Neural mechanobiology and neuronal vulnerability to traumatic loading

In order to understand the physical tolerance of neurons to traumatic insults, engineers and neuroscientists have attempted to reproduce the biomechanical environment during a traumatic event using in vitro injury systems with isolated components of the nervous system. This approach allows one to begin to unravel the underlying molecular and biochemical mechanisms that lead to cell dysfunction and death as a function of mechanical inputs. Excess mechanical force and deformation causes structural and functional breakdown, including several key deleterious cellular processes, such as membrane damage, an upset of calcium homeostasis, glutamate release, cell death, and caspase-mediated proteolysis. Understanding of the mechanotransduction events, however, that lead to cellular failure and dysfunction, are not well understood. Mechanically characterized cellular models of traumatic loading are critical to the improved understanding of mechanotransduction in the context of neural injury, the improvement of protective systems, and to provide a controlled setting for testing therapeutic interventions. In this review of the cellular mechanics of traumatic neural loading, we focus on the backdrop and motivation for studying mechanical thresholds in neurons and glial cells and discuss some of the acute responses that may help elucidate improved tolerance criteria and illuminate future research directions.     

Patient-specific bone modelling and remodelling simulation of hypoparathyroidism based on human iliac crest biopsies

We previously developed a load-adaptive bone modelling and remodelling simulation model that can predict changes in the bone micro-architecture as a result of changes in mechanical loading or cell activity. In combination with a novel algorithm to estimate loading conditions, this offers the possibility for patient-specific predictions of bone modelling and remodelling. Based on such models, the underlying mechanisms of bone diseases and/or the effects of certain drugs and their influence on the bone micro-architecture can be investigated. In the present study we test the ability of this approach to predict changes in bone micro-architecture during hypoparathyroidism (HypoPT), as an illustrative example. We hypothesize that, apart from reducing bone turnover, HypoPT must also lead to increased osteocyte mechanosensitivity in order to explain the changes in bone mass seen in patients. Healthy human iliac crest biopsies were used as the starting point for the simulations that mimic HypoPT conditions and the resultant micro-architectures were compared to age-matched clinical HypoPT biopsies. Simulation results were in good agreement with the clinical data when osteocyte mechanosensitivity was increased by 40%. In conclusion, the results confirm our hypothesis, and also demonstrate that patient-specific bone modelling and remodelling simulations are feasible.     

Mechanotransduction of stem cells for tendon repair

Tendon is a mechanosensitive tissue that transmits force from muscle to bone. Physiological loading contributes to maintaining the homeostasis and adaptation of tendon, but aberrant loading may lead to injury or failed repair. It is shown that stem cells respond to mechanical loading and play an essential role in both acute and chronic injuries, as well as in tendon repair. In the process of mechanotransduction, mechanical loading is detected by mechanosensors that regulate cell differentiation and proliferation via several signaling pathways. In order to better understand the stem-cell response to mechanical stimulation and the potential mechanism of the tendon repair process, in this review, we summarize the source and role of endogenous and exogenous stem cells active in tendon repair, describe the mechanical response of stem cells, and finally, highlight the mechanotransduction process and underlying signaling pathways.     

A sclerostin-based theory for strain-induced bone formation

Bone formation responds to mechanical loading, which is believed to be mediated by osteocytes. Previous theories assumed that loading stimulates osteocytes to secrete signals that stimulate bone formation. In computer simulations this 'stimulatory' theory successfully produced load-aligned trabecular structures. In recent years, however, it was discovered that osteocytes inhibit bone formation via the protein sclerostin. To reconcile this with strain-induced bone formation, one must assume that sclerostin secretion decreases with mechanical loading. This leads to a new 'inhibitory' theory in which loading inhibits osteocytes from inhibiting bone formation. Here we used computer simulations to show that a sclerostin-based model is able to produce a load-aligned trabecular architecture. An important difference appeared when we compared the response of the stimulatory and inhibitory models to loss of osteocytes, and found that the inhibitory pathway prevents the loss of trabeculae that is seen with the stimulatory model. Further, we demonstrated with combined stimulatory/inhibitory models that the two pathways can work side-by-side to achieve a load-adapted bone architecture.     

Mechanoregulation of gene expression in fibroblasts

Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This article reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested.     

Structural differences in the osteocyte network between the calvaria and long bone revealed by three-dimensional fluorescence morphometry, possibly reflecting distinct mechano-adaptations and sensitivities

The structural features of osteocytes and their cellular process network are thought to allow for mechanotransduction from the bone tissue to these cells. This study applied three-dimensional fluorescence microscopy to fixed and decalcified bone specimens to quantitatively compare the osteocytes and their networks between mouse parietal bone and tibia that are physiologically enforced by distinct mechanical loads. The subsequent morphometric analysis by the surface rendering of osteocyte cell bodies revealed the tibia to have relatively enriched cytoplasm in the osteocyte cell body in comparison to the parietal bone. Furthermore, quantitative tracing of the cellular processes in silico demonstrated that the numbers of the cellular processes and their bifurcation points per osteocyte in the tibia were significantly higher than those in the parietal bone. Though the total length of the processes per osteocyte in the tibia was two times longer, its total surface area and total volume were smaller than those in the parietal bone, due to its thinner diameter. These architectural differences in the osteocytes and their networks are thus implicated in the adaptation to physiologically different loading, and may also induce distinct mechanosensitivities.     

Mechanobiology of tendon

Tendons are able to respond to mechanical forces by altering their structure, composition, and mechanical properties--a process called tissue mechanical adaptation. The fact that mechanical adaptation is effected by cells in tendons is clearly understood; however, how cells sense mechanical forces and convert them into biochemical signals that ultimately lead to tendon adaptive physiological or pathological changes is not well understood. Mechanobiology is an interdisciplinary study that can enhance our understanding of mechanotransduction mechanisms at the tissue, cellular, and molecular levels. The purpose of this article is to provide an overview of tendon mechanobiology. The discussion begins with the mechanical forces acting on tendons in vivo, tendon structure and composition, and its mechanical properties. Then the tendon's response to exercise, disuse, and overuse are presented, followed by a discussion of tendon healing and the role of mechanical loading and fibroblast contraction in tissue healing. Next, mechanobiological responses of tendon fibroblasts to repetitive mechanical loading conditions are presented, and major cellular mechanotransduction mechanisms are briefly reviewed. Finally, future research directions in tendon mechanobiology research are discussed.     

Craniofacial sutures: Signaling centres integrating mechanosensation,cell signaling,and cell differentiation

Cranial sutures are dynamic structures in which stem cell biology, bone formation, and mechanical forces interface, influencing the shape of the skull throughout development and beyond. Over the past decade, there has been significant progress in understanding mesenchymal stromal cell (MSC) differentiation in the context of suture development and genetic control of suture pathologies, such as craniosynostosis. More recently, the mechanosensory function of sutures and the influence of mechanical signals on craniofacial development have come to the forefront. There is currently a gap in understanding of how mechanical signals integrate with MSC differentiation and ossification to ensure appropriate bone development and mediate postnatal growth surrounding sutures. In this review, we discuss the role of mechanosensation in the context of cranial sutures, and how mechanical stimuli are converted to biochemical signals influencing bone growth, suture patency, and fusion through mediation of cell differentiation. We integrate key knowledge from other paradigms where mechanosensation forms a critical component, such as bone remodeling and orthodontic tooth movement. The current state of the field regarding genetic, cellular, and physiological mechanisms of mechanotransduction will be contextualized within suture biology.     

Estrogen receptor and Wnt signaling interact to regulate early gene expression in response to mechanical strain in osteoblastic cells

Bone mass homeostasis is regulated by an interaction of various factors, including growth factors, systemic hormones and mechanical loading. Two signal transduction pathways, the estrogen receptor (ER) and the Wnt/β-catenin signal transduction pathway, have been shown to have an important role in regulating osteoblast and osteoclast function and to be involved in mechanotransduction. Therefore, dysfunction of these pathways can lead to osteoporotic bone loss. However, less is known about the modulation of gene expression by the interaction of these pathways in response to mechanical strain. We performed in vitro stretch experiments using osteoblastic MC3T3-E1 cells to study the effect of both pathways and mechanical strain on the expression of cyclooxygenase-2 (Cox-2), which is involved in the synthesis of prostaglandins, modulators of bone formation and resorption. Using specific agonists and antagonists, we demonstrated a regulation by an interaction of these pathways in mechantransduction. Estradiol (E2) had a sensitizing effect on mechanically induced Cox-2 expression, which seemed to be ligand-specific as it could be abolished using the antiestrogen ICI182,780. However, mechanical strain in the presence of Wnt signaling activators diminished both the E2 sensitizing effect and the stimulatory effect of Wnt signaling in the absence of strain. This interaction might be one regulatory mechanism by which mechanical loading exerts its role in bone mass homeostasis.     

Directional dependence of osteoblastic calcium response to mechanical stimuli

In adaptive bone remodeling, mechanical signals such as stress/strain caused by loading/deformation are believed to play important roles as regulators of the process in which osteoclastic resorption and osteoblastic formation are coordinated under a local mechanical environment. The mechanism by which cells sense and transduce mechanical signals to the intracellular biochemical signaling cascade is still unclear, however to address this issue, the present study investigated the characteristic response of a single osteoblastic cell, MC3T3-E1, to a well-defined mechanical stimulus and the involvement of the cytoskeletal actin fiber structure in the mechanotransduction pathway. First, by mechanically perturbing to a single cell using a microneedle, a change in the intracellular calcium ion concentration [Ca²⁺]_i was observed as a primal signaling response to a mechanical stimulus, and the threshold value of the perturbation as the mechanical stimulus was evaluated quantitatively. Second, to study directional dependence of the response to the mechanical stimulus, the effect of actin fiber orientation on the threshold value of the calcium response was investigated at various magnitudes and directions of the stimulus. It was found that the osteoblastic response to the perturbation exhibited a directional dependence. That is, the sensitivity of osteoblastic cells to a mechanical stimulus depends on the angle of the applied deformation with respect to the cytoskeletal actin fiber orientation. This finding is phenomenological evidence that cytoskeletal actin fiber structures are involved in the mechanotransduction mechanism, which may be related to cell polarization behaviors such as cellular alignment caused by mechanical stimulation.     

Cell mechanics and mechanotransduction: pathways, probes, and physiology

Cells face not only a complex biochemical environment but also a diverse biomechanical environment. How cells respond to variations in mechanical forces is critical in homeostasis and many diseases. The mechanisms by which mechanical forces lead to eventual biochemical and molecular responses remain undefined, and unraveling this mystery will undoubtedly provide new insight into strengthening bone, growing cartilage, improving cardiac contractility, and constructing tissues for artificial organs. In this article we review the physical bases underlying the mechanotransduction process, techniques used to apply controlled mechanical stresses on living cells and tissues to probe mechanotransduction, and some of the important lessons that we are learning from mechanical stimulation of cells with precisely controlled forces. cytoskeleton; micromanipulation; cell signaling     

Mechanotransduction in blood cells

Blood cells are subjected to various mechanical forces; including pressure, flow, shear force, gravity, and forces acting against them with varying stiffness (eg. blood vessel wall). Scientists have discovered that these forces have profound effects on cellular growth, differentiation, secretion of cytokines, cell death, and migration. These processes are called mechanotransduction, a conversion of mechanical forces to biochemical signals. In this article the author reviews biophysical forces that affect biological functions of blood cells and their responses in normal physiology and pathophysiology. Although input (forces) and output (cellular responses) have been well studied by utilizing recently developed various force-generating devices, the molecular mechanism of mechanotransudction is still a mystery. This is because reconstructing molecular interaction in the presence of mechanical forces in vitro is highly challenging and until now the molecular dynamics involved in structural changes caused by these forces are largely unknown. Nevertheless, the author has reviewed a few examples of potential structural effects on the molecular mechanism of mechanotransduction.     

The mechanics of the primary cilium: an intricate structure with complex function

The primary cilium is a non-motile singular cellular structure that extends from the surface of nearly every cell in the body. The cilium has been shown to play numerous roles in maintaining tissue homeostasis, through regulating signaling pathways and sensing both biophysical and biochemical changes in the extracellular environment. The structural performance of the cilium is paramount to its function as defective cilia have been linked to numerous pathologies. In particular, the cilium has demonstrated a mechanosensory role in tissues such as the kidney, liver, endothelium and bone, where cilium deflection under mechanical loading triggers a cellular response. Understanding of how cilium structure and subsequent mechanical behavior contributes to the roles that cilium plays in regulating cellular behavior is a compelling question, yet is a relatively untouched research area. Recent advances in biophysical measurements have demonstrated the cilium to be a structurally intricate organelle containing an array of load bearing proteins. Furthermore advances in modeling of this organelle have revealed the importance of these proteins at regulating the cilium's mechanosensitivity. Remarkably, the cilium is capable of adapting its mechanical state, altering its length and possibly it's bending resistance, to regulate its mechanosensitivity demonstrating the importance of cilium mechanics in cellular responses. In this review, we introduce the cilium as a mechanosensor; discuss the advances in the mechanical modeling of cilia; explore the structural features of the cilium, which contribute to its mechanics and finish with possible mechanisms in which alteration in structure may affect ciliary mechanics, consequently affecting ciliary based mechanosensing.     

Phenotypic plasticity and mechano‐transduction in the teleost skeleton

The proper formation, growth and maintenance of many bones depends on the mechanical loads generated by gravity and muscles. Mechanical loading by muscle forces does not only affect bone growth and maintenance in adult and juvenile vertebrates, but also affects larval and embryonic bone development. We have reviewed the current understanding of mechanotransduction in birds and mammals and compared it to teleosts. The major mechanosensing cells in the adult mammalian and avian skeleton are osteocytes. They are interconnected via cell processes and are contained within a canalicular network. Basal teleosts have osteocytes but their connectivity is questionable and the presence of a functional canalicular network is unlikely. Advanced teleosts have acellular bone and therefore lack osteocytes. Yet the skeleton of teleosts does show adaptive responses to changes in mechanical load. In these animals it is likely that osteoblasts, bone surface cells and chondrocytes act as mechanosensors. The factors expressed by osteocytes upon mechanical stimulation have been extensively investigated in vitro and in vivo in adult mammals and birds. Less is, however, known about the mechanotransduction pathway during embryonic bone development. The zebrafish presents new opportunities to analyze the mechanotransduction pathway during early (larval) bone formation due to the ex utero development and genetic analyses.     

Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation

Indian hedgehog (Ihh), a member of the vertebrate hedgehog morphogen family, is a key signaling molecule that controls chondrocyte proliferation and differentiation. In this study, we show a novel function of Ihh. Namely, it acts as an essential mediator of mechanotransduction in cartilage. Cyclic mechanical stress greatly induces the expression of Ihh by chondrocytes. This induction is abolished by gadolinium, an inhibitor of stretch-activated channels. This suggests that the IHH gene is mechanoresponsive. The mechano-induction of Ihh is essential for stimulating chondrocyte proliferation by mechanical loading. The presence of an Ihh functional blocking antibody during loading completely abolishes the stimulatory effect of mechanical load on proliferation. Furthermore, Ihh mediates the mechanotransduction process in a bone morphogenic protein (BMP)-dependent and parathyroid hormone-related peptide-independent manner. BMP 2/4 are up-regulated by mechanical stress through the induction of Ihh, and BMP antagonist noggin inhibits mechanical stimulation of chondrocyte proliferation. This suggests BMP lies downstream of Ihh in mechanotransduction pathway. Our data suggest that Ihh may transduce mechanical signals during cartilage growth and repair processes.     

A 3-D model of ligand transport in a deforming extracellular space

Cells communicate through shed or secreted ligands that traffic through the interstitium. Force-induced changes in interstitial geometry can initiate mechanotransduction responses through changes in local ligand concentrations. To gain insight into the temporal and spatial evolution of such mechanotransduction responses, we developed a 3-D computational model that couples geometric changes observed in the lateral intercellular space (LIS) of mechanically loaded airway epithelial cells to the diffusion-convection equations that govern ligand transport. By solving the 3-D fluid field under changing boundary geometries, and then coupling the fluid velocities to the ligand transport equations, we calculated the temporal changes in the 3-D ligand concentration field. Our results illustrate the steady-state heterogeneities in ligand distribution that arise from local variations in interstitial geometry, and demonstrate that highly localized changes in ligand concentration can be induced by mechanical loading, depending on both local deformations and ligand convection effects. The occurrence of inhomogeneities at steady state and in response to mechanical loading suggest that local variations in ligand concentration may have important effects on cell-to-cell variations in basal signaling state and localized mechanotransduction responses.     

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.     

Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes

The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or "biomarkers," of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations.     

Remodeling of actin cytoskeleton in mouse periosteal cells under mechanical loading induces periosteal cell proliferation during bone formation

Background

The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.

Principal Findings

We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells.

Conclusions

These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.