provide a better assessment and understanding of their individual roles, as well as their contribution to bone health overall.     

Effect of bioactive glass granules and polytetrafluoroethylene membrane on repair of cortical bone defect

The effect of bioactive glass (BG) granules and nonresorbable polytetrafluoroethylene (PTFE) membrane on the repair of cortical bone defects was studied. Monocortical holes (diameter 3.0 mm) were drilled in rabbit tibia. Sixteen holes were filled with BG granules (diameter 630–800 m). Twelve holes were left empty and covered with PTFE membrane. No material was used at ten control holes. All experiment areas were covered with periosteum attached to the soft tissue flap. Histomorphometric evaluation of resection specimens showed that new bone and glass particles formed a continuous bridge in the BG group at the upper part of the hole, occupying 73.6% and 61.7% of the defect at 6 and 12 weeks, respectively. If only the amount of bone but not glass particles was included in the measurements the corresponding figures were 31.4% and 41.5%. The bone repair in the PTFE group was 12.1% and 11.3% and in the control group 25.1% and 23.3% at 6 and 12 weeks, respectively. The results indicate that BG granules improve repair of cortical bone defects and PTFE membrane seems to impair bone formation in these defects.     

Mechanical properties and bone densities of canine trabecular bone

The purposes of this study were (1) to evaluate the mechanical properties of canine epiphyseal cancellous bones from adult canine femoral heads, femoral condyles, tibial plateau, and humeral heads, using indentation and compression tests, and (2) to measure bone densities (apparent density and ash density) of these cancellous bones so as to develop a normal data base of mechanical strength and bone density. The correlations between the two mechanical tests and between these tests and bone densities were also considered. The results showed all of the three mechanical parameters, ultimate load, stiffness, and ultimate strength, measured by the indentation test were higher than those measured by the compression test. Correlation analysis showed that the two sets of mechanical values correlated well (r=0.823–0.952, p<0.01). The apparent density and ash density correlated well with the mechanical parameters determined by the two types of mechanical tests (r=0.737–0.966, p<0.05). © 1998 Chapman & Hall     

Ultrasonic assessment of cortical bone thickness in vitro and in vivo

In osteoporosis, total bone mass decreases and the thickness of the cortical layer diminishes in the shafts of the long bones. In this study, a simple ultrasonic in vivo method for determining the thickness of the cortical bone layer was applied, and the suitability of two different signal analysis techniques, i.e., envelope and cepstral methods, for measuring cortical thickness was compared. The values of cortical thickness, as determined with both techniques, showed high linear correlations (r ges 0.95) with the thickness values obtained from in vitro measurements with a caliper or in vivo measurements by peripheral quantitative CT (pQCT). No systematic errors that could be related to the cortical thickness were found. The in vivo accuracy of the measurements was 6.6% and 7.0% for the envelope and cepstral methods, respectively. Further, the in vivo precision for the envelope and cepstral methods was 0.26 mm and 0.28 mm, respectively. Although the results are similar for both of the techniques, the simplicity of the envelope method makes it more attractive for clinical applications. In conclusion, a simple ultrasound measurement provides an accurate estimate of the cortical bone thickness. The techniques investigated may have clinical potential for osteoporosis screening and therefore warrant more extensive clinical investigations with healthy and osteoporotic individuals.     

Orientation and loading condition dependence of fracture toughness in cortical bone

The fracture toughness at crack initiation were determined for bovine cortical bone under tension (mode I), shear (mode II), and tear (mode III). A total of 140 compact tension specimens, compact shear specimens and triple pantleg (TP) specimens were used to measure fracture toughness under tension, shear, and tear, respectively. Multiple-sample compliance method was utilized to measure the critical strain energy release rate (G_c) at the a/W=0.55 (crack length, a, to specimen width, W, ratio). The critical stress intensity factor (K_c) was also calculates from the critical loading (P_c) of the specimens at the a/W=0.55. The effect of the anisotropy of bone on its resistance to crack initiation under shear and tear loading was investigated as well. Fracture toughness of bone with precrack orientations parallel (designed as longitudinal fracture) and vertical (designed as transverse fracture) to the longitudinal axis of bone were compared. In longitudinal fracture, the critical strain energy release rate (G_c) of cortical bone under tension, shear, and tear was 644±102, 2430±836, and 1723±486 N/m, respectively. In transverse fracture, the critical strain energy release rate (G_c) of cortical bone under tension, shear, and tear was 1374±183, 4710±1284, and 4016±948 N/m, respectively. An unpaired t-test analysis demonstrated that the crack initiation fracture toughness of bone under shear and tear loading were significantly greater than that under tensile loading in both longitudinal and transverse fracture (P<0.0001 for all). Our results also suggest that cortical bone has been “designed” to prevent crack initiation in transverse fracture under tension, shear, and tear.     

猪股骨常应变率动态压缩试验方法研究

准确的骨骼材料响应对于模拟交通事故、战争和跌落中的人体损伤机制作用重大,但上述工况所对应的皮质骨动态力学响应还没有在常应变率条件下被规范测量。为在动态压缩试验中实现和维持不同的恒定应变率,建立针对皮质骨的重复性良好的霍普金森杆动态材料测试方法,并测试大应变率范围内的骨骼材料力学参数。首先测试纯铝整形器尺寸和子弹撞击入射杆速度对入射脉冲形状的改变效果,并在此基础上不断改变入射脉冲形状直至满足常应变率条件,随后对32个加工规则的猪股骨中轴皮质骨试样按照应变率范围进行分组测量。当入射波平台部分斜率与透射波上升沿斜率相等时,试样以常应变率变形,此时常应变率产生的变形超过总应变量的70%。猪股骨皮质骨在200～1 500 s~(-1)应变率间极限应力和极限应变增大(分别为10.2%与25.0%),弹性模量减小(7%)。结果表明,相对于杆材料更软的塑性金属作为整形器能使骨骼在需要的应变率范围内保持应变率恒定,且重复性良好,适宜进一步用于人骨测试。     

Analysis of heat-treated bovine cortical bone by thermal gravimetric and nanoindentation

Xenograft bone has been widely used as a bone grafting material because it gains advantages in biological and mechanical properties as compare with the use of an allograft bone. Heat-treatment of bone is recognized as one of the simple and practical methods to lower the human immunodeficiency virus (HIV) infection and overcome the risks of rejection and disease transfer during the bone transplantation. Therefore, understanding the change of bone’s organic matrix after heat treatment has become a significant topic. In this study, thermal gravimetric analysis (TGA) was used to investigate the condition of organic constituents of a bovine cortical bone. In order to well characterize the microstructural and mechanical property of the bone after heat treatment, nanoindention technique was also employed to measure the localized elastic modulus (E) and hardness (H) of its interstitial lamellae and osteons lamellae at the temperatures of 23 °C (RT), 37 °C, 90 °C, 120 °C and 160 °C, respectively.The TGA results demonstrated that heat-treated bones had three stages of weight loss. The first stage was the loss of water, which started from RT to 160 °C. Follow by a weight loss of organic constituents starting from 200 °C to 600 °C. Upon reaching 600 °C, the organic constituents were decomposed and mineral phase loss started taking place until 850 °C. From the nanoindentation results, it showed the values of E and H measured for the interstitial lamellae were higher than that of the osteons lamellae. This phenomenon indicates that the interstitial lamellae are stiffer and easy to be mineralized than osteons lamellae. For a specimen heat-treated at 90 °C, the values of E and H of interstitial lamellae and osteons lamellae were similar to a non-heat-treated specimen. For a specimen heat-treated at 120 °C, its interstitial lamellae had higher E and H values than osteons lamellae. When a specimen was heat-treated at 160 °C, both interstitial lamellae and osteons lamellae demonstrated a slight decrease of their E and H values. An ANOVA statistical analysis was used to analyze the difference in elastic properties and hardness in various temperature ranges.     

Structural differences in the cortical bone of Turkey tibia

Different regions namely anterior, posterior, medial and lateral, of the cortical bone in six sections in the diaphysis from proximal to the distal end of a Turkey tibia were evaluated for structural differences. The predominant feature, Haversian canals showed high area fraction (can be viewed as high porosity) in the posterior region as compared to the anterior region and this difference is attributed to the high compressive and tensile stress states in the respective regions. With increasing size, the Haversian canals deviated from circular shapes in cross section. The distance from the lacunae to the center of the Haversian canal showed an increase with the size of the canals. However, the canal to lacunae-canal distance ratio is independent of the anterior and posterior regions. It was found that majority of the lacunae are placed at a distance of about 2.5 to 3 times the Haversian canal radius. At the ultrastructural level, periodic banding of the collagen fibers showed a bimodal banding in the anterior region while only one finer banding distribution in the posterior region and could probably the result of the different stress states in the two regions. The Haversian canals in the three dimensional reconstruction showed branching, twisted canals and Haversian space and resemble Cohen and Harris model. Differences in stress in different regions cause structural changes in bone.     

Formation of nanometer-scale gap electrodes based on a plasma ashing technique

We propose a new and reproducible method to fabricate metal electrodes with a nanometer-scale gap and width, which have been considered as one of the basic building blocks for future nano-devices. The techniques used in this study were the conventional photolithography, e-beam lithography and plasma ashing techniques. Specifically, the plasma ashing process was used to easily form the nanometer-sized gap (10 nm or less) that is difficult to realize by e-beam lithography only. Using the method investigated in this work, we demonstrated that Au electrodes with a nanometer-sized gap and width could be easily fabricated.