Age-related properties at the microscale affect crack propagation in cortical bone

The increased risk for fracture with age is associated not only with reduced bone mass but also with impaired bone quality. At the microscale, bone quality is related to porosity, microstructural organization, accumulated microdamage and intrinsic material properties. However, the link between these characteristics and fracture behavior is still missing. Bone tissue has a complex structure and as age-related compositional and structural changes occur at all hierarchical length scales it is difficult to experimentally identify and discriminate the effect of each mechanism. The aim of this study was therefore to use computational models to analyze how microscale characteristics in terms of porosity, intrinsic toughness properties and microstructural organization affect the mechanical behavior of cortical bone. Tensile tests were simulated using realistic microstructural geometries based on microscopy images of human cortical bone. Crack propagation was modelled using the extended finite element method where cement lines surrounding osteons were modelled with an interface damage law to capture crack deflections along osteon boundaries. Both increased porosity and impaired material integrity resulted in straighter crack paths with cracks penetrating osteons, similar to what is seen experimentally for old cortical bone. However, only the latter predicted a more brittle failure behavior. Furthermore, the local porosity influenced the crack path more than the macroscopic porosity. In conclusion, age-related changes in cortical bone affect the crack path and the mechanical response. However, increased porosity alone was not driving damage in old bone, but instead impaired tissue integrity was required to capture brittle failure in aging bone.     

Material heterogeneity,microstructure, and microcracks demonstrate differential influence on crack initiation and propagation in cortical bone

The recent studies have shown that long-term bisphosphonate use may result in a number of mechanical alterations in the bone tissue including a reduction in compositional heterogeneity and an increase in microcrack density. There are limited number of experimental and computational studies in the literature that evaluated how these modifications affect crack initiation and propagation in cortical bone. Therefore, in this study, the entire crack growth process including initiation and propagation was simulated at the microscale by using the cohesive extended finite element method. Models with homogeneous and heterogeneous material properties (represented at the microscale capturing the variability in material property values and their distribution) as well as different microcrack density and microstructure were compared. The results showed that initiation fracture resistance was higher in models with homogeneous material properties compared to heterogeneous ones, whereas an opposite trend was observed in propagation fracture resistance. The increase in material heterogeneity level up to 10 different material property sets increased the propagation fracture resistance beyond which a decrease was observed while still remaining higher than the homogeneous material distribution. The simulation results also showed that the total osteonal area influenced crack propagation and the local osteonal area near the initial crack affected the crack initiation behavior. In addition, the initiation fracture resistance was higher in models representing bisphosphonate treated bone (low material heterogeneity, high microcrack density) compared to untreated bone models (high material heterogeneity, low microcrack density), whereas an opposite trend was observed at later stages of crack growth. In summary, the results demonstrated that tissue material heterogeneity, microstructure, and microcrack density influenced crack initiation and propagation differently. The findings also elucidate how possible modifications in material heterogeneity and microcrack density due to bisphosphonate treatment may influence the initiation and propagation fracture resistance of cortical bone.     

Fracture toughness and fatigue crack propagation rate of short fiber reinforced epoxy composites for analogue cortical bone

Third-generation mechanical analogue bone models and synthetic analogue cortical bone materials manufactured by Pacific Research Laboratories, Inc. (PRL) are popular tools for use in mechanical testing of various orthopedic implants and biomaterials. A major issue with these models is that the current third-generation epoxy-short fiberglass based composite used as the cortical bone substitute is prone to crack formation and failure in fatigue or repeated quasistatic loading of the model. The purpose of the present study was to compare the tensile and fracture mechanics properties of the current baseline (established PRL "third-generation" E-glass-fiber-epoxy) composite analogue for cortical bone to a new composite material formulation proposed for use as an enhanced fourth-generation cortical bone analogue material. Standard tensile, plane strain fracture toughness, and fatigue crack propagation rate tests were performed on both the third- and fourth-generation composite material formulations using standard ASTM test techniques. Injection molding techniques were used to create random fiber orientation in all test specimens. Standard dog-bone style tensile specimens were tested to obtain ultimate tensile strength and stiffness. Compact tension fracture toughness specimens were utilized to determine plane strain fracture toughness values. Reduced thickness compact tension specimens were also used to determine fatigue crack propagation rate behavior for the two material groups. Literature values for the same parameters for human cortical bone were compared to results from the third- and fourth-generation cortical analogue bone materials. Tensile properties of the fourth-generation material were closer to that of average human cortical bone than the third-generation material. Fracture toughness was significantly increased by 48% in the fourth-generation composite as compared to the third-generation analogue bone. The threshold stress intensity to propagate the crack was much higher for the fourth-generation material than for the third-generation composite. Even at the higher stress intensity threshold, the fatigue crack propagation rate was significantly decreased in the fourth-generation composite compared to the third-generation composite. These results indicate that the bone analogue models made from the fourth-generation analogue cortical bone material may exhibit better performance in fracture and longer fatigue lives than similar models made of third-generation analogue cortical bone material. Further fatigue testing of the new composite material in clinically relevant use of bone models is still required for verification of these results. Biomechanical test models using the superior fourth-generation cortical analogue material are currently in development.     

Diversity of cortical bone morphology in anuran amphibians

The cortical bones of mammals, birds, and reptiles are composed of a complex of woven bone and lamellar bone (fibrolamellar bone) organized into a variety of different patterns; however, it remains unclear whether amphibians possess similar structures. Importantly, to understand the evolutionary process of limb bones in tetrapods, it is necessary to compare the bone structure of amphibians (aquatic to terrestrial) with that of amniotes (mostly terrestrial). Therefore, this study compared the cortical bones in the long bones of several frog species before and after metamorphosis. Using micro-computed tomography (CT), we found that the cortical bones in the fibrolamellar bone of Xenopus tropicalis (Pipoidea superfamily) and Lithobates catesbeianus (Ranoidea superfamily) froglets are dense, whereas those of Ceratophrys cranwelli (Hyloidea superfamily) are porous. To clarify whether these features are common to their superfamily or sister group, four other frog species were examined. Histochemical analyses revealed porous cortical bones in C. ornata and Lepidobatrachus laevis (belonging to the same family, Ceratophryidae, as C. cranwelli). However, the cortical bones of Dryophytes japonicus (Hylidae, a sister group of Ceratophryidae in the Hyloidea superfamily), Microhyla okinavensis (Microhylidae, independent of the Hyloidea superfamily), and Pleurodeles waltl, a newt as an outgroup of anurans, are dense with no observed cavities. Our findings demonstrate that at least three members of the Ceratophryidae family have porous cortical bones similar to those of reptiles, birds, and mammals, suggesting that the process of fibrolamellar bone formation arose evolutionarily in amphibians and is conserved in the common ancestor of amniotes.     

Mechanics of linear microcracking in trabecular bone

Microcracking in trabecular bone is responsible both for the mechanical degradation and remodeling of the trabecular bone tissue. Recent results on trabecular bone mechanics have demonstrated that bone tissue microarchitecture, tissue elastic heterogeneity and tissue-level mechanical anisotropy all should be considered to obtain detailed information on the mechanical stress state. The present study investigated the influence of tissue microarchitecture, tissue heterogeneity in elasticity and material separation properties and tissue-level anisotropy on the microcrack formation process. Microscale bone models were executed with the extended finite element method. It was demonstrated that anisotropy and heterogeneity of the bone tissue contribute significantly to bone tissue toughness and the resistance of trabecular bone to microcrack formation. The compressive strain to microcrack initiation was computed to increase by a factor of four from an assumed homogeneous isotropic tissue to an assumed anisotropic heterogenous tissue.     

Age-related differences in the morphology of microdamage propagation in trabecular bone

Microdamage density has been shown to increase with age in trabecular bone and is associated with decreased fracture toughness. Numerous studies of crack propagation in cortical bone have been conducted, but data in trabecular bone is lacking. In this study, propagation of severe, linear, and diffuse damage was examined in trabecular bone cores from the femoral head of younger (61.3±3.1 years) and older (75.0±3.9 years) men and women. Using a two-step mechanical testing protocol, damage was first initiated with static uniaxial compression to 0.8% strain then propagated at a normalized stress level of 0.005 to a strain endpoint of 0.8%. Coupling mechanical testing with a dual-fluorescent staining technique, the number and length/area of propagating cracks were quantified. It was found that the number of cycles to the test endpoint was substantially decreased in older compared to younger samples (younger: 77,372±15,984 cycles; older: 34,944±11,964 cycles, p=0.06). This corresponded with a greater number of severely damaged trabeculae expanding in area during the fatigue test in the older group. In the younger group, diffusely damaged trabeculae had a greater damage area, which illustrates an efficient energy dissipation mechanism. These results suggest that age-related differences in fatigue life of human trabecular bone may be due to differences in propagated microdamage morphology.     

Vascular invasion of cartilage: Correlation of morphology with lysozyme,glycosaminoglycans, protease,and protease-inhibitory activity during endochondral bone development

Changes in the levels of lysozyme, patterns of glycosaminoglycans, and activities of proteases and protease inhibitors were studied during matrix-induced cartilage, bone, and bone marrow development. The morphological transitions were correlated with the biochemical parameters. There was a peak in lysozyme content on Day 3, during mesenchymal cell proliferation, followed by a decline during endochondral bone formation. The lysozyme levels increased again and attained maximal values during hematopoiesis on Day 21. Protease-inhibitory activity was maximal during chondrogenesis and diminished during osteogenesis. Protease activity was maximal on Day 3 during mesenchymal cell proliferation and was apparently present as an enzyme-inhibitor complex. Vascularization and bone formation were accompanied by an increase in protease activity. Chondroitin-4-sulfate was the predominant glycosaminoglycan detected in the matrix-induced cartilage and bone.     

Characteristics of rickettsial morphology during intracellular development

The normal anatomy of rickettsiae has been characterized with the use of R. prowazekii, R. conorii and R. akari in continuous cell cultures L-929, Al, FL and in primary chick embryo fibroblast culture. Rickettsiae are short rod-shaped cells with the dense cytoplasm and the regular structure of the cell wall--cytoplasmic membrane complex. The study has shown the absence of polymorphism in rickettsiae growing under permissive conditions, but at the same time these organisms easily develop into pathological forms. Pathological forms can be detected alongside normal rickettsiae in the same cells. The classification of the pathological forms of rickettsiae is presented. In this classification the compensating (reversible) and destructive (irreversible) forms of alterations, as well as hypertrophic and dystrophic processes on the level of the whole rickettsial cell or its organelles, are pointed out.     

The effects of damage and microcracking on the impact strength of bone

Microcracking has been shown to occur when bone is 'damaged' as shown by a loss of stiffness. The effect on bone's toughness of the types of damage produced at low losses of stiffness are not known. We loaded bovine bone specimens in bending and tension to stiffness losses of up to 27%, and examined the microcracking produced. The tensile specimens had diffuse arrays of microcracks of 2-20 microm in length, characteristic of tensile loading, on all surfaces. The bending specimens showed tensile microcracking on the tensile surface and characteristic long, straight, cross-hatched compression cracks on the compressive surface. Specimens were then broken in impact. Those that had been damaged in bending were divided into two groups, in one group the part of the specimen which had undergone compression damage was placed in tension, and in the other group the tensile damage was placed in tension. Tensile damage loaded in tension did not reduce the bone's energy-absorbing ability in impact until a modulus reduction of over 20%. However compression damage loaded in tension did severely reduce the bone's energy absorption capabilities (by an average of about 40%).     

Induction of somatopause in adult mice compromises bone morphology and exacerbates bone loss during aging

Somatopause refers to the gradual declines in growth hormone (GH) and insulin‐like growth factor‐1 throughout aging. To define how induced somatopause affects skeletal integrity, we used an inducible GH receptor knockout (iGHRKO) mouse model. Somatopause, induced globally at 6 months of age, resulted in significantly more slender bones in both male and female iGHRKO mice. In males, induced somatopause was associated with progressive expansion of the marrow cavity leading to significant thinning of the cortices, which compromised bone strength. We report progressive declines in osteocyte lacunar number, and increases in lacunar volume, in iGHRKO males, and reductions in lacunar number accompanied by ~20% loss of overall canalicular connectivity in iGHRKO females by 30 months of age. Induced somatopause did not affect mineral/matrix ratio assessed by Raman microspectroscopy. We found significant increases in bone marrow adiposity and high levels of sclerostin, a negative regulator of bone formation in iGHRKO mice. Surprisingly, however, despite compromised bone morphology, osteocyte senescence was reduced in the iGHRKO mice. In this study, we avoided the confounded effects of constitutive deficiency in the GH/IGF‐1 axis on the skeleton during growth, and specifically dissected its effects on the aging skeleton. We show here, for the first time, that induced somatopause compromises bone morphology and the bone marrow environment.     

Unlocalized crack initiation and propagation in staggered biomaterials

Many types of tissues in living organisms exhibit a combination of different properties to fulfil their mechanical functions in complex environments. Nacre with more than 90% brittle and hard phase and a little protein matrix, exhibits high strength and toughness, which is difficult to achieve in artificial materials. Researchers have shown that the toughness of nacre is related to the cracking process. Most of them, however, assume an obvious pre-existing crack on the model and the initiation of the microscopical pre-existing crack is not considered yet. Based on fracture mechanics with the cohesive zone model, we reveal the mechanism of the crack initiation and propagation pattern in staggered biomaterials without any pre-existing crack. The simulation result shows that there are two crack propagation modes: localized mode and unlocalized mode. A crack initiates and propagates in a small area in the localized mode, while cracks initiate at different points and propagate in various paths in the unlocalized mode. The crack initiation mechanism from the intrinsic properties of the material is clarified using energy based stability analysis. The result shows that the shear interfacial mechanism significantly delays the crack initiation.     

The elastic moduli of human subchondral, trabecular, and cortical bone tissue and the size-dependency of cortical bone modulus

The elastic moduli of human subchondral, trabecular, and cortical bone tissue from a proximal tibia were experimentally determined using three-point bending tests on a microstructural level. The mean modulus of subchondral specimens was 1.15 GPa, and those of trabecular and cortical specimens was 4.59 GPa and 5.44 GPa respectively. Significant differences were found in the modulus values between bone tissues, which may have mainly resulted from the differences in the microstructures of each bone tissue rather than in the mineral density. Furthermore, the size-dependency of the modulus was examined using eight different sizes of cortical specimens (heights h = 100-1000 microns). While the modulus values for relatively large specimens (h greater than 500 microns) remained fairly constant (approximately 15 GPa), the values decreased as the specimens became smaller. A significant correlation was found between the modulus and specimen size. The surface area to volume ratio proved to be a key variable to explain the size-dependency.     

Computational simulation of simultaneous cortical and trabecular bone change in human proximal femur during bone remodeling

In this study, we developed a numerical framework that computationally determines simultaneous and interactive structural changes of cortical and trabecular bone types during bone remodeling, and we investigated the structural correlation between the two bone types in human proximal femur. We implemented a surface remodeling technique that performs bone remodeling in the exterior layer of the cortical bone while keeping its interior area unchanged. A micro-finite element (μFE) model was constructed that represents the entire cortical bone and full trabecular architecture in human proximal femur. This study simulated and compared the bone adaptation processes of two different structures: (1) femoral bone that has normal cortical bone shape and (2) perturbed femoral bone that has an artificial bone lump in the inferomedial cortex. Using the proposed numerical method in conjunction with design space optimization, we successfully obtained numerical results that resemble actual human proximal femur. The results revealed that actual cortical bone, as well as the trabecular bone, in human proximal femur has structurally optimal shapes, and it was also shown that a bone abnormality that has little contribution to bone structural integrity tends to disappear. This study also quantitatively determined the structural contribution of each bone: when the trabecular adaptation was complete, the trabecular bone supported 54% of the total load in the human proximal femur while the cortical bone carried 46%.     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.