Sclerostin-antibody treatment of glucocorticoid-induced osteoporosis maintained bone mass and strength.

UNLABELLED: This study was to determine if antibody against sclerostin (Scl-Ab) could prevent glucocorticoid (GC)-induced osteoporosis in mice. We found that Scl-Ab prevented GC-induced reduction in bone mass and bone strength and that the anabolic effects of Scl-Ab might be partially achieved through the preservation of osteoblast activity through autophagy. INTRODUCTION: Glucocorticoids (GCs) inhibit bone formation by altering osteoblast and osteocyte cell activity and lifespan. A monoclonal antibody against sclerostin, Scl-Ab, increased bone mass in both preclinical animal and clinical studies in subjects with low bone mass. The objectives of this study were to determine if treatment with the Scl-Ab could prevent loss of bone mass and strength in a mouse model of GC excess and to elucidate if Scl-Ab modulated bone cell activity through autophagy. METHODS: We generated reporter mice that globally expressed dsRed fused to LC3, a protein marker for autophagosomes, and evaluated the dose-dependent effects of GCs (0, 0.8, 2.8, and 4 mg/kg/day) and Scl-Ab on autophagic osteoblasts, bone mass, and bone strength. RESULTS: GC treatment at 2.8 and 4 mg/kg/day of methylprednisolone significantly lowered trabecular bone volume (Tb-BV/TV) at the lumbar vertebrae and distal femurs, cortical bone mass at the mid-shaft femur (FS), and cortical bone strength compared to placebo (PL). In mice treated with GC and Scl-Ab, Tb-BV/TV increased by 60-125 %, apparent bone strength of the lumbar vertebrae by 30-70 %, FS-BV by 10-18 %, and FS-apparent strength by 13-15 %, as compared to GC vehicle-treated mice. GC treatment at 4 mg/kg/day reduced the number of autophagic osteoblasts by 70 % on the vertebral trabecular bone surface compared to the placebo group (PL, GC 0 mg), and GC + Scl-Ab treatment. CONCLUSIONS: Treatment with Scl-Ab prevented GC-induced reduction in both trabecular and cortical bone mass and strength and appeared to maintain osteoblast activity through autophagy.

Effects of sequential osteoporosis treatments on trabecular bone in adult rats with low bone mass.

UNLABELLED: We used an osteopenic adult ovariectomized (OVX) rat model to evaluate various sequential treatments for osteoporosis, using FDA-approved agents with complementary tissue-level mechanisms of action. Sequential treatment for 3 months each with alendronate (Aln), followed by PTH, followed by resumption of Aln, created the highest trabecular bone mass, best microarchitecture, and highest bone strength. INTRODUCTION: Individual agents used to treat human osteoporosis reduce fracture risk by ∼ 50-60%. As agents that act with complementary mechanisms are available, sequential therapies that mix antiresorptive and anabolic agents could improve fracture risk reduction, when compared with monotherapies. METHODS: We evaluated bone mass, bone microarchitecture, and bone strength in adult OVX, osteopenic rats, during different sequences of vehicle (Veh), parathyroid hormone (PTH), Aln, or raloxifene (Ral) in three 90-day treatment periods, over 9 months. Differences among groups were evaluated. The interrelationships of bone mass and microarchitecture endpoints and their relationship to bone strength were studied. RESULTS: Estrogen deficiency caused bone loss. OVX rats treated with Aln monotherapy had significantly better bone mass, microarchitecture, and bone strength than untreated OVX rats. Rats treated with an Aln drug holiday had bone mass and microarchitecture similar to the Aln monotherapy group but with significantly lower bone strength. PTH-treated rats had markedly higher bone endpoints, but all were lost after PTH withdrawal without follow-up treatment. Rats treated with PTH followed by Aln had better bone endpoints than those treated with Aln monotherapy, PTH monotherapy, or an Aln holiday. Rats treated initially with Aln or Ral, then switched to PTH, also had better bone endpoints, than monotherapy treatment. Rats treated with Aln, then PTH, and returned to Aln had the highest values for all endpoints. CONCLUSION: Our data indicate that antiresorptive therapy can be coupled with an anabolic agent, to produce and maintain better bone mass, microarchitecture, and strength than can be achieved with any monotherapy.

Elastic strain-induced amorphization in high-entropy alloys.

Elastic stability is the basis for understanding structural responses to external stimuli in crystalline solids, including melting, incipient plasticity and fracture. In this work, elastic stability is investigated in a series of high-entropy alloys (HEAs) using in situ mechanical tests and atomic-resolution characterization in transmission electron microscopy. Under tensile loading, the HEA lattices are observed to undergo a sudden loss of ordering as the elastic strain reached ∽ 10%. Such elastic strain-induced amorphization stands in intrinsic contrast to previously reported dislocation-mediated elastic instability and defect accumulation-mediated amorphization, introducing a form of elastic instability. Together with the first principle calculations and atomic-resolution chemical mapping, we identify that the elastic strain-induced amorphization is closely related to the depressed dislocation nucleation due to the local atomic environment inhomogeneity of HEAs. Our findings provide insights for the understanding of the fundamental nature of physical mechanical phenomena like elastic instability and incipient plasticity.

Changes to the cell, tissue and architecture levels in cranial suture synostosis reveal a problem of timing in bone development.

Premature fusion of cranial sutures is a common problem with an incidence of 3-5 per 10,000 live births. Despite progress in understanding molecular/genetic factors affecting suture function, the complex process of premature fusion is still poorly understood. In the present study, corresponding excised segments of nine patent and nine prematurely fused sagittal sutures from infants (age range 3-7 months) with a special emphasis on their hierarchical structural configuration were compared. Cell, tissue and architecture characteristics were analysed by transmitted and polarised light microscopy, 2D-histomorphometry, backscattered electron microscopy and energy-dispersive-x-ray analyses. Apart from wider sutural gaps, patent sutures showed histologically increased new bone formation compared to reduced new bone formation and osseous edges with a more mature structure in the fused portions of the sutures. This pattern was accompanied by a lower osteocyte lacunar density and a higher number of evenly mineralised osteons, reflecting pronounced lamellar bone characteristics along the prematurely fused sutures. In contrast, increases in osteocyte lacunar number and size accompanied by mineralisation heterogeneity and randomly oriented collagen fibres predominantly signified woven bone characteristics in patent, still growing suture segments. The already established woven-to-lamellar bone transition provides evidence of advanced bone development in synostotic sutures. Since structural and compositional features of prematurely fused sutures did not show signs of pathological/defective ossification processes, this supports the theory of a normal ossification process in suture synostosis - just locally commencing too early. These histomorphological findings may provide the basis for a better understanding of the pathomechanism of craniosynostosis, and for future strategies to predict suture fusion and to determine surgical intervention.

Changes in cortical bone response to high-fat diet from adolescence to adulthood in mice.

UNLABELLED: Diabetic obesity is associated with increased fracture risk in adults and adolescents. We find in both adolescent and adult mice dramatically inferior mechanical properties and structural quality of cortical bone, in agreement with the human fracture data, although some aspects of the response to obesity appear to differ by age. INTRODUCTION: The association of obesity with bone is complex and varies with age. Diabetic obese adolescents and adult humans have increased fracture risk. Prior studies have shown reduced mechanical properties as a result of high-fat diet (HFD) but do not fully address size-independent mechanical properties or structural quality, which are important to understand material behavior. METHODS: Cortical bone from femurs and tibiae from two age groups of C57BL/6 mice fed either HFD or low-fat diet (LFD) were evaluated for structural and bone turnover changes (SEM and histomorphometry) and tested for bending strength, bending stiffness, and fracture toughness. Leptin, IGF-I, and non-enzymatic glycation measurements were also collected. RESULTS: In both young and adult mice fed on HFD, femoral strength, stiffness, and toughness are all dramatically lower than controls. Inferior lamellar and osteocyte alignment also point to reduced structural quality in both age groups. Bone size was largely unaffected by HFD, although there was a shift from increasing bone size in obese adolescents to decreasing in adults. IGF-I levels were lower in young obese mice only. CONCLUSIONS: While the response to obesity of murine cortical bone mass, bone formation, and hormonal changes appear to differ by age, the bone mechanical properties for young and adult groups are similar. In agreement with human fracture trends, adult mice may be similarly susceptible to bone fracture to the young group, although cortical bone in the two age groups responds to diabetic obesity differently.

Carbon nanotubes as nanoscale mass conveyors.

The development of manipulation tools that are not too 'fat' or too 'sticky' for atomic scale assembly is an important challenge facing nanotechnology. Impressive nanofabrication capabilities have been demonstrated with scanning probe manipulation of atoms and molecules on clean surfaces. However, as fabrication tools, both scanning tunnelling and atomic force microscopes suffer from a loading deficiency: although they can manipulate atoms already present, they cannot efficiently deliver atoms to the work area. Carbon nanotubes, with their hollow cores and large aspect ratios, have been suggested as possible conduits for nanoscale amounts of material. Already much effort has been devoted to the filling of nanotubes and the application of such techniques. Furthermore, carbon nanotubes have been used as probes in scanning probe microscopy. If the atomic placement and manipulation capability already demonstrated by scanning probe microscopy could be combined with a nanotube delivery system, a formidable nanoassembly tool would result. Here we report the achievement of controllable, reversible atomic scale mass transport along carbon nanotubes, using indium metal as the prototype transport species. This transport process has similarities to conventional electromigration, a phenomenon of critical importance to the semiconductor industry.

The true toughness of human cortical bone measured with realistically short cracks.

Bone is more difficult to break than to split. Although this is well known, and many studies exist on the behaviour of long cracks in bone, there is a need for data on the orientation-dependent crack-growth resistance behaviour of human cortical bone that accurately assesses its toughness at appropriate size scales. Here, we use in situ mechanical testing to examine how physiologically pertinent short (<600 microm) cracks propagate in both the transverse and longitudinal orientations in cortical bone, using both crack-deflection/twist mechanics and nonlinear-elastic fracture mechanics to determine crack-resistance curves. We find that after only 500 microm of cracking, the driving force for crack propagation was more than five times higher in the transverse (breaking) direction than in the longitudinal (splitting) direction owing to major crack deflections/twists, principally at cement sheaths. Indeed, our results show that the true transverse toughness of cortical bone is far higher than previously reported. However, the toughness in the longitudinal orientation, where cracks tend to follow the cement lines, is quite low at these small crack sizes; it is only when cracks become several millimetres in length that bridging mechanisms can fully develop leading to the (larger-crack) toughnesses generally quoted for bone.

Measurement of the toughness of bone: a tutorial with special reference to small animal studies.

Quantitative assessment of the strength and toughness of bone has become an integral part of many biological and bioengineering studies on the structural properties of bone and their degradation due to aging, disease and therapeutic treatment. Whereas the biomechanical techniques for characterizing bone strength are well documented, few studies have focused on the theory, methodology, and various experimental procedures for evaluating the fracture toughness of bone, i.e., its resistance to fracture, with particular reference to whole bone testing in small animal studies. In this tutorial, we consider the many techniques for evaluating toughness and assess their specific relevance and application to the mechanical testing of small animal bones. Parallel experimental studies on wild-type rat and mouse femurs are used to evaluate the utility of these techniques and specifically to determine the coefficient of variation of the measured toughness values.

Adhesion between biodegradable polymers and hydroxyapatite: Relevance to synthetic bone-like materials and tissue engineering scaffolds.

Many studies are currently underway on the quest to make synthetic bone-like materials with composites of polymeric materials and hydroxyapatite (HA). In the present work, we use wetting experiments and surface tension measurements to determine the work of adhesion between biodegradable polymers and HA, with specific reference to the role of humid environments. All the polymers are found to exhibit low contact angles (

A fracture-mechanics-based approach to fracture control in biomedical devices manufactured from superelastic Nitinol tube.

Several key fracture-mechanics parameters associated with the onset of subcritical and critical cracking, specifically the fracture toughness, crack-resistance curve, and fatigue threshold, have recently been reported for the superelastic alloy Nitinol, in the product form of the thin-walled tube that is used to manufacture several biomedical devices, most notably endovascular stents. In this study, we use these critical parameters to construct simple decision criteria for assessing the quantitative effect of crack-like defects in such Nitinol devices with respect to their resistance to failure by deformation or fracture. The criteria are based on the (equivalent) crack-initiation fracture toughness and fatigue threshold stress-intensity range, together with the general yield strength and fatigue endurance strength, and are used to construct a basis for design against single-event (overload) failures as well as for time-/cycle-delayed failures associated with fatigue.

Fatigue of dentin-composite interfaces with four-point bend.

OBJECTIVES: The objective was to determine the fracture and cyclic fatigue properties of composite-dentin beams bonded with a self-etching adhesive in four-point bend. METHODS: Beams of rectangular cross-section were shaped to a size of approximately 0.87mmx0.87mmx10mm and placed in a four-point bending apparatus, with the loading points 1.8 and 7.2mm apart, with the interface centered between the inner rollers. Cyclical loading was performed in Hanks' Balanced Salt Solution at 25 degrees C, with forces between 54% and 99% of the bending strength of the bonded beams. RESULTS: Solid dentin and solid composite beams [n=6] had bending strengths of 164.4 and 164.6MPa, respectively, under monotonically increasing loads. Bonded beams [n=6] had strengths of 90.6MPa. No significant difference was found between solid composite and solid dentin beams, the bonded beams were different (ANOVA, p<0.0001) With long-term cycling, stresses below 49MPa were tolerated for 10(6) cycles, but with increasing stress up to 90MPa, beams failed earlier, demonstrating that subcritical fatigue cycling will eventually cause failure. SIGNIFICANCE: Fatigue may be a significant mechanism of dentin-composite bond degradation.

Electrically reversible cracks in an intermetallic film controlled by an electric field.

Cracks in solid-state materials are typically irreversible. Here we report electrically reversible opening and closing of nanoscale cracks in an intermetallic thin film grown on a ferroelectric substrate driven by a small electric field (~0.83 kV/cm). Accordingly, a nonvolatile colossal electroresistance on-off ratio of more than 108 is measured across the cracks in the intermetallic film at room temperature. Cracks are easily formed with low-frequency voltage cycling and remain stable when the device is operated at high frequency, which offers intriguing potential for next-generation high-frequency memory applications. Moreover, endurance testing demonstrates that the opening and closing of such cracks can reach over 107 cycles under 10-µs pulses, without catastrophic failure of the film.

Fatigue-crack growth properties of thin-walled superelastic austenitic Nitinol tube for endovascular stents.

Over the past 10 years, the supereleastic nickel-titanium alloy Nitinol has found widespread application in the manufacture of small-scale biomedical devices, such as self-expanding endovascular stents. Although conventional stress/strain-life (S/N) analyses are invariably used as the primary method for design against fatigue loading and for predicting safe lifetimes, fracture mechanics-based methodologies provide a vital means of assessing the quantitative effect of defects on such lifetimes. Unfortunately, fracture mechanics studies on fatigue in Nitinol are scarce, and most results do not pertain to the (thin-walled tube) product forms that are typically used in the manufacture of endovascular stents. In the current work, we document the basic fatigue-crack growth properties of flattened thin-walled ( approximately 400 microm thick) Nitinol tubing in a 37 degrees C air environment. Crack-growth behavior is characterized over a wide range of growth rates ( approximately 6 orders of magnitude) and load ratios, that is, as a function of the alternating and maximum stress intensities, at 50 Hz. Limited experiments at both 5 and 50 Hz were also performed in 37 degrees C air and simulated body fluid to determine whether the cyclic frequency affects the fatigue behavior. Fatigue-crack growth-rate properties in such thin-walled Nitinol tube are found to be quite distinct from limited published data on other (mainly bulk) product forms of Nitinol, for example, bar and strip, both in terms of the relative fatigue thresholds and the variation in steady-state growth rates.

Multiscale structure and damage tolerance of coconut shells.

We investigated the endocarp of the fruit of Cocos nucifera (i.e., the inner coconut shell), examining the structure across multiple length scales through advanced characterization techniques and in situ testing of mechanical properties. Like many biological materials, the coconut shell possesses a hierarchical structure with distinct features at different length scales that depend on orientation and age. Aged coconut was found to have a significantly stronger (ultimate tensile strength, UTS = 48.5MPa), stiffer (Young's modulus, E = 1.92GPa), and tougher (fracture resistance (R-curve) peak of KJ = 3.2MPa m1/2) endocarp than the younger fruit for loading in the latitudinal orientation. While the mechanical properties of coconut shell were observed to improve with age, they also become more anisotropic: the young coconut shell had the same strength (17MPa) and modulus (0.64GPa) values and similar R-curves for both longitudinal and latitudinal loading configurations, whereas the old coconut had 82% higher strength for loading in the latitudinal orientation, and >50% higher crack growth toughness for cracking on the latitudinal plane. Structural aspects affecting the mechanical properties across multiple length scales with aging were identified as improved load transfer to the cellulose crystalline nanostructure (identified by synchrotron x-ray diffraction) and sclerification of the endocarp, the latter of which included closing of the cell lumens and lignification of the cell walls. The structural changes gave a denser and mechanically superior micro and nanostructure to the old coconut shell. Additionally, the development of anisotropy was attributed to the formation of an anisotropic open channel structure throughout the shell of the old coconut that affected both crack initiation during uniaxial tensile tests and the toughening mechanisms of crack trapping and deflection during crack propagation.

Re-evaluating the toughness of human cortical bone.

Data for fracture in human humeral cortical bone are re-analyzed to assess the validity for this material of linear-elastic fracture mechanics (LEFM), which is the standard method of analyzing toughness and one basis for analyzing clinical data relating to bone quality. A nonlinear fracture model, which is based on representing the damage zone in the bone by a cohesive model, is calibrated against a number of sets of test data for normal (not diseased or aged) human cortical bone taken from cadavers. The data consist of load vs. load-point displacement measurements from standard compact-tension fracture tests. Conventional LEFM is unable to account for the shape of the load-displacement curves, but the nonlinear model overcomes this deficiency. Calibration of the nonlinear model against one data curve leads to predictions of the peak load and the displacement to peak load for two other data curves that are, for this limited test set, more accurate than those made using LEFM. Furthermore, prior observations of damage mechanisms in bone are incompatible with the modeling assumption of LEFM that all nonlinearity is confined to a zone much smaller than the specimen and the crack length. The predictions of the cohesive model and the prior observations concur that the length of the nonlinear zone in human cortical bone varies in the range 3-10 mm, which is comparable to or larger than naturally-occurring bones and the specimens used to test them. We infer that LEFM is not an accurate model for cortical bone. The fracture toughness of bone deduced via LEFM from test data will not generally be a material constant, but will take different values for different crack lengths and test configurations. The accuracy of using LEFM or single-parameter fracture toughness for analyzing the significance of data from clinical studies is called into question. The nonlinear cohesive zone model is proposed to be a more accurate model of bone and the traction-displacement or cohesive law is hypothesized to be a material property. The cohesive law contains a more complete representation of the mechanics of material failure than the single-parameter fracture toughness and may therefore provide a superior measure of bone quality, e.g., for assessing the efficacy of therapy for osteoporosis.

Fatigue and life prediction for cobalt-chromium stents: A fracture mechanics analysis.

To design against premature mechanical failure, most implant devices such as coronary and endovascular stents are assessed on the basis of survival, i.e., if a fatigue life of 10(8) cycles is required, testing is performed to ascertain whether the device will survive 10(8) cycles under accelerated in vitro loading conditions. This is a far from satisfactory approach as the safety factors, which essentially tell you how close you are to failure, remain unknown; rather, the probability of fatigue failure should instead be assessed on the basis of testing to failure. In this work, a new damage-tolerant analysis of a cardiovascular stent is presented, where the design life is conservatively evaluated using a fracture mechanics methodology. In addition to enabling estimates of safe in vivo lifetimes to be made, this approach serves to quantify the effect of flaws in terms of their potential effect on device failure, and as such provides a rational basis for quality control.

Fracture length scales in human cortical bone: the necessity of nonlinear fracture models.

Recently published data for fracture in human humeral cortical bone are analyzed using cohesive-zone models to deal with the nonlinear processes of material failure. Such models represent the nonlinear deformation processes involved in fracture by cohesive tractions exerted by the failing material along a fracture process zone, rather than attributing all damage to a process occurring at a single point, as in conventional linear-elastic fracture mechanics (LEFM). The relationship between the tractions and the net displacement discontinuity across the process zone is hypothesized to be a material property for bone. To test this hypothesis, the cohesive law was evaluated by analyzing published load vs. load-point displacement data from one laboratory; the calibrated law was then used to predict similar data taken for a different source of bone using a different specimen geometry in a different laboratory. Further model calculations are presented to illustrate more general characteristics of the nonlinear fracture of bone and to demonstrate in particular that LEFM is not internally consistent for all cases of interest. For example, the fracture toughness of bone deduced via LEFM from test data is not necessarily a material constant, but will take different values for different crack lengths and test configurations. LEFM is valid when the crack is much longer than a certain length scale, representative of the length of the process zone in the cohesive model, which for human cortical bone ranges from 3 to 10mm. Since naturally occurring bones and the specimens used to test them are not much larger than this dimension for most relevant orientations, it is apparent that only nonlinear fracture models can give an internally consistent account of their fracture. The cohesive law is thus a more complete representation of the mechanics of material failure than the single-parameter fracture toughness and may therefore provide a superior measure of bone quality. The analysis of fracture data also requires proper representation of the approximately orthotropic elasticity of the bone specimen; if the specimen is incorrectly assumed to be isotropic, the initial measured compliance cannot be reproduced to within a factor of four and the fracture toughness deduced from the measured work of fracture will be overestimated by approximately 30%.

Role of alcohol in the fracture resistance of teeth.

Healthy dentin, the mineralized tissue that makes up the bulk of the tooth, is naturally hydrated in vivo; however, it is known that various chemical reagents, including acetone and ethanol, can induce dehydration and thereby affect its properties. Here, we sought to investigate this in light of the effect of alcohol on the mechanical properties of dentin, specifically by measuring the stiffness, strength, and toughness of dentin in simulated body fluid and Scotch whisky. Results indicated that chemical dehydration induced by the whisky had a significant beneficial effect on the elastic modulus, strength, and fracture toughness of dentin. Although this made teeth more resistant to fracture, the change in properties was fully reversible upon rehydration. This effect is considered to be associated with increased cross-linking of the collagen molecules from intermolecular hydrogen-bonding, where water is replaced with weaker hydrogen-bond-forming solvents such as alcohol.

Kitagawa-Takahashi diagrams define the limiting conditions for cyclic fatigue failure in human dentin.

As cyclic fatigue is considered to be a major cause of clinical tooth fractures, achieving a comprehensive understanding of the fatigue behavior of dentin is of importance. In this note, the fatigue behavior of human dentin is examined in the context of the Kitagawa-Takahashi diagram to define the limiting conditions for fatigue failure. Specifically, this approach incorporates two limiting threshold criteria for fatigue: (i) a threshold stress for fatigue failure, specifically the smooth-bar (unnotched) fatigue endurance strength, at small crack sizes and (ii) a threshold stress-intensity range for fatigue-crack growth at larger crack sizes. The approach provides a "bridge" between the traditional fatigue life and fracture mechanics based damage-tolerant approaches to fatigue-life estimation, and as such defines a "failure envelope" of applied stresses and flaw sizes where fatigue failure is likely in dentin This approach may also be applied to fatigue failure in human cortical bone (i.e. clinical "stress fractures"), which exhibits similar fatigue behavior characteristics, and in principle may aid clinicians in making quantitative evaluations of the risk of fractures in mineralized tissues.

Propagation of surface fatigue cracks in human cortical bone.

An understanding of how fatigue cracks grow in bone is of importance as fatigue is thought to be the main cause of clinical stress fractures. This study presents new results on the fatigue-crack growth behavior of small surface cracks (approximately 75-1000 microm in size) in human cortical bone, and compares their growth rates with data from other published studies on the behavior of both surface cracks and many millimeter, through-thickness large cracks. Results are obtained with a cyclically loaded cantilever-beam geometry using optical microscopy to examine for crack growth after every 100-500 cycles. Based on the current and previous results, small fatigue cracks appear to become more resistant to fatigue-crack growth with crack extension, analogous to the way the fracture resistance of cortical bone increases with crack growth. Mechanistically, a theory attributing such behavior to the development of bridges in the wake of the crack with crack growth is presented. The existence of such bridges is directly confirmed using optical microscopy.