Analysis of bone architecture sensitivity for changes in mechanical loading, cellular activity, mechanotransduction, and tissue properties

Bone has an architecture which is optimized for its mechanical environment. In various conditions, this architecture is altered, and the underlying cause for this change is not always known. In the present paper, we investigated the sensitivity of the bone microarchitecture for four factors: changes in bone cellular activity, changes in mechanical loading, changes in mechanotransduction, and changes in mechanical tissue properties. The goal was to evaluate whether these factors can be the cause of typical bone structural changes seen in various pathologies. For this purpose, we used an established computational model for the simulation of bone adaptation. We performed two sensitivity analyses to evaluate the effect of the four factors on the trabecular structure, in both developing and adult bone. According to our simulations, alterations in mechanical load, bone cellular activities, mechanotransduction, and mechanical tissue properties may all result in bone structural changes similar to those observed in various pathologies. For example, our simulations confirmed that decreases in loading and increases in osteoclast number and activity may lead to osteoporotic changes. In addition, they showed that both increased loading and decreased bone matrix stiffness may lead to bone structural changes similar to those seen in osteoarthritis. Finally, we found that the model may help in gaining a better understanding of the contribution of individual disturbances to a complicated multi-factorial disease process, such as osteogenesis imperfecta.     

Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells

Numerous cellular biochemical responses to mechanical loading are transient, indicating a cell's ability to adapt its behavior to a new mechanical environment. Since load-induced cellular deformation can initiate these biochemical responses, the overall goal of this study was to investigate the adaptation of global, or whole-cell, mechanical behavior, i.e., cellular deformability, in response to mechanical loading for osteoblastic cells. Confluent cell cultures were subjected to 1 or 2 Pa flow-induced shear stress for 2 h. Whole-cell mechanical behavior was then measured for individual cells using an atomic force microscope. Compared to cells maintained under static conditions, whole-cell stiffness was 1.36-fold (p=0.006) and 1.70-fold (p<0.001) greater for cells exposed to 1 and 2 Pa shear loading, respectively. The increase in shear stress magnitude from 1 to 2 Pa also caused a statistically significant, 1.25-fold increase in cell stiffness (p=0.02). Increases in cell stiffness were not altered in either flow group for 70 min after flow was terminated (p=0.15). Flow-induced rearrangement of the actin cytoskeleton was also maintained for at least 90 min after flow was terminated. Taken together, these findings support the hypothesis that cells become mechanically adapted to their mechanical environment via cytoskeletal modifications. Accordingly, cellular mechanical adaptation may play a key role in regulation of cellular mechanosensitivity and the related effects on tissue structure and function.     

The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

Background  

In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level.     

In vivo investigation of tendon responses to mechanical loading

Tendons transmit skeletal muscle forces to bone and are essential in all voluntary movement. In turn, movement appears to affect tendon properties, and in recent years considerable effort has been put into discovering how tendon tissue responds to mechanical stimuli in vivo. Months and years of mechanical loading can influence the gross morphology of tendon, seen as an increase tendon cross sectional area (CSA). Similarly, tendon stiffness appears to be affected by weeks to months of loading. Increased stiffness can relate to changes in CSA and/or tendon material properties (modulus), though the relative contribution of these parameters is largely unclear. The possible mechanisms behind alterations in tendon material properties include changes in collagen fibril morphology and levels of cross-linking between collagen molecules. Furthermore, increased levels of collagen synthesis and expression are seen as a response to acute exercise and training, and may be a central parameter in tendon adaptation to loading. There are indications that this collagen-induction relates to the auto-/paracrine action of collagen-stimulating growth factors, such as TGFβ-1 and IGF-I, which are expressed in response to mechanical stimuli.     

Neuromuscular and mechanical responses to inspiratory resistive loading during sleep

The purposes of this study were 1) to characterize the immediate inspiratory muscle and ventilation responses to inspiratory resistive loading during sleep in humans and 2) to determine whether upper airway caliber was compromised in the presence of a resistive load. Ventilation variables, chest wall, and upper airway inspiratory muscle electromyograms (EMG), and upper airway resistance were measured for two breaths immediately preceding and immediately following six applications of an inspiratory resistive load of 15 cmH2O.l-1 X s during wakefulness and stage 2 sleep. During wakefulness, chest wall inspiratory peak EMG activity increased 40 +/- 15% (SE), and inspiratory time increased 20 +/- 5%. Therefore, the rate of rise of chest wall EMG increased 14 +/- 10.9% (NS). Upper airway inspiratory muscle activity changed in an inconsistent fashion with application of the load. Tidal volume decreased 16 +/- 6%, and upper airway resistance increased 141 +/- 23% above pre-load levels. During sleep, there was no significant chest wall or upper airway inspiratory muscle or timing responses to loading. Tidal volume decreased 40 +/- 7% and upper airway resistance increased 188 +/- 52%, changes greater than those observed during wakefulness. We conclude that 1) the immediate inspiratory muscle and timing responses observed during inspiratory resistive loading in wakefulness were absent during sleep, 2) there was inadequate activation of upper airway inspiratory muscle activity to compensate for the increased upper airway inspiratory subatmospheric pressure present during loading, and 3) the alteration in upper airway mechanics during resistive loading was greater during sleep than wakefulness.     

Tensional homeostasis in dermal fibroblasts: Mechanical responses to mechanical loading in three-dimensional substrates

Many soft connective tissues are under endogenous tension, and their resident cells generate considerable contractile forces on the extracellular matrix. The present work was aimed to determine quantitatively how fibroblasts, grown within three-dimensional collagen lattices, respond mechanically to precisely defined tensional loads. Forces generated in response to changes in applied load were measured using a tensional culture force monitor. In a number of variant systems, resident cells consistently reacted to modify the endogenous matrix tension in the opposite direction to externally applied loads. That is, increased external loading was followed immediately by a reduction in cell-mediated contraction whilst decreased external loading elicited increased contraction. Responses were cell-mediated and not a result of material properties of the matrices. This is the first detailed characterisation of a tensional homeostatic response in cells. The maintained force, after 8 h in culture, was typically around 40–60 dynes/million cells. Maintenance of an active tensional homeostasis has widespread implications for cells in culture and forwhole tissue function. J. Cell. Physiol. 175:323–332, 1998. © 1998 Wiley-Liss, Inc.     

The contribution of cellular mechanotransduction to cardiomyocyte form and function

Myocardial development is regulated by an elegantly choreographed ensemble of signaling events mediated by a multitude of intermediates that take a variety of forms. Cellular differentiation and maturation are a subset of vertically integrated processes that extend over several spatial and temporal scales to create a well-defined collective of cells that are able to function cooperatively and reliably at the organ level. Early efforts to understand the molecular mechanisms of cardiomyocyte fate determination focused primarily on genetic and chemical mediators of this process. However, increasing evidence suggests that mechanical interactions between the extracellular matrix (ECM) and cell surface receptors as well as physical interactions between neighboring cells play important roles in regulating the signaling pathways controlling the developmental processes of the heart. Interdisciplinary efforts have made it apparent that the influence of the ECM on cellular behavior occurs through a multitude of physical mechanisms, such as ECM boundary conditions, elasticity, and the propagation of mechanical signals to intracellular compartments, such as the nucleus. In addition to experimental studies, a number of mathematical models have been developed that attempt to capture the interplay between cells and their local microenvironment and the influence these interactions have on cellular self-assembly and functional behavior. Nevertheless, many questions remain unanswered concerning the mechanism through which physical interactions between cardiomyocytes and their environment are translated into biochemical cellular responses and how these signaling modalities can be utilized in vitro to fabricate myocardial tissue constructs from stem cell-derived cardiomyocytes that more faithfully represent their in vivo counterpart. These studies represent a broad effort to characterize biological form as a conduit for information transfer that spans the nanometer length scale of proteins to the meter length scale of the patient and may yield new insights into the contribution of mechanotransduction into heart development and disease.     

Integrin activation and matrix binding mediate cellular responses to mechanical stretch

Mechanical tension is a critical determinant of cell growth, differentiation, apoptosis, migration, and development. Integrins have been implicated in sensing force but little is known about how forces are transduced to biochemical signals. We now show that mechanical strain stimulates conformational activation of integrin alphavbeta3 in NIH3T3 cells. Integrin activation is mediated by phosphoinositol 3-kinase and is followed by an increase in integrin binding to extracellular matrix proteins. Mechanical stretch stimulation of JNK was dependent on new integrin binding to extracellular matrix. These data define a molecular mechanism for the role of integrins in mechanotransduction.     

Mitochondrial requirement for endothelial responses to cyclic strain: implications for mechanotransduction

Mechanical strain triggers a variety of cellular responses, but the underlying mechanotransduction process has not been established. Endothelial cells (EC) respond to mechanical strain by upregulating adhesion molecule expression through a signaling process involving reactive oxygen species (ROS), but the site of their generation is unknown. Mitochondria anchor to the cytoskeleton and could function as mechanotransducers by releasing ROS during cytoskeletal strain. In human umbilical vein EC (HUVEC), ROS production increased 221 +/- 17% during 6 h of cyclic strain vs. unstrained controls. Mitochondrial inhibitors diphenylene iodonium or rotenone abrogated this response, whereas inhibitors of nitric oxide (NO) synthase (L-nitroarginine), xanthine oxidase (allopurinol), or NAD(P)H oxidase (apocynin) had no effect. The antioxidants ebselen and diethyldithiocarbamate inhibited the increase in ROS, but the NO scavenger Hb had no effect. Thus strain induces ROS release from mitochondria. In other studies, HUVEC were rendered mitochondria deficient (rho0 EC) to determine the requirement for electron transport in the response to strain. Strain-induced 2'7'-dichlorofluorescein fluorescence was attenuated by >80% in rho0 EC compared with HUVEC (43 +/- 7 vs. 221 +/- 17%). Treatment with cytochalasin D abrogated strain-induced ROS production, indicating a requirement for the actin cytoskeleton. Cyclic strain (6 h) increased VCAM-1 expression in wild-type but not rho0 EC. Increases in NF-kappaB activation and VCAM-1 mRNA expression during strain were prevented by antioxidants. These findings demonstrate that mitochondria function as mechanotransducers in endothelium by increasing ROS signaling, which is required for strain-induced increase in VCAM-1 expression via NF-kappaB.     

Articular area responses to mechanical loading: effects of exercise, age, and skeletal location.

How reliable are reconstructions of body mass and joint function based on articular surface areas? While the dynamic relationship between mechanical loading and cross‐sectional geometry in long bones is well‐established, the effect of loading on the subchondral articular surface area of epiphyses (hereafter, articular surface area, or ASA) has not been experimentally tested. The degree to which ASA can change in size and shape is important, because articular dimensions are frequently used to estimate body mass and positional behavior in fossil species. This study tests the hypothesis that mechanical loading influences ASA by comparing epiphyses of exercised and sedentary sheep from three age categories: juvenile, subadult, and adult (n = 44). ASA was measured on latex molds of subchondral articular surfaces of 10 epiphyses from each sheep. Areas were standardized by body mass, and compared to diaphyseal cross‐sectional geometrical data. Nonparametric statistical comparisons of exercised and control individuals found no increases in ASA in response to mechanical loading in any age group. In contrast, significant differences in diaphyseal cross‐sectional geometry were detected between exercised and control groups, but mostly in juveniles. The conservatism of ASA supports the hypothesis that ASA is ontogenetically constrained, and related to locomotor behavior at the species level and to body mass at the individual level, while variations in diaphyseal cross‐sectional geometry are more appropriate proxies for individual variations in activity level. Am J Phys Anthropol 116:266–277, 2001. © 2001 Wiley‐Liss, Inc.     

Microarray analysis of regional cellular responses to local mechanical stress in acute lung injury

Human acute lung injury is characterized by heterogeneous tissue involvement, leading to the potential for extremes of mechanical stress and tissue injury when mechanical ventilation, required to support critically ill patients, is employed. Our goal was to establish whether regional cellular responses to these disparate local mechanical conditions could be determined as a novel approach toward understanding the mechanism of development of ventilator-associated lung injury. We utilized cross-species genomic microarrays in a unilateral model of ventilator-associated lung injury in anesthetized dogs to assess regional cellular responses to local mechanical conditions that potentially contribute pathogenic mechanisms of injury. Highly significant regional differences in gene expression were observed between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the base, with 367 and 1,544 genes differentially regulated between these regions, respectively. Major functional groupings of differentially regulated genes included inflammation and immune responses, cell proliferation, adhesion, signaling, and apoptosis. Expression of genes encoding both acute lung injury-associated inflammatory cytokines and protective acute response genes were markedly different in the nondependent compared with the dependent regions of the lung base. We conclude that there are significant differences in the local responses to stress within the lung, and consequently, insights into the cellular responses that contribute to ventilator-associated lung injury development must be sought in the context of the mechanical heterogeneity that characterizes this syndrome.     

The endoplasmic reticulum: a sensor of cellular stress that modulates immune responses

Many inflammatory and infectious diseases are characterized by the activation of signaling pathways steaming from the endoplasmic reticulum (ER). These pathways, primarily associated with loss of ER homeostasis, are emerging as key regulators of inflammation and infection. Recent advances shed light on the mechanisms linking ER-stress and immune responses.     

Non-antisense cellular responses to oligonucleotides

Oligonucleotides induce various cellular responses which are not due to the blockade of protein synthesis by an antisense mechanism. Oligonucleotides presenting double-stranded or G-quartet structures (ribo- or deoxyribonucleotides, phosphodiester or phosphorothioated) induce retraction of neurites and aggregation of chicken retinal cells within 10–20 h. This effect is reversible, non-toxic; it appears to require internalization and can be mimicked by treatment of the cells with an RGDS peptide. The oligonucleotides appear to trigger a cascade of intracellular events, affecting the adhesive properties of integrins. In addition, a subset of oligonucleotides induced platelet aggregation, probably through their interaction with membrane receptors. Recognition of these effects is important for the design and interpretation of antisense experiments.     

A finite element model predicts the mechanotransduction response of tendon cells to cyclic tensile loading

The importance of fluid-flow-induced shear stress and matrix-induced cell deformation in transmitting the global tendon load into a cellular mechanotransduction response is yet to be determined. A multiscale computational tendon model composed of both matrix and fluid phases was created to examine how global tendon loading may affect fluid-flow-induced shear stresses and membrane strains at the cellular level. The model was then used to develop a quantitative experiment to help understand the roles of membrane strains and fluid-induced shear stresses on the biological response of individual cells. The model was able to predict the global response of tendon to applied strain (stress, fluid exudation), as well as the associated cellular response of increased fluid-flow-induced shear stress with strain rate and matrix-induced cell deformation with strain amplitude. The model analysis, combined with the experimental results, demonstrated that both strain rate and strain amplitude are able to independently alter rat interstitial collagenase gene expression through increases in fluid-flow-induced shear stress and matrix-induced cell deformation, respectively.