Cortical bone tissue resists fatigue fracture by deceleration and arrest of microcrack growth

Knowledge of kinetics of fatigue crack growth of microcracks is important so as to understand the dynamics of bone adaptation, remodeling, and the etiology of fatigue-based failures of cortical bone tissue. In this respect, theoretical models (Taylor, J. Biomech., 31 (1998) 587-592; Taylor and Prendergast, Proc. Instn. Mech. Engrs. Part H 211 (1997) 369-375) of microcrack growth in cortical bone have predicted a decreasing microcrack growth rate with increasing microcrack length. However, these predictions have not been observed directly. This study investigated microcrack growth and arrest through observations of surface microcracks during cyclic loading (R=0.1, 50-80MPa) of human femoral cortical bone (male, n=4, age range: 37-40yr) utilizing a video microscopy system. The change in crack length and orientation of eight surface microcracks were measured with the number of fatigue cycles from four specimens. At the applied cyclic stresses, the microcracks propagated and arrested in generally less than 10,000 cycles. The fatigue crack growth rate of all microcracks decreased with increasing crack length following initial identification, consistent with theoretical predictions. The growth rate of the microcracks was observed to be in the range of 5x10(-5) to 5x10(-7)mmcycle(-1). In addition, many of the microcracks were observed not to grow beyond 150 microm and a cyclic stress intensity factor of 0.5MNm(-3/2). The results of this study suggest that cortical bone tissue may resist fracture at the microscale by deceleration of fatigue crack growth and arrest of microcracks.     

Novel mechanical bioreactor for concomitant fluid shear stress and substrate strain

The two main types of mechanical stimuli used in cellular-level bone mechanotransduction studies are substrate strain and flow-induced shear stress. A subset of studies has investigated which of these stimuli induces the primary mechanotransduction effect on bone cells. The shortcomings of these experiments are twofold. First, in some experiments the magnitude of one loading type is able to be quantitatively measured while the other loading mode is only estimated. Second, the two loading modes are compared using different bioreactors, representing different cellular environments and substrates to which the cells are attached. In addition, none of these studies utilized bioreactors which apply controlled magnitudes of substrate strain and flow-induced shear stress differentially and simultaneously. This study presents the design of a multimodal loading device which can apply substrate stretch and fluid flow simultaneously while allowing for real-time cell imaging. The mechanical performance of the bioreactor is validated in this study by correlating the output levels of flow-induced shear stress and substrate strain with the input levels of displacement and displacement rate. The magnitudes of cross-talk loading (i.e. flow-induced strain, and strain-induced fluid flow) are also characterized and shown to be magnitudes lower than physiological levels of loading estimated to occur in bone in vivo.     

The behaviour of microcracks in compact bone

This paper summarises four separate studies carried out by our group over the past number of years in the area of bone microdamage. The first study investigated the manner by which microcracks accumulate and interact with bone microstructure during fatigue testing of compact bone specimens. In a series of fatigue tests carried out at four different stress ranges between 50 and 80 MPA, crack density increased with loading cycles at a rate determined by the applied stress. Variations in the patterns of microdamage accumulation suggest that that at low stress levels, larger amounts of damage can build up without failure occurring. In a second study using a series of four-pont bending tests carried out on ovine bone samples, it was shown that bone microstructure influenced the ability of microcracks to propagate, with secondary osteons acting as barriers to crack growth. In a third study, the manner by which crack growth disrupts the canalicular processes connecting osteocytes was investigated. Analysis of individual cracks showed that disruption of the canalicular processes connecting osteocytes occurred due to shear displacement at the face of propagating microcracks, suggesting that this may play some role in the mechanism that signals bone remodelling. In a fourth in vivo study, it was shown that altering the mechanical load applied to the long bones of growing rats causes microcrack formation. In vivo microdamage was present in rats subjected to hindlimb suspension with a higher microcrack density found in the humeri than the femora. Microdamage was also found in control animals. This is the first study to demonstrate in vivo microcracks in normally loaded bones in a rat model.     

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm² at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF₁₆₄, play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.     

Influence of interstitial bone microcracks on strain-induced fluid flow

It is well known that microcracks act as a stimulus for bone remodelling, initiating resorption by osteoclasts and new bone formation by osteoblasts. Moreover, microcracks are likely to alter the fluid flow and convective transport through the bone tissue. This paper proposes a quantitative evaluation of the strain-induced interstitial fluid velocities developing in osteons in presence of a microcrack in the interstitial bone tissue. Based on Biot theory in the low-frequency range, a poroelastic model is carried out to study the hydro-mechanical behaviour of cracked osteonal tissue. The finite element results show that the presence of a microcrack in the interstitial osteonal tissue may drastically reduce the fluid velocity inside the neighbouring osteons. This fluid inactive zone inside osteons can cover up to 10% of their surface. Consequently, the fluid environment of bone mechano-sensitive cells is locally modified.     

Non-overlapping functions for Pyk2 and FAK in osteoblasts during fluid shear stress-induced mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2(+/+) and Pyk2(-/-) primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2(+/+) and Pyk2(-/-) osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK(-/-) osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts.     

The role of the lamellar interface during torsional yielding of human cortical bone

Fragility fractures are a result of alterations in bone quantity, tissue properties, applied loads, or a combination of these factors. The current study addresses the contribution of cortical bone tissue properties to skeletal fragility by characterizing the shear damage accumulation processes which occur during torsional yielding in normal bone. Samples of human femoral cortical bone were loaded in torsion and damaged at a post-yield twist level. The number of microcracks within osteons, interstitial tissue, and along cement lines were assessed using basic fuchsin staining. Damage density measures (number of cracks/mm2) were correlated with stiffness degradation and changes in relaxation. Damaged samples exhibited a wide variation in total microcrack density, ranging from 1.1 to 43.3 cracks/mm2 with a mean density of 19.7 +/- 9.8 cracks/mm2. Lamellar interface cracks comprised more than 75% of the total damage, indicating that the lamellar interface is weak in shear and is a principal site of shear damage accumulation. Damage density was positively correlated with secant stiffness degradation, but only explained 22% of the variability in degradation. In contrast, damage density was uncorrelated with the changes in relaxation, indicating that a simple crack counting measure such as microcrack density was not an appropriate measure of relaxation degradation. Finally, a nonuniform microcrack density distribution was observed, suggesting that internal shear stresses were redistributed within the torsion samples during post-yield loading. The results suggested that the lamellar interface in human cortical bone plays an important role in torsional yielding by keeping cracks physically isolated from each other and delaying microcrack coalescence in order to postpone the inevitable formation of the fatal crack.     

Response of osteoblasts to low fluid shear stress is time dependent

The process of mechanotransduction of bone, the conversion of a mechanical stimulus into a biochemical response, is known to occur in osteoblasts in response to fluid shear stress. In order to understand the reaction of osteoblasts to various times of flow perfusion, osteoblasts were seeded on three-dimensional scaffolds, and cultured in the following conditions: continuous flow perfusion, intermittent flow perfusion, and static condition. We collected samples on day 4, 8 and 12 for analysis. Osteoblast proliferation was demonstrated by cell proliferation and scanning electron microscopy assay. Additionally, the expression of known markers of differentiation, including alkaline phosphatase and osteocalcin, were tested by qRT-PCR and alkaline phosphatase activity assay, and the deposition of calcium was used as an indicator of mineralization demonstrated by calcium content assay. The results supported that low fluid shear stress plays an important role in the activation of osteoblasts: enhance cell proliferation, increase calcium deposition, and promote the expression of osteoblastic markers. Furthermore, the continuous flow perfusion is a more favorable environment for the initiation of osteoblast activity compared with intermittent flow perfusion. Therefore, the force and time of fluid shear stress are important parameters for osteoblast activation.     

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.     

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE(2) but no change in mineralization.

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE(2) production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE(2) is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 +/- 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE(2) production. After 7 days of static culture (0 dyn/cm(2)) or low (0.06 +/- 0.05 Pa, 0.3 Hz) or high (0.6 +/- 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference (P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE(2) levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.     

Effect of LIMK2 RNAi on reorganization of the actin cytoskeleton in osteoblasts induced by fluid shear stress

The biomechanical characteristics of bone tissue and its cells under mechanical stress are significant for bone biomechanics research, but the mechanism of mechanotransduction is still unknown. It has been established that the actin cytoskeleton of osteoblasts plays an important role in this process. However, the structure of the actin cytoskeleton is reorganized when loaded with mechanical stress, which results in changes in cell stiffness. These phenomena suggest that an actin-cytoskeleton-induced feedback regulation mechanism may be involved in the mechanotransduction of osteoblasts, but this has not yet been proven. The aim of this study was to explore the role of LIMK2 in the reorganization of the actin cytoskeleton induced by fluid shear stress in osteoblasts by using RNA interference. Balb/c mouse primary osteoblasts were divided into four groups. Cells in Groups 1 and 3 were transfected with negative control RNA, while cells in Groups 2 and 4 were transfected with a specific siRNA designed to silence the LIMK2 gene. Twenty-four hours after transfection, cells in Groups 1 and 2 were loaded with fluid shear stress at 12 dyne/cm2 while cells in Groups 3 and 4 were not. Compared with Group 1, the mean fluorescence density of the actin cytoskeleton in the other three groups was 28.9%, 45.7%, and 33.0%, respectively. These results indicate that LIMK2 plays an important role in the reorganization of the actin cytoskeleton induced by fluid shear stress.     

Do microcracks decrease or increase fatigue resistance in cortical bone?

Fatigue of cortical bone produces microcracks; it has been hypothesized that these cracks are analogous to those occurring in engineered composite materials and constitute a similar mechanism for fatigue resistance. However, the numbers of these linear microcracks increase substantially with age, suggesting that they contribute to increased fracture incidence among the elderly. To test these opposing hypotheses, we fatigued 20 beams of femoral cortical bone from elderly men and women in load-controlled four point bending having initial strain ranges of 3000 or 5000 microstrain. Loading was stopped at fracture or 10(6) cycles, whichever occurred first, and microcrack density and length were measured in the loaded region and in a control region that was not loaded. We studied the dependence of fatigue life and induced microdamage on initial microdamage, cortical region, subject gender and age, and several other variables. When the effect of modulus variability was controlled, longer fatigue life was associated with higher rather than lower initial crack density, particularly in the medial cortex. The increase in crack density following fatigue loading was greater in specimens from older individuals and those initially having longer microcracks. Crack density increased as much in specimens fatigued short of the failure point as in those that fractured, and microcracks were, on average, shorter in specimens with greater numbers of resorption spaces, a measure of remodeling rate.     

Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport

Fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. In addition to membrane shear stress, loading-induced fluid flow will enhance chemotransport due to convection or mass transport thereby affecting the biochemical environment surrounding the cell. This study investigated the role of oscillating fluid flow induced shear stress and chemotransport in cellular mechanotransduction mechanisms in bone. Intracellular calcium mobilization and prostaglandin E(2) (PGE(2)) production were studied with varying levels of shear stress and chemotransport. In this study MC3T3-E1 cells responded to oscillating fluid flow with both an increase in intracellular calcium concentration ([Ca(2+)](i)) and an increase in PGE(2) production. These fluid flow induced responses were modulated by chemotransport. The percentage of cells responding with an [Ca(2+)](i) oscillation increased with increasing flow rate, as did the production of PGE(2). In addition, depriving the cells of nutrients during fluid flow resulted in an inhibition of both [Ca(2+)](i) mobilization and PGE(2) production. These data suggest that depriving the cells of a yet to be determined biochemical factor in media affects the responsiveness of bone cells even at a constant peak shear stress. Chemotransport alone will not elicit a response, but it appears that sufficient nutrient supply or waste removal is needed for the response to oscillating fluid flow induced shear stress.     

Microdamage propagation in trabecular bone due to changes in loading mode

Microdamage induced by falls or other abnormal loads that cause shear stress in trabecular bone could impair the mechanical properties of the proximal femur or spine. Existing microdamage may also increase the initiation and propagation of further microdamage during subsequent normal, on-axis, loading conditions, resulting in atraumatic or "spontaneous" fractures. Microdamage formation due to shear and compressive strains was studied in 14 on-axis cylindrical bovine tibial trabecular bone specimens. Microdamage was induced by a torsional overload followed by an on-axis compressive overload and quantified microscopically. Fluorescent agents were used to label microdamage and differentiate damage due to the two loading modes. Both the microcrack density and diffuse damage area caused by the torsional overload increased with increasing shear strain from the center to the edge of the specimen. However, the mean microcrack length was uniform across the specimen, suggesting that microcrack length is limited by microstructural features. The mean density of microcracks caused by compressive overloading was slightly higher near the center of the specimen, and the diffuse damage area was uniform across the specimen. Over 20% of the microcracks formed in the initial torsional overloading propagated during compression. Moreover the propagating microcracks were, on average, longer than microcracks formed by a single overload. As such, changes in loading mode can cause propagation of microcracks beyond the microstructural barriers that normally limit the length. Damage induced by in vivo off-axis loads such as falls may similarly propagate during subsequent normal loading, which could affect both remodeling activity and fracture susceptibility.     

Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO-Y4 osteocytes

Mechanical loads are required for optimal bone mass. One mechanism whereby mechanical loads are transduced into localized cellular signals is strain-induced fluid flow through lacunae and canaliculi of bone. Gap junctions (GJs) between osteocytes and osteoblasts provides a mechanism whereby flow-induced signals are detected by osteocytes and transduced to osteoblasts. We have demonstrated the importance of GJ and gap junctional intercellular communication (GJIC) in intracellular calcium and prostaglandin E(2) (PGE(2)) increases in response to flow. Unapposed connexons, or hemichannels, are themselves functional and may constitute a novel mechanotransduction mechanism. Using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes, we examined the time course and mechanism of hemichannel activation in response to fluid flow, the composition of the hemichannels, and the role of hemichannels in flow-induced ATP release. We demonstrate that fluid flow activates hemichannels in MLO-Y4, but not MC3T3-E1, through a mechanism involving protein kinase C, which induces ATP and PGE(2) release.