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.     

Effects of deproteinization and ashing on site-specific properties of cortical bone

Buffered sodium hypochlorite (NaOCl) solution was used to remove selectively the collagen phase from bovine cortical bone. Changes in the mechanical behaviour and material properties were studied over a wide range of resolution (from 5 m to 3 mm) using an integrated combination of experimental techniques. Optical microscopy indicated that timed immersion in NaOCl results in cortical bone specimens that consist of a mineralized tissue core surrounded by a layer of deproteinized or anorganic bone. With increased NaOCl treatment, the mechanical behaviour in three-point flexure of the intact specimens became increasingly characteristic of a brittle ceramic material. Localized material properties were evaluated using histology, scanning electron microscopy and microhardness testing. The site-specific properties and the mineralization of the cores were not significantly affected by the treatment; however, the interactions and structural framework of the hydroxyapatite crystallites within the anorganic material were compromised. This destruction of crystallite interlocking was not observed in samples in which the organic phase was removed by ashing at 800°C. The ashed samples maintained microhardness values three times those of the bleached samples. Because of its damaging effects on cortical bone structural integrity, the NaOCl treatment did not provide a reasonable means of studying, as a function of the phasic mass fraction, incremental changes in bone mechanical behaviour or the relative roles of collagen and mineral within the structural hierarchy.     

Distribution of hydroxyapatite crystallite orientation and ultrasonic wave velocity in ring-shaped cortical bone of bovine femur

At the nanoscopic level, bone consists of calcium phosphate, which forms incomplete hydroxyapatite (HAp) crystals. The preferred orientation of the c-axis of HAp crystallites induces anisotropy and inhomogeneity of elastic properties in bone. In this study, the effect of the preferred orientation of HAp crystallites on the spatial distribution of ultrasonic wave velocity was experimentally investigated, considering bone mineral density (BMD) and microstructure. Three ring-shaped cortical bone samples were made from a 36-month-old bovine femur. Longitudinal wave velocity was measured by a conventional ultrasonic pulse system, using self-made polyvinylidene fluoride transducers. The integrated intensity of the (0002) peak obtained using X-ray diffraction was estimated to evaluate the amount of preferred orientation. The velocity distribution pattern was similar to the distribution of integrated intensity of (0002). The effect of the preferred orientation of HAp crystallites on velocity was clearly observed in the plexiform structure, despite the fact that the BMD value was almost independent of the preferred orientation of HAp crystallites. Velocity measurement of cortical bone can reveal information about HAp crystallite orientation.     

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.     

Effects of drying on the mechanical properties of bovine femur measured by nanoindentation

Effects of drying on the measurement of mechanical properties of bone by nanoindentation methods have been examined. Tests were conducted to measure the elastic modulus and hardness of two cross-sectional cortical specimens obtained from adjacent areas of bovine femur. One specimen was thoroughly dried in air prior to testing while the other was stored in deionized water. The properties of osteons and interstitial lamellae showed statistically significant differences (plt; 0.0001) and were therefore investigated separately. Drying was found to increase the elastic modulus by 9.7% for interstitial lamellae and 15.4% for osteons. The hardness was also found to increase by 12.2% for interstitial lamellae and 17.6% for osteons.     

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.     

Immuno-SEM characterization of developing bovine cartilage

Collagen is a vital material in the tissues of living organisms. Found almost everywhere in the human body, collagen is important in connective tissues, bone growth, and cartilage. Collagen XI makes up a very small portion of the cartilaginous tissue; however, it plays a key role in cartilaginous tissue. Collagen XI and two collagen XI isoforms, V1b and V2, are critical in the ossification process. The location of collagen XI, V1b, V2, and their specific functions in the ossification process within developing bovine cartilage are not well characterized. In this work, the location of collagens I, II, XI and two collagen XI isoforms, V1b and V2, present in developing bovine cartilage are investigated using the immuno-SEM technique. The results for the locations of collagen I and II indicate a high level of consistency with previous work, thus showing that the technique of immuno-SEM can be used with confidence to determine the location of various collagen types within cartilaginous and mineralized tissue. This work has shown that collagen XI is present in the lower hypertrophic region and also in a pericellular arrangement, within about two microns of cell walls, throughout the cartilaginous tissue. V1b is expressed in the articular surface, mineralized region, resting zone, and the distal edge of the diaphysis. The V2 isoform is most strongly expressed in areas of newly forming cartilage, and disappears with chondrocyte maturation. V2 is present in the distal edge of the epiphysis, as well as in mineralized tissue. Collagen XI and two of its isoforms, V1b and V2, are thought to play a critical role in the ossification process. However, this role is not well understood, and is still being characterized. The detection of collagen XI and two of its isoforms in the osteo-chondral junction as well as at a joint surface further point to collagen XI, V1b, and V2 playing a vital role in the ossification process, and warrants further research as to their specific function within the ossification process.     

Non-uniqueness of cohesive-crack stress-separation law of human and bovine bones and remedy by size effect tests

It is shown that if the bilinear stress-separation law of the cohesive crack model is identified from the complete softening load-deflection curve of a notched human bone specimen of only one size, the problem is ill-conditioned and the result is non-unique. The same measured load-deflection curve can be fitted with values of initial fracture energy and tensile strength differing, respectively, by up to 100 and 72.4 % (of the lower value). The material parameters, however, give very different load-deflection curves when the specimen is scaled up or down significantly. This implies that the aforementioned non-uniqueness could be avoided by testing human bone specimens of different sizes. To demonstrate it, tests of notched bovine bone beams of sizes in the ratio of 1: $\sqrt{6}$ :6 are conducted. To minimize random scatter, all the specimens are cut from one and the same bovine bone, even though this limits the number of specimens to 8. A strong size effect is found, but an anomaly in the size effect data trend is obtained, probably due to random scatter and too small a number of specimens. Further it is shown that the optimum range of size effect testing based on Ba?ant’s size effect law approximately coincides with the size range of beams that can be cut from one bovine bone. By size effect fitting of previously published data on human bone, it is shown that the optimum size range calls for beam depths under 10 mm, which is too small for the standard equipment of mechanics of materials labs and would require a special miniaturized precision equipment.     

Nanoscale characterization of the biomechanical hardening of bovine zona pellucida

The zona pellucida (ZP) is an extracellular membrane surrounding mammalian oocytes. The so-called zona hardening plays a key role in fertilization process, as it blocks polyspermy, which may also be caused by an increase in the mechanical stiffness of the ZP membrane. However, structural reorganization mechanisms leading to ZP''s biomechanical hardening are not fully understood yet. Furthermore, a correct estimate of the elastic properties of the ZP is still lacking. Therefore, the aim of the present study was to investigate the biomechanical behaviour of ZP membranes extracted from mature and fertilized bovine oocytes to better understand the mechanisms involved in the structural reorganization of the ZP that may lead to the biomechanical hardening of the ZP. For that purpose, a hybrid procedure is developed by combining atomic force microscopy nanoindentation measurements, nonlinear finite element analysis and nonlinear optimization. The proposed approach allows us to determine the biomechanical properties of the ZP more realistically than the classical analysis based on Hertz''s contact theory, as it accounts for the nonlinearity of finite indentation process, hyperelastic behaviour and material heterogeneity. Experimental results show the presence of significant biomechanical hardening induced by the fertilization process. By comparing various hyperelastic constitutive models, it is found that the Arruda–Boyce eight-chain model best describes the biomechanical response of the ZP. Fertilization leads to an increase in the degree of heterogeneity of membrane elastic properties. The Young modulus changes sharply within a superficial layer whose thickness is related to the characteristic distance between cross-links in the ZP filamentous network. These findings support the hypothesis that biomechanical hardening of bovine ZP is caused by an increase in the number of inter-filaments cross-links whose density should be higher in the ZP inner side.     

Kinetic and chemical characterization of thermal decomposition of dicumylperoxide in cumene

Dicumylperoxide (DCP) is one of the most used peroxides in the polymer industry. It has been reported that its thermal decomposition can result in runaway phenomena and thermal explosions with significant economic losses and injuries to people. In the present paper thermal behaviour of dicumylperoxide in cumene was investigated over the temperature range of 393-433 K under aerated and de-aerated conditions. The results indicated that when oxygen was present, the decomposition rate did not follow a simple pseudo-first order kinetic as previously reported in literature. A satisfactory fit of the experimental data was, in this case, achieved by means of kinetic expression derived under the assumption of an autocatalytic scheme of reaction. The reaction rate was, on the contrary, correctly described by a pseudo-first order kinetic in absence of oxygen. Under both aerated and de-aerated conditions, chemical analysis showed that the decomposition mainly resulted in the formation of acetophenone and dimethylphenylcarbinol with minor occurrence of 2,3-dimethyl-2,3-diphenylbutane. The formation of methane and ethane was also invariably observed while the appearance of cumylhydroperoxide as a reaction intermediate was detected under only aerated conditions. Therefore, two reaction schemes were proposed to explain system behaviour in the presence of oxygen and after its purging.     

Effects of structural anisotropy of cancellous bone on speed of ultrasonic fast waves in the bovine femur

Ultrasonic waves in cancellous bone change dramatically depending on its structural complexity. One good example is the separation of an ultrasonic longitudinal wave into fast and slow waves during propagation. In this study, we examined fast wave propagation in cancellous bone obtained from the head of the bovine femur, taking the bone structure into consideration. We investigated the wave propagation perpendicular to the bone axis and found the two-wave phenomenon. By rotating the cylindrical cancellous bone specimen, changes in the fast wave speed due to the rotation angle then were observed. In addition to the ultrasonic evaluation, the structural anisotropy of each specimen was measured by X-ray micro-computed tomography (CT). From the CT images, we obtained the mean intercept length (MIL), degree of anisotropy (DA), and angle of insonification relative to the trabecular orientation. The ultrasonic and CT results showed that the fast wave speed was dependent on the structural anisotropy, especially on the trabecular orientation and length. The fast wave speeds always were higher for propagation parallel to the trabecular orientation. In addition, there was a strong correlation between the DA and the ratio between maximum and minimum speeds (V(max)/V(min)) (R(2) = 0.63).     

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.     

2D and 3D mapping of microindentations in hydrated and dehydrated cortical bones using confocal laser scanning microscopy

We report on responses of hydrated and dehydrated cortical bone tissues to mechanical loading applied by a Vickers indenter. The Vickers indentations were imaged in two- and three-dimensions (2D and 3D) using confocal laser scanning microscopy (CLSM) to understand mechanical behavior of bone tissues. Serial optical sections of indentation patterns of dry and wet bones were collected using CLSM. The indention surface structures were mapped using topographical CLSM imaging. The observation of CLSM shows the fundamental indentation responses for both the hydrated and dehydrated bone tissues were plastic deformation. No visible fracture was observed in the Vickers indentation patterns in the wet bone tissue, while non-propagating lamellar microcracks occurred in the dry bone tissue. This indicates that drying resulted in increased brittleness of the bone tissue. The Vickers hardness values of dry bone tissue were significantly higher than those of wet bone tissue at any applied loads (analysis of variation, ANOVA, p < 0.05). The resolution limits of confocal microscopy were also discussed for bone tissue scanning.     

Kinetic characterization and comparison of various protein crosslinking reagents for matrix modification

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; l-threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP ? LT. Most of the crosslinkers displayed a pH dependence and were more effective at more alkaline pH. At optimal pH and saturating conditions, the relative reaction rates of the crosslinkers were: PA = GA > EDC > GP > MG ? LT.     

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.     

Structural and functional characterization of glutaraldehyde-polymerized bovine hemoglobin and its isolated fractions

Glutaraldehyde-polymerized bovine hemoglobin (PolyHbBv, trade name Oxyglobin), is a non-site-specific modified hemoglobin-based oxygen-carrying solution, developed for use in veterinary medicine. PolyHbBv was fractionated into four distinct tetrameric and multiple polytetrameric forms ranging in molecular mass (87.2-502.3 kDa) using size exclusion chromatography (SEC) and verified by laser light scattering. We evaluated the structural modification occurring in the fractionated mixture of PolyHbBv and assessed the functionality and redox stability of each fraction in relation to the mixture as a whole. Intramolecular cross-linking evaluation as performed by MALDI-MS and SEC under dissociating conditions revealed no-site-specific tetramer stabilization within the fractions; Intermolecular cross-linking was highly correlated with lysine and histidine modification as determined by amino acid composition analysis. While native unmodified hemoglobin, HbBv, PolyHbBv, and PolyHbBv fractions (F1-F4) revealed significant methionine oxidation, modification, or both, the critical betaMet55 located in the functionally plastic domains (alpha1-beta1 interface) of HbBv was unaltered. Moreover, neither of the two betaCys93 located in the highly plastic alpha1-beta2 interface were modified in PolyHbBv or in F1-F4. Our structural analysis also revealed that the reported loss in sensitivity to chloride in PolyHbBv could not be attributed to direct alteration of chloride ion binding amino acids. Structural modification imparted by glutaraldehyde resulted in nearly identical functional characteristics of PolyHbBv and its fractions with regard to oxygen equilibrium, ligand binding, and autoxidative kinetics.     

Atomic force microscopic studies on the structure of bovine femoral cortical bone at the collagen fibril-mineral level

The structure of cortical bone at the collagen-mineral level was investigated by means of atomic force microscopy. Surfaces of the specimens treated with collagenase and ethylenediaminetetraacetic acid (EDTA) were examined. Images of blob-like objects observed in intact specimen became clearly outlined after collagenase treatment; the sizes of the blob decreased, suggesting that each blob had been fragmented by the collagenase treatment. Following EDTA treatment of an intact specimen, an image of thread-like objects appeared; the thread was partly constructed by trains of blobs and the other parts of the threads had a periodic pattern along its longer axis. The period was almost equal to the collagen D-period of the Hodge–Petruska model, indicating that the threads are collagen fibrils and that the blobs are related to the mineral phase in bone. It was concluded that minerals were deposited on and along collagen fibrils. A decorated collagen fibril model for the spatial relationship between mineral and collagen fibril was proposed. According to our model, the mineral inside the collagen fibril is about one forth of the extrafibrillar mineral.     

基于超声导波反演参数定征皮质骨骨质状况研究

骨质疏松发生时，人体皮质骨层的孔隙度将增大。为研究皮质骨参数（厚度、横波速度、纵波速度）与骨质疏松症的关系，文章利用有限元方法对不同孔隙度（0∶3%∶27%）的单层皮质骨进行仿真，以3个周期的高斯包络正弦波作为激励，将采集到的超声导波信号先后经过二维傅里叶变换和Burg算法处理后得到频散数据，与基于FloquetBloch理论建立的理论频散曲线数据库进行匹配反演，得到皮质骨厚度、横波速度、纵波速度参数。结果显示皮质骨厚度反演准确，皮质骨孔隙度与横波速度和纵波速度呈负相关，横波速度敏感度为19.0%，纵波速度敏感度为5.5%。横波速度敏感度更高，临床诊断潜力更大。并对6组牛胫骨进行了离体实验，结果显示，反演得到的皮质骨厚度与其实际测量值的平均相对误差为4.0%，且实验频散曲线与理论频散曲线相吻合，验证了文中算法在真实皮质骨参数反演上的可行性和准确性。文中的研究在骨质疏松超声检测中具有应用潜力。