Adaptation of connexin 43-hemichannel prostaglandin release to mechanical loading

Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE(2), a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE(2) by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels. The opening of hemichannels and release of PGE(2) are magnitude-dependent on the level of shear stress. Insertion of a rest period between stress enhances this response. Hemichannels gradually close after 24 h of continuous shear stress corresponding with reduced Cx43 expression on the cell surface, thereby reducing any potential negative effects of channels staying open for extended periods. These data suggest that Cx43-hemichannel activity associated with PGE(2) release is adaptively regulated by mechanical loading to provide an effective means of regulating levels of extracellular signaling molecules responsible for initiation of bone (re)modeling.     

Intercellular mechanotransduction: cellular circuits that coordinate tissue responses to mechanical loading

Physical forces play an important role in modulating cell function and shaping tissue structure. Mechanotransduction, the process by which cells transduce physical force-induced signals into biochemical responses, is critical for mediating adaptations to mechanical loading in connective tissues. While much is known about mechanotransduction in cells involving forces delivered through extracellular matrix proteins and integrins, there is limited understanding of how mechanical signals are propagated through the interconnected cellular networks found in tissues and organs. We propose that intercellular mechanotransduction is a critical component for achieving coordinated remodeling responses to force application in connective tissues. We examine here recent evidence on different pathways of intercellular mechanotransduction and suggest a general model for how multicellular structures respond to mechanical loading as an integrated unit.     

Altered mechanical behavior of epicardium under isothermal biaxial loading

Most soft tissues that are treated clinically via heating experience multiaxial states of stress and strain in vivo and are subject to complex constraints during treatment. Remarkably, however, there are no prior data on changes in the multiaxial mechanical behavior of a collagenous tissue subjected to isometric constraints during heating. This paper presents the first biaxial stress-stretch data on a collagenous membrane (epicardium) before and after heating while subjected to various biaxial isometric constraints. It was found that isometric heating does not allow the increase in stiffness at low strains that occurs following isotonic heating. Moreover increasing the degree of stretch prior to heating increased the thermal stability of the tissue consistent with the concept that mechanical loading primarily affects the activation entropy, not the activation energy.     

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.     

Adaptation of the tendon to mechanical usage

Tendons primarily function as contractile force transmitters, but their mechanical properties may change dependent upon their level of mechanical usage. Using an ultrasound-based technique we have assessed tendon mechanical properties in vivo in a number of conditions representing different levels of mechanical usage. Ageing alters tendon mechanical properties; stiffness and modulus were lower in older adults by 10 and 14%, respectively, compared to young adults. Increased levels of exercise loading in old age can however partly reverse this process, as tendon stiffness and modulus were found to increase by 65 and 69%, respectively. Complete unloading due to bed rest or spinal cord injury both reduce tendon stiffness and modulus, however, only chronic unloading due to spinal cord injury seems to cause tendon atrophy. Alterations in tendon mechanical properties due to changes in the levels loading have implications for the speed of force transmission, the muscle's operating range and the likelihood of tendon strain injury.     

Analysis of the mechanical behavior of chondrocytes in unconfined compression tests for cyclic loading

Experimental evidence indicates that the biosynthetic activity of chondrocytes is associated with the mechanical environment. For example, excessive, repetitive loading has been found to induce cell death, morphological and cellular damage, as seen in degenerative joint disease, while cyclic, physiological-like loading has been found to trigger a partial recovery of morphological and ultrastructural aspects in osteoarthritic human articular chondrocytes. Mechanical stimuli are believed to influence the biosynthetic activity via the deformation of cells. However, the in situ deformation of chondrocytes for cyclic loading conditions has not been investigated experimentally or theoretically. The purpose of the present study was to simulate the mechanical response of chondrocytes to cyclic loading in unconfined compression tests using a finite element model. The material properties of chondrocytes and extracellular matrix were considered to be biphasic. The time-histories of the shape and volume variations of chondrocytes at three locations (i.e., surface, center, and bottom) within the cartilage were predicted for static and cyclic loading conditions at two frequencies (0.02 and 0.1 Hz) and two amplitudes (0.1 and 0.2 MPa). Our results show that cells at different depths within the cartilage deform differently during cyclic loading, and that the depth dependence of cell deformation is influenced by the amplitude of the cyclic loading. Cell deformations under cyclic loading of 0.02 Hz were found to be similar to those at 0.1 Hz. We conclude from the simulation results that, in homogeneous cartilage layers, cell deformations are location-dependent, and further are affected by load magnitude. In physiological conditions, the mechanical environment of cells are even more complex due to the anisotropy, depth-dependent inhomogeneity, and tension-compression non-linearity of the cartilage matrix. Therefore, it is feasible to speculate that biosynthetic responses of chondrocytes to cyclic loading depend on cell location and load magnitude.     

Development of a cell culture system loading cyclic mechanical strain to chondrogenic cells

Mechanical stimulation is considered to be one of the major epigenetic factors regulating the metabolism, proliferation, survival and differentiation of cells in the skeletal tissues. It is generally accepted that the cytoskeleton can undergo remodeling in response to mechanical stimuli such as tensile strain or fluid flow. Mechanically induced cell deformation is one of the possible mechanotransduction pathways by which chondrocytes sense and respond to changes in their mechanical environment. Mechanical strain has a variety of effects on the structure and function of their cells in the skeletal tissues, such as chondrocytes, osteoblasts and fibroblasts. However, little is known about the effect of the quality and quantity of mechanical strain and the timing of mechanical loading on the differentiation of these cells. The present study was designed to investigate the effect of the deformation of chondrogenic cells, and cyclic compression using a newly developed culture device, by analyzing mechanobiological response to the differentiating chondrocytes. Cyclic compression between 0 and 22% strains, at 23 microHz was loaded on chondrogenic cell line ATDC5 by seeding in a mass mode on PDMS membrane, assuming direct transfer of cyclic deformation from the membrane to the cells at the same frequency. The compressive strain, induced within the membrane, was characterized based on the analysis of the finite element modeling (FEM). The results showed that the tensile strain inhibits the chondrogenic differentiation of ATDC5 cells, whereas the compressive strain enhances the chondrogenic differentiation, suggesting that the differentiation of the chondrogenic cells could be controlled by the amount and the mode of strain. In conclusion, we have developed a unique strain loading culture system to analyze the effect of various types of mechanical stimulation on various cellular activities.     

Signal transduction pathways involved in mechanical regulation of HB-GAM expression in osteoblastic cells

Protein kinase C (PKC), protein kinase A (PKA), prostaglandin synthesis, and various mitogen-activated protein kinases (MAPKs) have been reported to be activated in bone cells by mechanical loading. We studied the involvement of these signal transduction pathways in the downregulation of HB-GAM expression in osteoblastic cells after cyclic stretching. Specific antagonists and agonists of these signal transduction pathways were added to cells before loading and to non-loaded control cells. Quantitative RT-PCR was used to evaluate gene expression. The data demonstrated that the extracellular signal-regulated kinase (ERK) 1/2 pathway, PKC, PKA, p38, and c-Jun N-terminal kinase MAPK participated in the mechanical downregulation of HB-GAM expression, whereas prostaglandin synthesis did not seem to be involved.     

Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

Summary We report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments. After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited osteoblastic phenotype. Cultured cells show a regular arrangment in vitro and exhibited multiple interconnecting junctions on scanning electron microscopic examination. Immunocytochemical staining showed that the cells produced almost exclusively type I collagen. Bone-surface-derived cells responded to 1–34 human parathyroid hormone by increasing intracellular cyclic AMP. Cell cultures exhibited high alkaline phosphatase activity, which was unaffected by 1,25 (OH)₂ vitamin D. Untreated cells produced high levels of osteocalcin, a bone-specific protein, and they responded to 1,25(OH) vitamin D by increasing osteocalcin synthesis in a dose-dependent manner. Although cells cultured for up to 5 mo. still produced osteocalcin, the response to 1,25(OH)₂D decreased after multiple passages. This study shows that the bone cell populations isolated from trabecular bone surface are enriched in osteoblast precursors and mature osteoblstic cells.     

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.     

Cytoskeletal architecture and mechanical behavior of living cells

Conventional continuum mechanics models considering living cells as viscous fluid balloons are unable to explain some recent experimental observations. In contrast, new microstructural models provide the desirable explanations. These models emphasize the role of the cell cytoskeleton built of struts-microtubules and cables-microfilaments. A specific architectural model of the cytoskeletal framework called "tensegrity" deserved wide attention recently. Tensegrity models particularly account for the phenomenon of linear stiffening of living cells. These models are discussed from the structural mechanics perspective. Classification of structural assemblies is given and the meaning of "tensegrity" is pinpointed. Possible sources of non-linearity leading to cell stiffening are emphasized. The role of local buckling of microtubules and overall stability of the cytoskeleton is stressed. Computational studies play a central role in the development of the microstructural theoretical framework allowing for the prediction of the cell behavior from "first principles". Algorithms of computer analysis of the cytoskeleton that consider unilateral response of microfilaments and deep postbuckling of microtubules are addressed.     

Focal contact clustering in osteoblastic cells under mechanical stresses: microgravity and cyclic deformation

We quantitatively compared vinculin-related adhesion parameters in osteoblastic cells submitted to two opposing mechanical stresses: low deformation and frequency strain regimens (stretch conditions) and microgravity exposure (relaxed conditions). In both ROS 17/2.8 cells and rat primary osteoblastic cells, 1% cyclic deformations at 0.05 Hz for 10 min per day for seven days stimulated cell growth compared to static culture conditions, while relaxed ROS cells proliferated in a similar way to static cultures (BC). We studied the short-term (up to 24 h) adaptation of focal contact reorganization under these two conditions. Cyclic deformation induced a biphasic response comprising the formation of new focal contacts followed by clustering of these focal contacts in both ROS cells and primary osteoblasts. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. To evaluate whether the proliferation (stretch) or survival (relaxed) status of ROS cells influences focal contact organization, we inhibited the ERK proliferative-dependent pathway. Inhibition of proliferation by PD98059 was partially reversed, but not fully restored by stretch. Stretch-induced clustering of vinculin-positive contacts also persisted in the presence of PD98059, whereas the increase in focal contact number was abolished. In conclusion, we show that focal contacts are mechanoeffectors, and we suggest that their morphologic organization might serve as a discriminant functional parameter between survival and proliferation status in ROS 17/2.8 osteoblastic cells.     

Site specific bone adaptation response to mechanical loading

Over 25 million Americans suffer from osteoporosis. Bone size and strength depends both upon the level of adaptation due to physical activity (applied load), and genetics. We hypothesized that bone adaptation to loads differs among mice breeds and bone sites. Forty-five adult female mice from three inbred strains (C57BL/6 [B6], C3H/HeJ [C3], and DBA/2J [D2]) were loaded at the right tibia and ulna in vivo with non-invasive loading devices. Each loading session consisted of 99 cycles at a force range that induced approximately 2000 microstrain (microepsilon) at the mid-shaft of the tibia (2.5 to 3.5 N force) and ulna (1.5 to 2 N force). The right and left ulnae and tibiae were collected and processed using protocols for histological undecalcified cortical bone slides. Standard histomorphometry techniques were used to quantify new bone formation. The histomorphometric variables include percentage mineralizing surface (%MS), mineral apposition rate (MAR), and bone formation rate (BFR). Net loading response [right-left limb] was compared between different breeds at tibial and ulnar sites using two-way ANOVA with repeated measures (p<0.05). Significant site differences in bone adaptation response were present within each breed (p<0.005). In all the three breeds, the tibiae showed greater percentage MS, MAR and BFR than the ulna at similar in vivo load or mechanical stimulus (strain). These data suggest that the bone formation due to loading is greater in the tibiae than the ulnae. Although, no significant breed-related differences were found in response to loading, the data show greater trends in tibial bone response in B6 mice as compared to D2 and C3 mice. Our data indicate that there are site-specific skeletal differences in bone adaptation response to similar mechanical stimulus.     

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.     

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 mammalian cells to non-ammoniagenic media

Although glutamine is used as a major substrate for the growth of mammalian cells in culture, it suffers from some disadvantages. Glutamine is deaminated through storage or by cellular metabolism, leading to the formation of ammonia which can result in growth inhibition. Non-ammoniagenic alternatives to glutamine have been investigated in an attempt to develop strategies for obtaining improved cell yields for ammonia sensitive cell lines.Glutamate is a suitable substitute for glutamine in some culture systems. A period of adaptation to glutamate is required during which the activity of glutamine synthetase and the rate of transport of glutamate both increase. The cell yield increases when the ammonia accumulation is decreased following culture supplementation with glutamate rather than glutamine. However some cell lines fail to adapt to growth in glutamate and this may be due to a low efficiency transport system.The glutamine-based dipeptides, ala-gln and gly-gln can substitute for glutamine in cultures of antibody-secreting hybridomas. The accumulation of ammonia in these cultures is less and cell yields in dipeptide-based media may be improved compared to glutamine-based controls. In murine hybridomas, a higher concentration of gly-gln is required to obtain comparable cell growth to ala-gln or gln-based cultures. This is attributed to a requirement for dipeptide hydrolysis catalyzed by an enzyme with higher affinity for ala-gln than gly-gln.