- Email: [email protected]
Fabrication of a pneumatically-driven tensile stimulator
Microelectronic Engineering 98 (2012) 715–719
Contents lists available at SciVerse ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Fabrication of a pneumatically-driven tensile stimulator Tae Kyung Kim, Ok Chan Jeong ⇑ Dept. Biomedical Eng., Inje university, Gimhae 621-749, Republic of Korea
a r t i c l e
i n f o
Article history: Available online 7 June 2012 Keywords: Single cell Pneumatic actuation Tensile stimulator Intracellular calcium
a b s t r a c t This paper introduces a pneumatically-driven tensile stimulator for investigating the effect of mechanical strain on intracellular calcium expression in MG-63 cell (human osteoblast-like bone cell). An optically transparent micro-tensile stimulator array, consisting of deformable diaphragms and micro-fluidic channels, was fabricated with polydimethylsiloxane (PDMS) and a glass substrate. A strain gradient generated by a three-dimensional dome-shaped deformation of the circular diaphragm was applied to live cells seeded on the deformable diaphragm of the pneumatic stimulator. During the operation, intracellular calcium responses were measured using a laser-scanning microscope. Based on the temporal responses of the fluorescent intensities, we discovered that the periods of the measured calcium expressions of cells in different strain regions were well matched with the applied signal waveforms of the external compressed air. Moreover, the magnitude of the intensities in a cell was proportional to the magnitude of the mechanical strain. We were able to conclude that the release/uptake of intracellular calcium in the endoplasmic reticulum may be activated by, and sensitive to the applied strain. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Mechanotransduction is the process by which mechanical energy is converted into biological and/or chemical response. Cells are mechanosensitive, and physical forces (including gravity, tension, compression, and shear) influence cell growth, differentiation, secretion, movement, signal transduction, gene expression and remodeling in all living tissues at the cellular level [1,2]. Thus, various kinds of macro-systems have been developed in an effort to provide mechanical stimuli for experimental studies on mechanotransduction. Unlike the systems used for shear and compressive stimuli, in the case of tensile stimulation in cells, a deformable substrate is required, since the typical substrates for cell cultures (such as flasks, dishes, plates, or cover slips) are not available for stretching cultured cells seeded on a substrate. The commercially available bioreactor Flexcell culture system [3] is one of the more popular systems used to stretch cells. However, this system is preferable for conventional biological experiments, as a bulk method. A more sophisticated substrate-bending system, which provides uniform and stable microenvironments, is necessary for the study of strain-induced cellular responses in a single cell. Recently, the rapid development of micro-fabrication and micro-fluidics technologies has made it possible to fabricate cell
⇑ Corresponding author. E-mail address: [email protected] (O.C. Jeong). 0167-9317/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mee.2012.05.012
stimulation chips [4–6] for mechanically stretching cells seeded on flexible membranes like polydimethylsiloxane (PDMS) [7–9]. Because the mechanical strain was applied to cells using the structural deformation of the flexible diaphragm, the structural analysis of the elastomeric membrane with highly nonlinear mechanical properties was essential for determining the magnitude of the strain. Moreover, the effective operational conditions based on the correlation between the duration of the well-defined mechanical strain and the refractory period of the cell should be carefully considered for the successful operation of the mechanical stimulation. One of the practical aims of such an understanding is to design and fabricate the effective micro-structures for stimulating cells. This paper describes the fabrication of a micro-stimulator for applying mechanical strain to a cell. We report our discoveries on mechanical strain-dependent intracellular calcium responses obtained using the proposed stimulator. The remarkable feature of our new micro-tensile stimulator is that a three-dimensional dome-shaped deformation of the single stimulator diaphragm can apply various mechanical strains to cells seeded on the deformable PDMS membrane. Structural analysis of the PDMS membrane was performed in order to investigate the strain distribution of the dome-shaped deformation of the stimulator diaphragm. The optically transparent cell-stimulation device was fabricated via the replica molding method and a structural bonding process. The intracellular calcium expression in a cell stimulated with various strains was observed and measured using a laserscanning microscope.
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T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
2. Method and structure 2.1. Operational method Fig. 1 shows the structure and operational method of the proposed micro-tensile stimulator. The device is comprised of a pneumatic actuator with a deformable diaphragm and a micro-fluidic channel. The technique is comprised of two steps: a typical twodimensional cell-seeding process, followed by the application of strain to the cells, using a three-dimensional deformation of the seeding substrate. After the cells have been seeded, the actuator diaphragm is deformed by a distributed load when external compressed air is supplied to the actuator. Strain can be applied to cells seeded on the diaphragm via a dome-shaped deformation of the circular actuator diaphragm. 2.2. Structural analysis of the PDMS diaphragm Fig. 2 shows the simulated deformation of the all-edge-clamped diaphragm of the pneumatic stimulator. A Mooney model [10] was used for the structural analysis of the PDMS elastomer, which has highly nonlinear material properties. Both the diameter and thickness of the PDMS diaphragm were 1 mm. The MarcMentact software package was used for this simulation. In order to investigate (a) the displacement and (b) the strain distributions of a dome-shaped deformation of the circular diaphragm, their A–A node paths were plotted (c). The displacement was maximized at the center of the diaphragm, as expected. However, the strain exhibited an inflection point. The strain was maximized at the clamped edges, decreased dramatically to the inflection point (I), and then gradually increased (II). If cells are present on the circular diaphragm, they should be affected by the strain gradient. Thus, a quantitative study of the strain-induced cellular responses of live cells could be carried out using the proposed single pneumatic stimulator. 3. Experiment 3.1. Fabrication of the micro-tensile stimulator Fig. 3 illustrates the schematic view of the proposed tensile stimulator array. The device consists of the glass substrate and
two PDMS layers like the fluid channel layer to introduce cell/media and stimulator layer to act as the pneumatic actuator for stimulating cells on actuator diaphragm. Two photo masks are used to fabricate SU8 molds for the replica molding process of the PDMS structure like fluid channel and the pneumatic stimulator. After the replica molding process, the fluid channel and stimulator layers are first bonded together (I), and then this structure is bonded again with the glass substrate (II). Fig. 4 shows the fabricated transparent tensile stimulator array. First, SU8 molds for the fluid channel and stimulator layers were fabricated. Then, the liquid PDMS (Sylgard 184, Dow–Corning) was prepared by mixing, stirring, and degassing in the vacuum chamber. In order to increase the flexibility of the PDMS structure, the mixing ratio of the curing agent and base polymer for stimulator layer having deformable diaphragm were 20:1. In the case of the fluid channel without moving part, it was 5:1. The prepared liquid PDMS mixture was poured into the SU8 molds, spun, and cured at 75 °C for 10 minutes. After the preparation of two PDMS structures for the fluidic channel and the pneumatic stimulator, through-holes were cut to serve as fluid and pressure ports. The two PDMS structures and the glass substrate were sequentially bonded, using the atmosphere plasma bonding method. The width and height of the micro-fluidic channel were 1 mm and 70 lm, respectively. The width and height of the pneumatic channel for supplying external compressed air were 100 lm and 50 lm, respectively. The thickness of the deformable diaphragm of the micro-tensile stimulator was 1 mm. After the fabrication process, the stimulator was sterilized by ultraviolet irradiation, and fibronectin was introduced through the micro-fluidic channel to enhance cell adhesion. MG-63 cells (1.0 105 cells/ml) were then seeded, incubated for 24 hours, and stained with fluorescent calcium indicator for measuring the intracellular calcium concentrations inside living cells (Fluo-4 AM, Invitrogen, USA). 3.2. Measurement system In this work, the intracellular calcium expressions in live cells were measured and examined in order to investigate the effects of mechanical strain on a cell. The measurement system was comprised of the pneumatic control system for actuating the vibrator array, and a laser-scanning microscope (LSM 510 META, Carl Zeiss, Germany) for measuring the intracellular calcium of the live cells. Compressed air generated by a compressor was adjusted by an on/ off gate valve and a precision regulator. The input waveform of the supplied air pressure was set by a computer-controlled electromagnetic valve. The magnitude and frequency of the supplied pressure were monitored with precision pressure sensors (SDX05D4, Honeywell, USA). During the actuation of the micro-stimulator, the fluorescent intensity of the intracellular calcium in the cell was recorded and analyzed. 4. Results and discussion
Fig. 1. Proposed tensile stimulator: (a) two-dimensional cell seeding, (b) threedimensional deformation of the actuator for applying the pure tensile stimulation to cells, (c) structure and method of the tensile stimulator.
Fig. 5 shows cell morphologies and three-dimensional surface plots of the fluorescent intensities of the intracellular calcium of cells, with and without mechanical strain induced by deformation of the stimulator diaphragm. The fluorescent intensity of the cells at rest (a) increased dramatically after applying compressed air to the stimulator (b). The interesting point is that the cells seeded on the diaphragm were affected by various mechanical strains because there was a strain gradient in the deformed actuator diaphragm. Compared with the strain distributions shown in Fig. 2(b), the strain-dependent intracellular calcium expression of the cells can be divided into two regions, using two contour lines: I for the strain-decrement
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
717
Fig. 2. Simulation results for the PDMS stimulator diaphragm: (a) displacement, (b) elastic strain, and (c) their node-path plots (A–A). The second-order Mooney model was used for structural analysis of the PDMS elastomer, which has highly nonlinear material properties. I and II represent the decrement and increment regions, respectively, of the percentage strain.
Fig. 3. Schematic view (left) and assemble sequence (right) of the tensile stimulator device.
region and II for the strain-increment region. In order to investigate the strain-dependent cellular responses to the applied strain, four groups of cells were selected, one from the outer region of the diaphragm (as a comparable strain-free area, C1) and the others from the three strain-contour lines (C2–C4) in the deformed region. C2 and C3 are contour lines of cells representing the maximum strain at the diaphragm edge and the minimum strain, respectively. In the case of C4, the mechanical strain of the deformed diaphragm near the central region of the stimulator was relatively uniform. Thus, the cells seeded along C2 and C3 were expected to show higher and lower intracellular calcium expressions, respectively. However, the intracellular calcium expression of the cells in R1 did not respond to the mechanical strain because there was no structural deformation of the substrate.
Fig. 6 shows the temporal responses of the fluorescent intensities of a single cell. The magnitude and frequency of the squarewave compressed air were 50 kPa and 0.05 Hz, respectively. We discovered that the periods of the measured calcium expressions of cells seeded on the diaphragm in various strain regions were well matched with the applied signal waveforms of the external compressed air. Moreover, the variations of the calcium expression in the cells were highly correlated with the magnitude of the applied strain. As the applied strain increased (C1C3C4C2), the release/uptake of intracellular calcium in the endoplasmic reticulum was activated and became more sensitive to applied strain. Thus, the intensities in the cell were proportional in magnitude to the magnitude of the mechanical strain. However, a minor intensity of the cell seed in the outer region of the stimulator diaphragm
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T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
Fig. 4. (a) Fabricated tensile stimulator array and (b) an enlarged view.
Fig. 5. Cell morphologies and three-dimensional surface plots of fluorescent intensities (a) at rest and (b) under mechanical strain. (C1: comparable strain-free region; C2 and C3: contour lines representing maximum strain at the diaphragm edge and minimum strain, respectively; C4: relatively uniform strain region near the central region of the stimulator diaphragm).
(C1) was also observed. There are two possible reasons for this. The fluid in the shallow channel was compressed toward the inlet/outlet port during the repeated deformations of the stimulator diaphragm. Therefore, the cells on the diaphragm could have been affected by the compressive stress of the diaphragm on the fluid film and the shear stress of the fluid flow in the thin fluidic channel. Nevertheless, considering the magnitudes of the shear (a few Pa) [11] and the compressive stress (several tens of kPa) [12], the effect of these stresses induced by the deformation of the stimulator diaphragm could be very minor. In order to understand this complex mechanical environment quantitatively, further multiphysical analysis will be necessary. 5. Conclusion In this paper, a pneumatic tensile stimulator was fabricated in order to investigate cellular responses to programmed mechanical strains. The proposed device consisted of a pneumatic stimulator (with a dome-shaped deformation of the circular diaphragm to apply a strain gradient to cells) and a fluidic channel to introduce
cells/media. The magnitude and frequency of the external compressed air for actuating the stimulator diaphragm were adjusted using a lab-designed pneumatic control system. To investigate the effect of mechanical strain on cells, the intracellular calcium expressions in MG-63 cells were observed and measured with a laser-scanning microscope. Based on the temporal responses of the fluorescent intensities of the cells, we found that the periods of the measured calcium expressions of cells in different strain regions were well matched with the waveforms of the applied compressed air. Moreover, the magnitude of the intracellular calcium in the cells was also highly correlated with the magnitude of the strain. Since these cells are very sensitive to the mechanical environment, it was confirmed experimentally that various mechanical strains could be applied to cells via three-dimensional deformation of the circular diaphragm of the proposed device. Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF),
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
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Fig. 6. Temporal responses of the fluorescent intensities of a single cell.
funded by the Ministry of Education, Science, and Technology (2009-0076333). References [1] [2] [3] [4] [5]
D.E. Ingber, Annu. Rev. Physiol. 59 (1997) 575–599. P.J. Ehrlich, L.E. Lanyon, Osteoporos. Int. 13 (2002) 688–700.
[6] W.W. Ahmed, M.H. Kural, T.A. Saif, Acta Biomaterialia 6 (2010) 2979–2990. [7] D. Wang, Y. Xie, B. Yuan, J. Xu, P. Gong, X. Jiang, Integr. Biol. 2 (2010) 288–293. [8] Y. Kamotani, T.B. Begey, N. Kato, Y.C. Tung, D. Huh, J.W. Song, S. Takayama, Biomaterials 29 (2008) 2646–2655. [9] K. Shimizu, A. Shunori, K. Morimoto, M. Hashida, S. Konishi, Sens. Actuators, B 156 (2011) 486–493. [10] T.K. Kim, O.C. Jeong, Microelectron. Eng. 88 (8) (2011) 1982–1985. [11] J.H. Jeon, T.K. Kim, S.H. Park, J.W. Shin, O.C. Jeong, Proc. Micro Total Analy. Syst. (2009) 1156–1568. [12] J.H. Jeon, T.K. Kim, O.C. Jeong, Proc. Micro Total Analy. Syst. (2010) 271–273.
Contents lists available at SciVerse ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Fabrication of a pneumatically-driven tensile stimulator Tae Kyung Kim, Ok Chan Jeong ⇑ Dept. Biomedical Eng., Inje university, Gimhae 621-749, Republic of Korea
a r t i c l e
i n f o
Article history: Available online 7 June 2012 Keywords: Single cell Pneumatic actuation Tensile stimulator Intracellular calcium
a b s t r a c t This paper introduces a pneumatically-driven tensile stimulator for investigating the effect of mechanical strain on intracellular calcium expression in MG-63 cell (human osteoblast-like bone cell). An optically transparent micro-tensile stimulator array, consisting of deformable diaphragms and micro-fluidic channels, was fabricated with polydimethylsiloxane (PDMS) and a glass substrate. A strain gradient generated by a three-dimensional dome-shaped deformation of the circular diaphragm was applied to live cells seeded on the deformable diaphragm of the pneumatic stimulator. During the operation, intracellular calcium responses were measured using a laser-scanning microscope. Based on the temporal responses of the fluorescent intensities, we discovered that the periods of the measured calcium expressions of cells in different strain regions were well matched with the applied signal waveforms of the external compressed air. Moreover, the magnitude of the intensities in a cell was proportional to the magnitude of the mechanical strain. We were able to conclude that the release/uptake of intracellular calcium in the endoplasmic reticulum may be activated by, and sensitive to the applied strain. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Mechanotransduction is the process by which mechanical energy is converted into biological and/or chemical response. Cells are mechanosensitive, and physical forces (including gravity, tension, compression, and shear) influence cell growth, differentiation, secretion, movement, signal transduction, gene expression and remodeling in all living tissues at the cellular level [1,2]. Thus, various kinds of macro-systems have been developed in an effort to provide mechanical stimuli for experimental studies on mechanotransduction. Unlike the systems used for shear and compressive stimuli, in the case of tensile stimulation in cells, a deformable substrate is required, since the typical substrates for cell cultures (such as flasks, dishes, plates, or cover slips) are not available for stretching cultured cells seeded on a substrate. The commercially available bioreactor Flexcell culture system [3] is one of the more popular systems used to stretch cells. However, this system is preferable for conventional biological experiments, as a bulk method. A more sophisticated substrate-bending system, which provides uniform and stable microenvironments, is necessary for the study of strain-induced cellular responses in a single cell. Recently, the rapid development of micro-fabrication and micro-fluidics technologies has made it possible to fabricate cell
⇑ Corresponding author. E-mail address: [email protected] (O.C. Jeong). 0167-9317/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mee.2012.05.012
stimulation chips [4–6] for mechanically stretching cells seeded on flexible membranes like polydimethylsiloxane (PDMS) [7–9]. Because the mechanical strain was applied to cells using the structural deformation of the flexible diaphragm, the structural analysis of the elastomeric membrane with highly nonlinear mechanical properties was essential for determining the magnitude of the strain. Moreover, the effective operational conditions based on the correlation between the duration of the well-defined mechanical strain and the refractory period of the cell should be carefully considered for the successful operation of the mechanical stimulation. One of the practical aims of such an understanding is to design and fabricate the effective micro-structures for stimulating cells. This paper describes the fabrication of a micro-stimulator for applying mechanical strain to a cell. We report our discoveries on mechanical strain-dependent intracellular calcium responses obtained using the proposed stimulator. The remarkable feature of our new micro-tensile stimulator is that a three-dimensional dome-shaped deformation of the single stimulator diaphragm can apply various mechanical strains to cells seeded on the deformable PDMS membrane. Structural analysis of the PDMS membrane was performed in order to investigate the strain distribution of the dome-shaped deformation of the stimulator diaphragm. The optically transparent cell-stimulation device was fabricated via the replica molding method and a structural bonding process. The intracellular calcium expression in a cell stimulated with various strains was observed and measured using a laserscanning microscope.
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T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
2. Method and structure 2.1. Operational method Fig. 1 shows the structure and operational method of the proposed micro-tensile stimulator. The device is comprised of a pneumatic actuator with a deformable diaphragm and a micro-fluidic channel. The technique is comprised of two steps: a typical twodimensional cell-seeding process, followed by the application of strain to the cells, using a three-dimensional deformation of the seeding substrate. After the cells have been seeded, the actuator diaphragm is deformed by a distributed load when external compressed air is supplied to the actuator. Strain can be applied to cells seeded on the diaphragm via a dome-shaped deformation of the circular actuator diaphragm. 2.2. Structural analysis of the PDMS diaphragm Fig. 2 shows the simulated deformation of the all-edge-clamped diaphragm of the pneumatic stimulator. A Mooney model [10] was used for the structural analysis of the PDMS elastomer, which has highly nonlinear material properties. Both the diameter and thickness of the PDMS diaphragm were 1 mm. The MarcMentact software package was used for this simulation. In order to investigate (a) the displacement and (b) the strain distributions of a dome-shaped deformation of the circular diaphragm, their A–A node paths were plotted (c). The displacement was maximized at the center of the diaphragm, as expected. However, the strain exhibited an inflection point. The strain was maximized at the clamped edges, decreased dramatically to the inflection point (I), and then gradually increased (II). If cells are present on the circular diaphragm, they should be affected by the strain gradient. Thus, a quantitative study of the strain-induced cellular responses of live cells could be carried out using the proposed single pneumatic stimulator. 3. Experiment 3.1. Fabrication of the micro-tensile stimulator Fig. 3 illustrates the schematic view of the proposed tensile stimulator array. The device consists of the glass substrate and
two PDMS layers like the fluid channel layer to introduce cell/media and stimulator layer to act as the pneumatic actuator for stimulating cells on actuator diaphragm. Two photo masks are used to fabricate SU8 molds for the replica molding process of the PDMS structure like fluid channel and the pneumatic stimulator. After the replica molding process, the fluid channel and stimulator layers are first bonded together (I), and then this structure is bonded again with the glass substrate (II). Fig. 4 shows the fabricated transparent tensile stimulator array. First, SU8 molds for the fluid channel and stimulator layers were fabricated. Then, the liquid PDMS (Sylgard 184, Dow–Corning) was prepared by mixing, stirring, and degassing in the vacuum chamber. In order to increase the flexibility of the PDMS structure, the mixing ratio of the curing agent and base polymer for stimulator layer having deformable diaphragm were 20:1. In the case of the fluid channel without moving part, it was 5:1. The prepared liquid PDMS mixture was poured into the SU8 molds, spun, and cured at 75 °C for 10 minutes. After the preparation of two PDMS structures for the fluidic channel and the pneumatic stimulator, through-holes were cut to serve as fluid and pressure ports. The two PDMS structures and the glass substrate were sequentially bonded, using the atmosphere plasma bonding method. The width and height of the micro-fluidic channel were 1 mm and 70 lm, respectively. The width and height of the pneumatic channel for supplying external compressed air were 100 lm and 50 lm, respectively. The thickness of the deformable diaphragm of the micro-tensile stimulator was 1 mm. After the fabrication process, the stimulator was sterilized by ultraviolet irradiation, and fibronectin was introduced through the micro-fluidic channel to enhance cell adhesion. MG-63 cells (1.0 105 cells/ml) were then seeded, incubated for 24 hours, and stained with fluorescent calcium indicator for measuring the intracellular calcium concentrations inside living cells (Fluo-4 AM, Invitrogen, USA). 3.2. Measurement system In this work, the intracellular calcium expressions in live cells were measured and examined in order to investigate the effects of mechanical strain on a cell. The measurement system was comprised of the pneumatic control system for actuating the vibrator array, and a laser-scanning microscope (LSM 510 META, Carl Zeiss, Germany) for measuring the intracellular calcium of the live cells. Compressed air generated by a compressor was adjusted by an on/ off gate valve and a precision regulator. The input waveform of the supplied air pressure was set by a computer-controlled electromagnetic valve. The magnitude and frequency of the supplied pressure were monitored with precision pressure sensors (SDX05D4, Honeywell, USA). During the actuation of the micro-stimulator, the fluorescent intensity of the intracellular calcium in the cell was recorded and analyzed. 4. Results and discussion
Fig. 1. Proposed tensile stimulator: (a) two-dimensional cell seeding, (b) threedimensional deformation of the actuator for applying the pure tensile stimulation to cells, (c) structure and method of the tensile stimulator.
Fig. 5 shows cell morphologies and three-dimensional surface plots of the fluorescent intensities of the intracellular calcium of cells, with and without mechanical strain induced by deformation of the stimulator diaphragm. The fluorescent intensity of the cells at rest (a) increased dramatically after applying compressed air to the stimulator (b). The interesting point is that the cells seeded on the diaphragm were affected by various mechanical strains because there was a strain gradient in the deformed actuator diaphragm. Compared with the strain distributions shown in Fig. 2(b), the strain-dependent intracellular calcium expression of the cells can be divided into two regions, using two contour lines: I for the strain-decrement
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
717
Fig. 2. Simulation results for the PDMS stimulator diaphragm: (a) displacement, (b) elastic strain, and (c) their node-path plots (A–A). The second-order Mooney model was used for structural analysis of the PDMS elastomer, which has highly nonlinear material properties. I and II represent the decrement and increment regions, respectively, of the percentage strain.
Fig. 3. Schematic view (left) and assemble sequence (right) of the tensile stimulator device.
region and II for the strain-increment region. In order to investigate the strain-dependent cellular responses to the applied strain, four groups of cells were selected, one from the outer region of the diaphragm (as a comparable strain-free area, C1) and the others from the three strain-contour lines (C2–C4) in the deformed region. C2 and C3 are contour lines of cells representing the maximum strain at the diaphragm edge and the minimum strain, respectively. In the case of C4, the mechanical strain of the deformed diaphragm near the central region of the stimulator was relatively uniform. Thus, the cells seeded along C2 and C3 were expected to show higher and lower intracellular calcium expressions, respectively. However, the intracellular calcium expression of the cells in R1 did not respond to the mechanical strain because there was no structural deformation of the substrate.
Fig. 6 shows the temporal responses of the fluorescent intensities of a single cell. The magnitude and frequency of the squarewave compressed air were 50 kPa and 0.05 Hz, respectively. We discovered that the periods of the measured calcium expressions of cells seeded on the diaphragm in various strain regions were well matched with the applied signal waveforms of the external compressed air. Moreover, the variations of the calcium expression in the cells were highly correlated with the magnitude of the applied strain. As the applied strain increased (C1C3C4C2), the release/uptake of intracellular calcium in the endoplasmic reticulum was activated and became more sensitive to applied strain. Thus, the intensities in the cell were proportional in magnitude to the magnitude of the mechanical strain. However, a minor intensity of the cell seed in the outer region of the stimulator diaphragm
718
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
Fig. 4. (a) Fabricated tensile stimulator array and (b) an enlarged view.
Fig. 5. Cell morphologies and three-dimensional surface plots of fluorescent intensities (a) at rest and (b) under mechanical strain. (C1: comparable strain-free region; C2 and C3: contour lines representing maximum strain at the diaphragm edge and minimum strain, respectively; C4: relatively uniform strain region near the central region of the stimulator diaphragm).
(C1) was also observed. There are two possible reasons for this. The fluid in the shallow channel was compressed toward the inlet/outlet port during the repeated deformations of the stimulator diaphragm. Therefore, the cells on the diaphragm could have been affected by the compressive stress of the diaphragm on the fluid film and the shear stress of the fluid flow in the thin fluidic channel. Nevertheless, considering the magnitudes of the shear (a few Pa) [11] and the compressive stress (several tens of kPa) [12], the effect of these stresses induced by the deformation of the stimulator diaphragm could be very minor. In order to understand this complex mechanical environment quantitatively, further multiphysical analysis will be necessary. 5. Conclusion In this paper, a pneumatic tensile stimulator was fabricated in order to investigate cellular responses to programmed mechanical strains. The proposed device consisted of a pneumatic stimulator (with a dome-shaped deformation of the circular diaphragm to apply a strain gradient to cells) and a fluidic channel to introduce
cells/media. The magnitude and frequency of the external compressed air for actuating the stimulator diaphragm were adjusted using a lab-designed pneumatic control system. To investigate the effect of mechanical strain on cells, the intracellular calcium expressions in MG-63 cells were observed and measured with a laser-scanning microscope. Based on the temporal responses of the fluorescent intensities of the cells, we found that the periods of the measured calcium expressions of cells in different strain regions were well matched with the waveforms of the applied compressed air. Moreover, the magnitude of the intracellular calcium in the cells was also highly correlated with the magnitude of the strain. Since these cells are very sensitive to the mechanical environment, it was confirmed experimentally that various mechanical strains could be applied to cells via three-dimensional deformation of the circular diaphragm of the proposed device. Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF),
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 715–719
719
Fig. 6. Temporal responses of the fluorescent intensities of a single cell.
funded by the Ministry of Education, Science, and Technology (2009-0076333). References [1] [2] [3] [4] [5]
D.E. Ingber, Annu. Rev. Physiol. 59 (1997) 575–599. P.J. Ehrlich, L.E. Lanyon, Osteoporos. Int. 13 (2002) 688–700.
[6] W.W. Ahmed, M.H. Kural, T.A. Saif, Acta Biomaterialia 6 (2010) 2979–2990. [7] D. Wang, Y. Xie, B. Yuan, J. Xu, P. Gong, X. Jiang, Integr. Biol. 2 (2010) 288–293. [8] Y. Kamotani, T.B. Begey, N. Kato, Y.C. Tung, D. Huh, J.W. Song, S. Takayama, Biomaterials 29 (2008) 2646–2655. [9] K. Shimizu, A. Shunori, K. Morimoto, M. Hashida, S. Konishi, Sens. Actuators, B 156 (2011) 486–493. [10] T.K. Kim, O.C. Jeong, Microelectron. Eng. 88 (8) (2011) 1982–1985. [11] J.H. Jeon, T.K. Kim, S.H. Park, J.W. Shin, O.C. Jeong, Proc. Micro Total Analy. Syst. (2009) 1156–1568. [12] J.H. Jeon, T.K. Kim, O.C. Jeong, Proc. Micro Total Analy. Syst. (2010) 271–273.