Mechanobiology of bone remodelling

Bone as most of living tissues is able, during its entire lifetime, to adapt its internal microstructure and subsequently its associated mechanical properties to the specific mechanical and physiological environment in a process commonly known as bone remodelling. Bone is therefore continuously renewed and microdamage removed minimizing the risk of fracture. Bone remodelling is controlled by mechanical and metabolical stimuli. In this paper, we introduce a new model of bone remodelling that takes into account both types of influences. The predicted results show a good correspondence with experimental and clinical data. For example, in disuse, bone porosity increases until an equilibrium situation, while, in overloading, decreases unless the damage rate is so high that causes resorption and "stress fracture". This model has been employed to predict bone adaptation in the proximal femur after total hip replacement proving its consistence and good correspondence with well-known clinical experiences.

Variability of nanoindentation responses of bone and artificial bone-like composites

Nanoindentation is a popular technique for measuring the intrinsic mechanical response of bone and has been used to measure a single-valued elastic modulus. However, bone is a composite material with 20-80 nm hydroxyapatite plates embedded in a collagen matrix, and modern instrumentation allows for measurements at these small length scales. The present study examines the indentation response of bone and artificial gelatin-apatite nanocomposite materials across three orders of magnitude of lengthscale, from nanometers to micrometers, to isolate the composite phase contributions to the overall response. The load-displacement responses were variable and deviated from the quadratic response of homogeneous materials at small depths. The distribution of apparent elastic modulus values narrowed substantially with increasing indentation load. Indentation of particulate nanocomposites was simulated using finite element analysis. Modeling results replicated the convergence in effective modulus seen in the experiments. It appears that the apatite particles are acting as the continuous ("matrix") phase in bone and nanocomposites. Copyright © 2004 by ASME.

Viscoelastic properties of bone as a function of hydration state determined by nanoindentation

Spherical indentation creep testing was used to examine the effect of hydration state on bone mechanical properties. Analysis of creep data was based on the elastic-viscoelastic correspondence principle and utilized a direct solution for the finite loading-rate experimental conditions. The zero-time shear modulus was computed from the creep compliance function and compared to the indentation modulus obtained via conventional indentation analysis, based on an elastic unloading response. The method was validated using a well-known polymer material under three different loading conditions. The method was applied to bone samples prepared with different water content by partial exchange with ethanol, where 70% ethanol was considered as the baseline condition. A hydration increase was associated with a 43% decrease in stiffness, while a hydration decrease resulted in a 20% increase in bone tissue stiffness.

Nanoindentation measurements of bone viscoelasticity as a function of hydration state

Bone is an anisotropic material, and its mechanical properties are determined by its microstructure as well as its composition. Mechanical properties of bone are a consequence of the proportions of, and the interactions between, mineral, collagen and water. Water plays an important role in maintaining the mechanical integrity of the composite, but the manner in which water interacts within the ultrastructure is unclear. Dentine being an isotropic two-dimensional structure presents a homogenous composite to examine the dehydration effects. Nanoindentation methods for determining the viscoelastic properties have recently been developed and are a subject of great interest. Here, one method based on elastic-viscoelastic correspondence for 'ramp and hold' creep testing (Oyen, J. Mater. Res., 2005) has been used to analyze viscoelastic behavior of polymeric and biological materials. The method of 'ramp and hold' allows the shear modulus at time zero to be determined from fitting of the displacement during the maximum load hold. Changes in the viscoelastic properties of bone and dentine were examined as the material was systematically dehydrated in a series of water:solvent mixes. Samples of equine dentine were sectioned and cryo-polished. Shear modulus was obtained by nanoindentation using spherical indenters with a maximum load hold of 120s. Samples were tested in different solvent concentrations sequentially, 70% ethanol to 50% ethanol, 70 % ethanol to 100% ethanol, 70% ethanol to 70% methanol to 100% methanol, and 70% ethanol to 100% acetone, after storage in each condition for 24h. By selectively removing and then replacing water from the composite, insights in to the ultrastructure of the tissue can be gained from the corresponding changes in the experimentally determined moduli, as well as an understanding of the complete reversibility of the dehydration process. © 2006 Materials Research Society.

Effects of dietary phosphorus and zinc levels on growth artd bone mineralization in fingerlings of rainbow trout, Oncorhynchus mykiss

A laboratory based 2 x 2 factorial experiment was conducted to investigate the influences of dietary phosphorus and zinc levels on growth and bone mineralization in fingerlings of rainbow trout for 21 weeks. Two levels of phosphorus (19 and 30 mg/g) and two levels of zinc (55 and 103 Ag/g) in the dry diets were tested. Duplicate tanks of 30 rainbow trout (average weight 1.56 ± 0.24 g) per 60L glass tank were fed experimental diets three times a day to apparent satiation level at 15 to 24°C water temperature. The results of the present study demonstrated that dietary phosphorus supplementation influenced the growth and bone mineralization whereas zinc levels significantly (p<0.05) influenced bone mineralization in rainbow trout. Further investigations in this area with different size and age groups of this fish are broadly needed.

Ultrastructural mechanical and material characterization of fossilized bone

Bone plays a key role in the paleontological and archeological records and can provide insight into the biology, ecology and the environment of ancient vertebrates. Examination of bone at the tissue level reveals a definitive relationship between nanomechanical properties and the local organic content, mineral content, and microstructural organization. However, it is unclear as to how these properties change following fossilization, or diagenesis, where the organic phase is rapidly removed and the remaining mineral phase is reinforced by the deposition of apatites, calcites, and other minerals. While the process of diagenesis is poorly understood, its outcome clearly results in the potential for dramatic alteration of the mechanical response of biological tissues. In this study, fossilized specimens of mammalian long bones, collected from Colorado and Wyoming, were studied for mechanical variations. Nanoindentation performed in both longitudinal and transverse directions revealed preservation of bone's natural anisotropy as transverse modulus values were consistently smaller than longitudinal values. Additionally, modulus values of fossilized bone from 35.0 to 89.1 GPa increased linearly with logarithm of the sample's age. Future studies will aim to clarify what mechanical and material elements of bone are retained during diagenesis as bone becomes part of the geologic milieu. © 2007 Materials Research Society.

SCALING OF LIMB PROPORTIONS AND LIMB BONE DIAMETERS IN 3 SPECIES OF CHINESE SNUB-NOSED LANGURS (GENUS RHINOPITHECUS)

In this study, aspects of the structural mechanics of the upper and lower limbs of the three Chinese species of Rhinopithecus were examined. Linear regression and reduced major axis (RMA) analyses of natural log-transformed data were used to examine the dimensions of limb bones and other relationships to body size and locomotion. The results of this study suggest that: (1) the allometry exponents of the lengths of long limbs deviate from isometry, being moderately negative, while the shaft diameters (both sagittal and transverse) show significantly positive allometry; (2) the sagittal diameters of the tibia and ulna show extremely significantly positive allometry - the relative enlargement of the sagittal, as opposed to transverse, diameters of these bones suggests that the distal segments of the fore- and hindlimbs of Rhinopithecus experience high bending stresses during locomotion; (3) observations of Rhinopithecus species in the field indicate that all species engage in energetic leaping during arboreal locomotion. The limbs experience rapid and dramatic decelerations upon completion of a leap. We suggest that these occasional decelerations produce high bending stresses in the distal limb segments and so account for the hypertrophy of the sagittal diameters of the ulna and tibia.

Ultra-structural defects cause low bone matrix stiffness despite high mineralization in osteogenesis imperfecta mice.

Bone is a complex material with a hierarchical multi-scale organization from the molecule to the organ scale. The genetic bone disease, osteogenesis imperfecta, is primarily caused by mutations in the collagen type I genes, resulting in bone fragility. Because the basis of the disease is molecular with ramifications at the whole bone level, it provides a platform for investigating the relationship between structure, composition, and mechanics throughout the hierarchy. Prior studies have individually shown that OI leads to: 1. increased bone mineralization, 2. decreased elastic modulus, and 3. smaller apatite crystal size. However, these have not been studied together and the mechanism for how mineral structure influences tissue mechanics has not been identified. This lack of understanding inhibits the development of more accurate models and therapies. To address this research gap, we used a mouse model of the disease (oim) to measure these outcomes together in order to propose an underlying mechanism for the changes in properties. Our main finding was that despite increased mineralization, oim bones have lower stiffness that may result from the poorly organized mineral matrix with significantly smaller, highly packed and disoriented apatite crystals. Using a composite framework, we interpret the lower oim bone matrix elasticity observed as the result of a change in the aspect ratio of apatite crystals and a disruption of the crystal connectivity.

Insight into differences in nanoindentation properties of bone.

Nanoindentation provides the ideal framework to determine mechanical properties of bone at the tissue scale without being affected by the size, shape, and porosity of the bone. However, the values of tissue level mechanical properties vary significantly between studies. Since the differences in the bone sample, hydration state, and test parameters complicate direct comparisons across the various studies, these discrepancies in values cannot be compared directly. The objective of the current study is to evaluate and compare mechanical properties of the same bones using a broad range of testing parameters. Wild type C56BL6 mice tibiae were embedded following different processes and tested in dry and rehydrated conditions. Spherical and Berkovich indenter probes were used, and data analysis was considered within the elasto-plastic (Oliver-Pharr), viscoelastic and visco-elastic-plastic frameworks. The mean values of plane strain modulus varied significantly depending on the hydration state, probe geometry and analysis method. Indentations in dry bone analyzed using a visco-elastic-plastic approach gave values of 34 GPa. After rehydrating the same bones and indenting them with a spherical tip and utilizing a viscoelastic analysis, the mean modulus value was 4 GPa, nearly an order of magnitude smaller. Results suggest that the hydration state, probe geometry and the limitations and assumptions of each analysis method influence significantly the measured mechanical properties. This is the first time that such a systematic study has been carried out and it has been concluded that the discrepancies in the mechanical properties of bone measured by nanoindentation found in the literature should not be attributed only to the differences between the bones themselves, but also to the testing and analysis protocols.

Multi-scale permeability of murine bone measured by nanoindentation

The determination of lacunar-canalicular permeability is essential to understand the mechano-transduction mechanism of bone. Murine models are widely used to investigate skeletal growth and regulation, but the value of lacunar-canalicular permeability is still unclear. To address this question, a poroelastic analysis based on nanoindentation data was used to calculate the lacunar-canalicular permeability of wild type C57BL/6 mice of 12 months. Cross-sections of three tibiae were indented using spherical fluid cell indenter tips of two sizes. Results suggest that the value of lacunar-canalicular intrinsic permeability of B6 female murine tibia is in the order of 10 -24 m2. The distribution of the values of intrinsic permeability suggests that with larger contact sizes, nanoindentation alone is capable of capturing the multi-scale permeability of bone. Multi-scale permeability of bone measured by nanoindentation will lead to a better understanding of the role of fluid flow in mechano-transduction. © 2013 American Society of Civil Engineers.

Age-related changes in mouse bone permeability.

The determination of lacunar-canalicular permeability is essential for understanding local fluid flow in bone, which may indicate how bone senses changes in the mechanical environment to regulate mechano-adaptation. The estimates of lacunar-canalicular permeability found in the literature vary by up to eight orders of magnitude, and age-related permeability changes have not been measured in non-osteonal mouse bone. The objective of this study is to use a poroelastic approach based on nanoindentation data to characterize lacunar-canalicular permeability in murine bone as a function of age. Nine wild type C57BL/6 mice of different ages (2, 7 and 12 months) were used. Three tibiae from each age group were embedded in epoxy resin, cut in half and indented in the longitudinal direction in the mid-cortex using two spherical fluid indenter tips (R=238 μm and 500 μm). Results suggest that the lacunar-canalicular intrinsic permeability of mouse bone decreases from 2 to 7 months, with no significant changes from 7 to 12 months. The large indenter tip imposed larger contact sizes and sampled larger ranges of permeabilities, particularly for the old bone. This age-related difference in the distribution was not seen for indents with the smaller radius tip. We conclude that the small tip effectively measured lacunar-canalicular permeability, while larger tip indents were influenced by vascular permeability. Exploring the age-related changes in permeability of bone measured by nanoindentation will lead to a better understanding of the role of fluid flow in mechano-transduction. This understanding may help indicate alterations in bone adaptation and remodeling.

Age-related changes in mouse bone permeability

The determination of lacunar-canalicular permeability is essential for understanding local fluid flow in bone, which may indicate how bone senses changes in the mechanical environment to regulate mechano-adaptation. The estimates of lacunar-canalicular permeability found in the literature vary by up to eight orders of magnitude, and age-related permeability changes have not been measured in non-osteonal mouse bone. The objective of this study is to use a poroelastic approach based on nanoindentation data to characterize lacunar-canalicular permeability in murine bone as a function of age. Nine wild type C57BL/6 mice of different ages (2, 7 and 12 months) were used. Three tibiae from each age group were embedded in epoxy resin, cut in half and indented in the longitudinal direction in the mid-cortex using two spherical fluid indenter tips (R=238 μm and 500 μm). Results suggest that the lacunar-canalicular intrinsic permeability of mouse bone decreases from 2 to 7 months, with no significant changes from 7 to 12 months. The large indenter tip imposed larger contact sizes and sampled larger ranges of permeabilities, particularly for the old bone. This age-related difference in the distribution was not seen for indents with the smaller radius tip. We conclude that the small tip effectively measured lacunar-canalicular permeability, while larger tip indents were influenced by vascular permeability. Exploring the age-related changes in permeability of bone measured by nanoindentation will lead to a better understanding of the role of fluid flow in mechano-transduction. This understanding may help indicate alterations in bone adaptation and remodeling. © 2013 Elsevier Ltd.