Abstract Modeling Biohybrid Devices Introduction Living Machines ...
Fabrication and Modeling Techniques for Development of Biohybrid Devices Victoria A. Webster1, Ozan Akkus1, Hillel J. Chiel2, and Roger D. Quinn1 1) Dept. of Mechanical and Aerospace Engineering and 2) Dept. of Biology, Case Western Reserve University
Abstract
Modeling Biohybrid Devices
Research in the area of biohybrid robotics, while currently a niche field, has been increasing over the past decade. The development of such devices, composed of biocompatible substrates and living tissues, has many far reaching applications including targeted drug delivery and vascular inspection or repair. However, designing these devices is complicated by the compliant nature of tissue based constructs and the stochastic nature of cell culture and function. Additionally, current fabrication techniques rely on non-organic bio-compatible polymers which must often be further processed with complicated micro patterning and chemical treatments to induce cellular alignment and attachment. In order to increase activity in the research and development of biohybrid devices, such barriers to entry must be decreased. To this end a modeling technique has been developed using the native capabilities of commercial Finite Element Analysis (FEA) tools that can serve as a foundation for further development of a biohybrid modeling toolbox. Additionally, fabrication and culture techniques are presented for manufacture of biohybrid devices using aligned collagen, which naturally induces cellular alignment and attachment without additional patterning steps. Using Electrochemically Compacted and Aligned Collagen (ELAC) scaffolds, devices have been fabricated and cultured with primary cardiomyocytes isolated from chick embryos. These devices are capable of locomotion when paced by external electrical stimulation.
Model Calibration
• Completely organic, locomoting, devices have been fabricated using electrochemically compacted collagen as a scaffold
Determine stress from experimental biohybrid cantilevers
Fabrication
,ν)
Cell Layer
Collagen
Load Mold
Compact Collagen
+
Substrate Layer ,ν) 6
1
1 min
1 Cells
Calculate Thermal Expansion Coefficient (TEC)
• Functional, completely organic, muscle powered living machines were fabricated and locomotion was achieved in response to electrical stimulation
Modeling Culture: 4-6 days
Seed Cells
Remove Collagen by Peeling
Collagen Alignment
Simulate Device using Commercial FEA Tools
• Improve model calibration by performing micropillar experiments in which substrate modulus and thickness are varied systematically and pillar deflection as a result of cell contraction is measured.
Living Machines • Develop multi-scale simulations of biohybrid devices and living machines by incorporating FEA simulations and discrete rigid body dynamic simulations • Optimize device geometry in simulation
Small Scale Actuation
• Calibrated 7 models from 3 studies in existing literature • Wide range of geometric and material properties • Resulting range of αΔT: -0.0086 to -0.4218
Collagen alignment as revealed by polarized light microscopy. Left) Leg of scaffold removed from cathode via peeling, blue indicates alignment along leg. Right) Leg of scaffold removed via ultrasonic vibration lacks alignment.
Cellular Alignment
Model Validation
Living Machines
Cells are isolated from chick embryos and seeded on a collagen scaffold. Tissue Force (μN)
Introduce collagen between two electrodes
E (kPa)
Thread Width (mm)
Model
k (μN/μm)
1
5
0.098
2
5
0.397
3
25
0.098
4
25
0.397
15 Exp Median Sim.
10 5 0 2
3
4
Top Left) FEA simulation (stress) of micropillar array based on Legant et al. 2009[2]. Bottom Left) A comparison of the tissue force reported by Legant et al. and simulation results
1 0.5
Polarized light microscopy of collagen sheets with blue coloration along the alignment axis (double headed arrow). A) Unaligned collagen sheet. B) Mechanically aligned collagen sheet. C) F-actin staining of unaligned cells. D) F-actin staining of aligned cells
Locomotion
1.5 1 0.5 0 25 50 75 100 Collagen Dilution (% collagen)
Acknowledgements The authors would like to thank Katherine Chapin for providing the collagen solution for scaffold fabrication. Additionally, the authors would like to thank Emma Hawley and Jill Patel for assistance with image analysis.
• Device stimulation (15 V for 100 ms at 1 Hz) results in device locomotion
References Active Tension (μN)
Forces compact collagen along isolelectric point
1.5
0.5 1 2 Compaction Time (min)
• Biohybrid Walker [4]
Collagen molecules become charged
2
0
20
1
• Improve device performance by varying scaffold modulus and thickness 2
• Micropillars [2]
ELAC Fabrication
Device geometries can be discretized and simulated in AnimatLab, combining muscle dynamics and neural control, in order to investigate the effect of geometry and control on device motion.
Thread Width (mm)
• Milli and micro-scale actuators are needed for the miniaturization of robotics • Miniature medical devices need to safely interact with living tissue • Traditional small robotic actuators: • Piezoelectrics –have small displacements and low compliance • Shape memory alloys – require temperature changes that are often not compatible with living tissue • Electrochemically compacted and aligned collagen (ELAC) can be used as a substrate in order to fabricate completely organic, living machines • Cells can be isolated from chick embryos and seeded on scaffolds • Stimulating constructs results in organized contraction
• An initial range of TEC values of -0.061056 to -0.151514 can be used to simulate cellular contraction • For models with higher cell moduli, a lower TEC values is needed • For models with low cell modulus and low substrate modulus a higher TEC value is needed
Future Work
∆ Stimulate
Introduction
Modeling
Living Machines
25V
1
1
Conclusions
Living Machines
300 200 100 0 1 Hz stimulation 4 Hz stimulation Sim. Range Exp. Sim. Median
Left) FEA simulation (deflection) of one half of a biohybrid walker based on Cvetkovic et al. 2014[4] . Right) a comparison of the active tension reported by Cvetkovic et al. and simulation results
Characteristic locomotion of an H-shaped ELAC living machine. Left) Initial position of device. Right) Final position of the device with the dashed outline indicating the initial location (Elapsed Time: 10 min).
[1] J. Park et al. Anal. Chem., vol. 77, pp. 6571–80, Oct. 2005. [2] W. Legant et al. PNAS, vol. 106, no. 25, pp. 10097–102, 2009. [3] A. Feinberg et al. Science, vol. 317, pp. 1366–70, Sep. 2007. [4] C. Cvetkovic et al. PNAS, vol. 111, no. 28, pp. 10125–30, Jun. 2014. [5] Chan, V et al. Sci. Rep. Jan. 2012 [6] Kim, J. et al. Lab Chip 7. Nov. 2007 [7] Nawroth, J.C. et al. Nat. Biotechnol. 30(8). Aug. 2012 [8] V. Chan et al. Lab Chip, vol. 12, pp. 88–98, Jan. 2012. [9] K. Wilson et al. PLoS One, vol. 5, no. 6, p. e11042, Jan. 2010. [10] A. Atkinson. Br. Ceram. Proc., vol. 54, no. 1, 1995. [11] C. Klein. J. Appl. Phys., vol. 88, no. 9, p. 5487, 2000. [12] K. Na et al. NSTI-Nanotech, 2008, vol. 3, pp. 737–740.
Abstract
Modeling Biohybrid Devices
Research in the area of biohybrid robotics, while currently a niche field, has been increasing over the past decade. The development of such devices, composed of biocompatible substrates and living tissues, has many far reaching applications including targeted drug delivery and vascular inspection or repair. However, designing these devices is complicated by the compliant nature of tissue based constructs and the stochastic nature of cell culture and function. Additionally, current fabrication techniques rely on non-organic bio-compatible polymers which must often be further processed with complicated micro patterning and chemical treatments to induce cellular alignment and attachment. In order to increase activity in the research and development of biohybrid devices, such barriers to entry must be decreased. To this end a modeling technique has been developed using the native capabilities of commercial Finite Element Analysis (FEA) tools that can serve as a foundation for further development of a biohybrid modeling toolbox. Additionally, fabrication and culture techniques are presented for manufacture of biohybrid devices using aligned collagen, which naturally induces cellular alignment and attachment without additional patterning steps. Using Electrochemically Compacted and Aligned Collagen (ELAC) scaffolds, devices have been fabricated and cultured with primary cardiomyocytes isolated from chick embryos. These devices are capable of locomotion when paced by external electrical stimulation.
Model Calibration
• Completely organic, locomoting, devices have been fabricated using electrochemically compacted collagen as a scaffold
Determine stress from experimental biohybrid cantilevers
Fabrication
,ν)
Cell Layer
Collagen
Load Mold
Compact Collagen
+
Substrate Layer ,ν) 6
1
1 min
1 Cells
Calculate Thermal Expansion Coefficient (TEC)
• Functional, completely organic, muscle powered living machines were fabricated and locomotion was achieved in response to electrical stimulation
Modeling Culture: 4-6 days
Seed Cells
Remove Collagen by Peeling
Collagen Alignment
Simulate Device using Commercial FEA Tools
• Improve model calibration by performing micropillar experiments in which substrate modulus and thickness are varied systematically and pillar deflection as a result of cell contraction is measured.
Living Machines • Develop multi-scale simulations of biohybrid devices and living machines by incorporating FEA simulations and discrete rigid body dynamic simulations • Optimize device geometry in simulation
Small Scale Actuation
• Calibrated 7 models from 3 studies in existing literature • Wide range of geometric and material properties • Resulting range of αΔT: -0.0086 to -0.4218
Collagen alignment as revealed by polarized light microscopy. Left) Leg of scaffold removed from cathode via peeling, blue indicates alignment along leg. Right) Leg of scaffold removed via ultrasonic vibration lacks alignment.
Cellular Alignment
Model Validation
Living Machines
Cells are isolated from chick embryos and seeded on a collagen scaffold. Tissue Force (μN)
Introduce collagen between two electrodes
E (kPa)
Thread Width (mm)
Model
k (μN/μm)
1
5
0.098
2
5
0.397
3
25
0.098
4
25
0.397
15 Exp Median Sim.
10 5 0 2
3
4
Top Left) FEA simulation (stress) of micropillar array based on Legant et al. 2009[2]. Bottom Left) A comparison of the tissue force reported by Legant et al. and simulation results
1 0.5
Polarized light microscopy of collagen sheets with blue coloration along the alignment axis (double headed arrow). A) Unaligned collagen sheet. B) Mechanically aligned collagen sheet. C) F-actin staining of unaligned cells. D) F-actin staining of aligned cells
Locomotion
1.5 1 0.5 0 25 50 75 100 Collagen Dilution (% collagen)
Acknowledgements The authors would like to thank Katherine Chapin for providing the collagen solution for scaffold fabrication. Additionally, the authors would like to thank Emma Hawley and Jill Patel for assistance with image analysis.
• Device stimulation (15 V for 100 ms at 1 Hz) results in device locomotion
References Active Tension (μN)
Forces compact collagen along isolelectric point
1.5
0.5 1 2 Compaction Time (min)
• Biohybrid Walker [4]
Collagen molecules become charged
2
0
20
1
• Improve device performance by varying scaffold modulus and thickness 2
• Micropillars [2]
ELAC Fabrication
Device geometries can be discretized and simulated in AnimatLab, combining muscle dynamics and neural control, in order to investigate the effect of geometry and control on device motion.
Thread Width (mm)
• Milli and micro-scale actuators are needed for the miniaturization of robotics • Miniature medical devices need to safely interact with living tissue • Traditional small robotic actuators: • Piezoelectrics –have small displacements and low compliance • Shape memory alloys – require temperature changes that are often not compatible with living tissue • Electrochemically compacted and aligned collagen (ELAC) can be used as a substrate in order to fabricate completely organic, living machines • Cells can be isolated from chick embryos and seeded on scaffolds • Stimulating constructs results in organized contraction
• An initial range of TEC values of -0.061056 to -0.151514 can be used to simulate cellular contraction • For models with higher cell moduli, a lower TEC values is needed • For models with low cell modulus and low substrate modulus a higher TEC value is needed
Future Work
∆ Stimulate
Introduction
Modeling
Living Machines
25V
1
1
Conclusions
Living Machines
300 200 100 0 1 Hz stimulation 4 Hz stimulation Sim. Range Exp. Sim. Median
Left) FEA simulation (deflection) of one half of a biohybrid walker based on Cvetkovic et al. 2014[4] . Right) a comparison of the active tension reported by Cvetkovic et al. and simulation results
Characteristic locomotion of an H-shaped ELAC living machine. Left) Initial position of device. Right) Final position of the device with the dashed outline indicating the initial location (Elapsed Time: 10 min).
[1] J. Park et al. Anal. Chem., vol. 77, pp. 6571–80, Oct. 2005. [2] W. Legant et al. PNAS, vol. 106, no. 25, pp. 10097–102, 2009. [3] A. Feinberg et al. Science, vol. 317, pp. 1366–70, Sep. 2007. [4] C. Cvetkovic et al. PNAS, vol. 111, no. 28, pp. 10125–30, Jun. 2014. [5] Chan, V et al. Sci. Rep. Jan. 2012 [6] Kim, J. et al. Lab Chip 7. Nov. 2007 [7] Nawroth, J.C. et al. Nat. Biotechnol. 30(8). Aug. 2012 [8] V. Chan et al. Lab Chip, vol. 12, pp. 88–98, Jan. 2012. [9] K. Wilson et al. PLoS One, vol. 5, no. 6, p. e11042, Jan. 2010. [10] A. Atkinson. Br. Ceram. Proc., vol. 54, no. 1, 1995. [11] C. Klein. J. Appl. Phys., vol. 88, no. 9, p. 5487, 2000. [12] K. Na et al. NSTI-Nanotech, 2008, vol. 3, pp. 737–740.
