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Mechanically loaded photopolymerized hydrogels as 3D models to probe mechanotransduction pathways in chondrocytes
Track 10. Cellular and Molecular Mechanics volume (>75% of total change in <2 minutes), which remained stable thereafter. Numerical modeling predicts that the concentration of shed autocrine ligands changes inversely to, and slightly lags the change in LIS width, depending on the effective diffusivity of the ligand. Our finding that the increase in EGFR activation closely follows the dynamics of LIS collapse supports a direct linkage between compressive stress, LIS collapse, increased ligand concentration, and autocrine signaling. Mechanical and geometric perturbation of local autocrine signaling represents a new mechanism for mechano-signaling with potentially broad implications. 5380 Th, 11:30-11:45 (P41) Effects of hydrostatic pressure on ERK signalling of chondrocytes cultured in agarose gel K. Mio 1, J. Kirkham 2, W.A. Bonass 2. 1Academic Unit efMuscule-Skeletal and
Rehabilitation Medicine, Bioengineering Division, University of Leeds, Leeds, UK, 2Department of Oral Biology, Leeds Dental Institute, University of Leeds, Leeds, UK Chondrocytes need appropriate mechanical stress for maintenance of phenotype, suggesting a role for intracellular mechanotransduction pathways, possibly including those associated with mitogen-activated protein kinases (MAPKs) [1]. However, the precise role of MAPK signalling in mechanotransduction remains controversial [2,3]. We hypothesize that the extracellular signal-regulated kinase (ERK) pathway (one of the MAPK pathways) participates in hydrostatic pressure-induced mechanotransduction. To test this hypothesis, we investigated ERK activation under hydrostatic pressure. Expression of sox9 mRNA (regarded as a "master switch" for chondrocyte differentiation) was determined as a possible downstream target of the pathway. 5 MPa hydrostatic pressure was applied to bovine chondrocyte-agarose constructs for 4 hours. ERK phosphorylation (associated with ERK activation), was determined using western blotting. Expression of sox9 mRNA was analysed by RT-PCR. The results showed a decrease in ERK phosphorylation with a concomitant increase in the sox9 mRNA expression under hydrostatic pressure. A similar increase in sox9 expression was seen in the absence of hydrostatic pressure when an inhibitor of ERK phosphorylation (PD98059) was added. These data suggest that the ERK pathway may participate in hydrostatic pressure-induced mechanotransduction and may also be a negative regulator of sox9 expression and therefore for the differentiation of chondrocytes. Hydrostatic pressure may modulate the negative effect of the ERK pathway in chondrocytes and contribute towards maintaining cell homeostasis. It is hoped that these data will contribute towards a better understanding of mechanotransduction in chondrocytes and the role of application of mechanical stress in tissue engineering and cartilage repair. References [1] Hung C.T., Henshaw D.R., Wang C.C., Mauck R.L., Raia R, Palmer G., Chao P.H., Mow V.C., Ratcliffe A., Valhmu W.B. J Biomech 2000; 33: 73-80. [2] Bobick B.E., Kulyk W.M. J Biol Chem 2004; 279: 4588-95. [3] Murakami S., Kan M., McKeehan W.L., de Crombrugghe B. Proc Natl Acad Sci USA 2000; 97: 1113-8. 7474 Th, 11:45-12:00 (P41) Mechanically loaded photopolymerized hydrogels as 3D models to probe mechanotransduction pathways in chondrocytes S. Bryant, I. Villanueva. Department of Chemical and Biological Engineering,
University of Colorado, Boulder, CO, USA During mechanical loading, chondrocytes sense changes in their local environment by resolving extracellular events (e.g., cell deformation) that trigger changes in intracellular signaling molecules (e.g., nitric oxide (NO)) and ultimately affect the overall tissue quality - a process termed mechanotransduction. In particular, NO has been implicated in cartilage degradation and osteoarthritis; however, many of the pathways associated with mechanotransduction remain unclear. We have previously developed a 3D model prepared from photopolymerized, crosslinked, neutral poly(ethylene glycol) (PEG) hydrogels to study cell deformation through changes in crosslinking density (px), i.e. an increase in Px leads to increased cell deformation (Bryant et al. J Orthep Res (2004)). The goal of this study was to elucidate the mechanotransduction pathways associated with NO and cell deformation using PEG gels. PEG hyrogels were fabricated with a range of px'S, 0.09 to 0.7 mol/I, by varying the PEG concentration to yield gels with the same chemistry but different network structures. When subjected to cyclic compressive strains (1 Hz, 15% amplitude strains, 48 hours), chondrocyte response was dependent on gel Px. NO was inhibited (-37±15% compared to unloaded controls) in gels with low Px (0.09 mol/I) and stimulated (+77±40% compared to unloaded controls) in gels with high px (0.7 mol/I) as measured by nitrite, the stable end product. Interestingly, the effects of loading on cell proliferation was reversed with a 59±18% increase in total DNA content in the low px gels while an 11±18% decrease in the high Px gels. A lower frequency (0.3Hz, 15% amplitude
10.6. Microstructural Modeling of Cells
$239
strains, 48 hours) resulted in decreased cell proliferation and increased NO production compared to higher frequencies for a given gel Px. There was not a direct relationship with NO levels to proteoglycan production. This study demonstrates that decreased cell deformation and/or higher frequencies (of 1 Hz) inhibit NO production. Furthermore, this study supports evidence in the literature that NO may be a key mediator in mechanically transduced signaling pathways involving cell proliferation. 5485 Th, 12:00-12:15 (P41) Evaluation of local strain magnitude at initiation point of calcium signaling response to mechanical stimuli in osteoblastic cells K. Sato 1, T. Adachi 2, D. Ueda 2, M. Hojo 2. 1Department ef Mechanical Engineering, Yamaguchi University, Ube, Japan, 2Department of Mechanical Engineering and Science, Kyoto University, Kyoto, Japan In adaptive bone remodeling process, it is believed that cells in bone such as osteoblasts, osteocytes, and osteoclasts can sense mechanical stimuli and modulate their activities. However, the mechanosensing mechanism by which these cells sense mechanical stimuli and transduce mechanical signals into biochemical signals is still not clearly understood. From the viewpoint of cell biomechanics, it is important to clarify the mechanical condition that activates the cellular mechanosensing mechanism. The aims of this study were to evaluate the mechanical condition, that is, the local strain magnitude on the cell membrane, at the initiation point of the intracellular calcium signaling response to the applied mechanical stimulus in osteoblast-like MC3T3-E1 cells, and to investigate the effect of deformation velocity on the characteristics of the cellular response. To apply a local deformation as mechanical stimuli to a single cell, a glass microneedle was directly indented to the cell and moved horizontally on the cell membrane. To observe the cellular response and the deformation of the cell membrane, the intracellular calcium ions and cell membrane were labeled using fluorescent dyes and simultaneously observed using confocal laser scanning microscopy. The strain distribution on the cell membrane due to the applied local deformation and the strain magnitude at the initiation point of the calcium signaling response were analyzed using the obtained fluorescence images. It was found, from the two-dimensionally projected images, that there is local compressive strain at the initiation point of calcium signaling. Moreover, the cellular response revealed velocity dependence, that is, the cells seemed to respond with the higher sensitivity to the higher deformation velocity. From the viewpoint of cell biomechanics, these results bring us a fundamental understanding of the mechanosensing mechanism of osteoblast-like cells.
10.6. Microstructural Modeling of Cells 4586 Th, 14:00-14:15 (P44) Active filament gels: towards modeling the plant microtubule cytoskeleton B. Mulder. FOM Institute for Atomic and Molecular Physics, Amsterdam, The
Netherlands Plant cells exhibit a remarkable sequence of highly structured microtubule arrangements during the cell cycle. From an initially fairly random radial pattern, centered on the nucleus, one sees the successive development of the transverse cortical array, the pre-prophaseband, the mitotic spindle and the phragmoplast. The structures can now be visualized in confocal microscopy using genetically modified cells expressing fluorescent protein tagged constructs. This allows e.g. the collection of quantitative data on the density and degree of orientational order of the microtubules. In spite of these experimental advances, there has been little progress in understanding these structures and their evolution from a molecular/mechanistic viewpoint. Although we have a fair idea on a functional level which physical and biological parameters should play a role in this problem, these systems are paradigmatic of the challenging multiparameter, multi-timescale, hierarchical systems that one encounters cellular biophysics. One intriguing aspect is that these systems "active" i.e. out of thermodynamic equilibrium, being in part driven by motor proteins, which act as force producing components. In my presentation I will focus on two complementary physics based approaches towards modeling active networks of microtubules. The first is mesoscopic and focuses on capturing the coarse grained behaviour on a supra-molecular scale using symmetry arguments, in the spirit of the Landau theory of phase transitions. I will present the first results obtained with this approach towards understanding the formation of the preprophase band. The second is microscopic and focuses on the details of the kinetic effects that accompany the interaction of filamentous proteins through tightly coupled motor protein complexes. In this case, we are so far only able to study the onset of instabilities of such systems that ultimately drive them into stable patterns. References [1] A. Zumdieck, M. Cosentino-Lagomarsino, C. Tanase, K. Kruse, B. Mulder, M. Dogterom and R Juelicher. A coarse grained approach to actively driven self
Rehabilitation Medicine, Bioengineering Division, University of Leeds, Leeds, UK, 2Department of Oral Biology, Leeds Dental Institute, University of Leeds, Leeds, UK Chondrocytes need appropriate mechanical stress for maintenance of phenotype, suggesting a role for intracellular mechanotransduction pathways, possibly including those associated with mitogen-activated protein kinases (MAPKs) [1]. However, the precise role of MAPK signalling in mechanotransduction remains controversial [2,3]. We hypothesize that the extracellular signal-regulated kinase (ERK) pathway (one of the MAPK pathways) participates in hydrostatic pressure-induced mechanotransduction. To test this hypothesis, we investigated ERK activation under hydrostatic pressure. Expression of sox9 mRNA (regarded as a "master switch" for chondrocyte differentiation) was determined as a possible downstream target of the pathway. 5 MPa hydrostatic pressure was applied to bovine chondrocyte-agarose constructs for 4 hours. ERK phosphorylation (associated with ERK activation), was determined using western blotting. Expression of sox9 mRNA was analysed by RT-PCR. The results showed a decrease in ERK phosphorylation with a concomitant increase in the sox9 mRNA expression under hydrostatic pressure. A similar increase in sox9 expression was seen in the absence of hydrostatic pressure when an inhibitor of ERK phosphorylation (PD98059) was added. These data suggest that the ERK pathway may participate in hydrostatic pressure-induced mechanotransduction and may also be a negative regulator of sox9 expression and therefore for the differentiation of chondrocytes. Hydrostatic pressure may modulate the negative effect of the ERK pathway in chondrocytes and contribute towards maintaining cell homeostasis. It is hoped that these data will contribute towards a better understanding of mechanotransduction in chondrocytes and the role of application of mechanical stress in tissue engineering and cartilage repair. References [1] Hung C.T., Henshaw D.R., Wang C.C., Mauck R.L., Raia R, Palmer G., Chao P.H., Mow V.C., Ratcliffe A., Valhmu W.B. J Biomech 2000; 33: 73-80. [2] Bobick B.E., Kulyk W.M. J Biol Chem 2004; 279: 4588-95. [3] Murakami S., Kan M., McKeehan W.L., de Crombrugghe B. Proc Natl Acad Sci USA 2000; 97: 1113-8. 7474 Th, 11:45-12:00 (P41) Mechanically loaded photopolymerized hydrogels as 3D models to probe mechanotransduction pathways in chondrocytes S. Bryant, I. Villanueva. Department of Chemical and Biological Engineering,
University of Colorado, Boulder, CO, USA During mechanical loading, chondrocytes sense changes in their local environment by resolving extracellular events (e.g., cell deformation) that trigger changes in intracellular signaling molecules (e.g., nitric oxide (NO)) and ultimately affect the overall tissue quality - a process termed mechanotransduction. In particular, NO has been implicated in cartilage degradation and osteoarthritis; however, many of the pathways associated with mechanotransduction remain unclear. We have previously developed a 3D model prepared from photopolymerized, crosslinked, neutral poly(ethylene glycol) (PEG) hydrogels to study cell deformation through changes in crosslinking density (px), i.e. an increase in Px leads to increased cell deformation (Bryant et al. J Orthep Res (2004)). The goal of this study was to elucidate the mechanotransduction pathways associated with NO and cell deformation using PEG gels. PEG hyrogels were fabricated with a range of px'S, 0.09 to 0.7 mol/I, by varying the PEG concentration to yield gels with the same chemistry but different network structures. When subjected to cyclic compressive strains (1 Hz, 15% amplitude strains, 48 hours), chondrocyte response was dependent on gel Px. NO was inhibited (-37±15% compared to unloaded controls) in gels with low Px (0.09 mol/I) and stimulated (+77±40% compared to unloaded controls) in gels with high px (0.7 mol/I) as measured by nitrite, the stable end product. Interestingly, the effects of loading on cell proliferation was reversed with a 59±18% increase in total DNA content in the low px gels while an 11±18% decrease in the high Px gels. A lower frequency (0.3Hz, 15% amplitude
10.6. Microstructural Modeling of Cells
$239
strains, 48 hours) resulted in decreased cell proliferation and increased NO production compared to higher frequencies for a given gel Px. There was not a direct relationship with NO levels to proteoglycan production. This study demonstrates that decreased cell deformation and/or higher frequencies (of 1 Hz) inhibit NO production. Furthermore, this study supports evidence in the literature that NO may be a key mediator in mechanically transduced signaling pathways involving cell proliferation. 5485 Th, 12:00-12:15 (P41) Evaluation of local strain magnitude at initiation point of calcium signaling response to mechanical stimuli in osteoblastic cells K. Sato 1, T. Adachi 2, D. Ueda 2, M. Hojo 2. 1Department ef Mechanical Engineering, Yamaguchi University, Ube, Japan, 2Department of Mechanical Engineering and Science, Kyoto University, Kyoto, Japan In adaptive bone remodeling process, it is believed that cells in bone such as osteoblasts, osteocytes, and osteoclasts can sense mechanical stimuli and modulate their activities. However, the mechanosensing mechanism by which these cells sense mechanical stimuli and transduce mechanical signals into biochemical signals is still not clearly understood. From the viewpoint of cell biomechanics, it is important to clarify the mechanical condition that activates the cellular mechanosensing mechanism. The aims of this study were to evaluate the mechanical condition, that is, the local strain magnitude on the cell membrane, at the initiation point of the intracellular calcium signaling response to the applied mechanical stimulus in osteoblast-like MC3T3-E1 cells, and to investigate the effect of deformation velocity on the characteristics of the cellular response. To apply a local deformation as mechanical stimuli to a single cell, a glass microneedle was directly indented to the cell and moved horizontally on the cell membrane. To observe the cellular response and the deformation of the cell membrane, the intracellular calcium ions and cell membrane were labeled using fluorescent dyes and simultaneously observed using confocal laser scanning microscopy. The strain distribution on the cell membrane due to the applied local deformation and the strain magnitude at the initiation point of the calcium signaling response were analyzed using the obtained fluorescence images. It was found, from the two-dimensionally projected images, that there is local compressive strain at the initiation point of calcium signaling. Moreover, the cellular response revealed velocity dependence, that is, the cells seemed to respond with the higher sensitivity to the higher deformation velocity. From the viewpoint of cell biomechanics, these results bring us a fundamental understanding of the mechanosensing mechanism of osteoblast-like cells.
10.6. Microstructural Modeling of Cells 4586 Th, 14:00-14:15 (P44) Active filament gels: towards modeling the plant microtubule cytoskeleton B. Mulder. FOM Institute for Atomic and Molecular Physics, Amsterdam, The
Netherlands Plant cells exhibit a remarkable sequence of highly structured microtubule arrangements during the cell cycle. From an initially fairly random radial pattern, centered on the nucleus, one sees the successive development of the transverse cortical array, the pre-prophaseband, the mitotic spindle and the phragmoplast. The structures can now be visualized in confocal microscopy using genetically modified cells expressing fluorescent protein tagged constructs. This allows e.g. the collection of quantitative data on the density and degree of orientational order of the microtubules. In spite of these experimental advances, there has been little progress in understanding these structures and their evolution from a molecular/mechanistic viewpoint. Although we have a fair idea on a functional level which physical and biological parameters should play a role in this problem, these systems are paradigmatic of the challenging multiparameter, multi-timescale, hierarchical systems that one encounters cellular biophysics. One intriguing aspect is that these systems "active" i.e. out of thermodynamic equilibrium, being in part driven by motor proteins, which act as force producing components. In my presentation I will focus on two complementary physics based approaches towards modeling active networks of microtubules. The first is mesoscopic and focuses on capturing the coarse grained behaviour on a supra-molecular scale using symmetry arguments, in the spirit of the Landau theory of phase transitions. I will present the first results obtained with this approach towards understanding the formation of the preprophase band. The second is microscopic and focuses on the details of the kinetic effects that accompany the interaction of filamentous proteins through tightly coupled motor protein complexes. In this case, we are so far only able to study the onset of instabilities of such systems that ultimately drive them into stable patterns. References [1] A. Zumdieck, M. Cosentino-Lagomarsino, C. Tanase, K. Kruse, B. Mulder, M. Dogterom and R Juelicher. A coarse grained approach to actively driven self