JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

Published: April 25th, 2013

DOI:

10.3791/50387

1Department of Orthopaedics, The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, 2Center for Restorative and Regenerative Medicine, VA Medical Center, Providence, RI, 3University of Texas Southwestern Medical Center

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We have designed a loading bioreactor that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. This device is primarily designed as a bioreactor for engineered replacements for articular cartilage; it could also be used for other load-bearing tissues in the human body. Our motivation in this bioreactor design stems from Drachman and Sokoloff 1, who made the seminal observation of abnormal formation of articular cartilage in paralyzed chick embryos due to absence of motion. Similarly, physical exercise is essential for development of normal muscle and bone. In keeping with....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Biaxial Loading Bioreactor Design

  1. The bioreactor employs two stages manufactured by Thor-labs (Newton, MA) for precision-guided laser applications for applying uniaxial or biaxial mechanical strain to engineered tissues, with great precision of loading dose (amplitude and frequency) and application to a wide variety of tissue culture conditions from single to 24 well plates (Figure 1).
  2. Bi-axial loading is accomplished by two TravelMax stages (LNR50SE). These stages are mounted or.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The device was tested by using agarose gels seeded with 20 million cells/ml chondrocytes and cultivated in the presence of uniaxial (compression) or biaxial (compression and shear) mechanical loading. Primary porcine chondrocytes were isolated from the articular cartilage of 2-4 month old pigs. 5 mm diameter and 1.5 mm thick samples were cultured in 2 ml of defined chondrogenic culture medium (High glucose DMEM, 1% ITS+ Premix, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 2.5 μg/ml amphotericin B,.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We have designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to tissue engineered constructs fabricated for transplantation. The device can be used as a bioreactor for in vitro cultivation of engineered biocomposites or as a testing device to describe the mechanical characteristics of the native tissue or after other treatments prior to. The device subjects engineered tissue constructs to biaxial mechanical loading with great precision of loading dose (amplitude and .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by the Office of Research and Development, RR&D Service, U.S. Department of Veterans Affairs, NIH COBRE 1P20RR024484, NIH K24 AR02128 and Department of Defense W81XWH-10-1-0643.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
REAGENTS
DMEM, High glucose, pyruvate Invitrogen 11995
Agarose Type II Sigma CAS 39346-81-1
Penicillin Streptomycin Glutamine 100X Invitrogen 10378-016
ITS+ Premix BD Biosciences 354352
Pen Strep Glutamine Invitrogen 10378-016
Amphotericin B Invitrogen 041-95780
Ascorbic Acid Sigma A-2218
Nonessential Amino Acid Solution 100x Sigma M-7145
L-proline Sigma P-5607
Dexamethasone Sigma D-2915
Recombinant Human Transforming Growth Factor β1 R&D Systems 240-B-010
EQUIPMENT
Model 31 Load Cell (1000 g) Honeywell AL311
Single Channel Display Honeywell SC500
50 mm Linear Encoded Travelmax Stage with Stepper Actuator Thorlabs LNR50SE/M
Two Channel Stepper Motor Controller Thorlabs BSC102
50 mm Trapezoidal Stepper Motor Drive (2) Thorlabs DRV014
Adjustable Kinematic Locator (4) Thorlabs KL02
Precision Right Angle Plate Thorlabs AP90/M
Vertical Mounting Bracket Thorlabs LNR50P2/M
Solid Aluminum Breadboard Thorlabs MB3030/M
Gel Casting System with 1.5 mm and 0.75 mm spacer plates BioRad #1653312 and #1653310
Disposable Biopsy Punch, 5 mm Miltex, Inc. 33-35
16 mm hollow punch Neiko Tools
Non-Tissue Culture Treated Plates, 24 Well, Flat Bottom BD Biosciences 351147
Ultra-Moisture-Resistant Polysulfone sheet for loading platens McMaster-Carr 86735k19 Custom-machined

  1. Drachman, D. B., Sokoloff, L. The role of movement in embryonic joint development. Devl. Biol. 14, 401-420 (1966).
  2. Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Hunziker, E. B. Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J. Cell Sci. 108, 1497-1508 (1995).
  3. Vunjak-Novakovic, G., et al. Bioreactor Cultivation Conditions Modulate the Composition and Mechanical Properties of Tissue-Engineered Cartilage. Journal of Orthopaedic Research. 17, 130-138 (1999).
  4. Gooch, K. J., et al. Effects of Mixing Intensity on Tissue-Engineered Cartilage. Biotechnology and Bioengineering. 72, 402-407 (2001).
  5. Carver, S. E., Heath, C. A. Increasing extracellular matrix production in regenerating cartilage with intermittent physiological pressure. Biotechnology and Bioengineering. 62, 166-174 (1999).
  6. Frank, E. H., Jin, M., Loening, A. M., Levenston, M. E., Grodzinsky, A. J. A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants. J. Biomech. 33, 1523-1527 (2000).
  7. Wagner, D. R., et al. Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium. Ann. Biomed. Eng. 36, 813-820 (2008).
  8. Butler, D. L., Goldstein, S. A., Guilak, F. Functional Tissue Engineering: The Role of Biomechanics. J. Biomech. Eng. 122, 570-575 (2000).
  9. Guilak, F., Butler, D. L., Goldstein, S. A. Functional Tissue Engineering. The role of biomechanics in articular cartilage repair. Clin. Orthop. 391S, S295-S305 (2001).
  10. Mauck, R. L., Byers, B. A., Yuan, X., Tuan, R. S. Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. Biomech. Model Mechanobiol. 6, 113-125 (2007).
  11. Rubin, C., Xu, G., Judex, S. The anabolic activity of bone tissue, suppressed by disuse, is normalized by brief exposure to extremely low-magnitude mechanical stimuli. FASEB J. 15, 2225-2229 (2001).
  12. Wimmer, M. A., et al. Tribology approach to the engineering and study of articular cartilage. Tissue Eng. 10, 1436-1445 (2004).
  13. Miyata, S., Tateishi, T., Ushida, T. Influence of cartilaginous matrix accumulation on viscoelastic response of chondrocyte/agarose constructs under dynamic compressive and shear loading. J. Biomech. Eng. 130, 051016 (2008).
  14. Heiner, A. D., Martin, J. A. Cartilage responses to a novel triaxial mechanostimulatory culture system. J. Biomech. 37, 689-695 (2004).
  15. Waldman, S. D., Couto, D. C., Grynpas, M. D., Pilliar, R. M., Kandel, R. A. Multi-axial mechanical stimulation of tissue engineered cartilage: review. Eur. Cell Mater. 13, 66-73 (2007).
  16. Wartella, K. A., Wayne, J. S. Bioreactor for biaxial mechanical stimulation to tissue engineered constructs. J. Biomech. Eng. 131, 044501 (2009).
  17. Bian, L., et al. Dynamic mechanical loading enhances functional properties of tissue-engineered cartilage using mature canine chondrocytes. Tissue Eng. Part A. 16, 1781-1790 (2010).
  18. Bilgen, B., et al. Design of a Biaxial Loading Device for Cartilage Tissue Engineering. , 1815 (2011).
  19. Mauck, R. L., Wang, C. C., Oswald, E. S., Ateshian, G. A., Hung, C. T. The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading. Osteoarthritis Cartilage. 11, 879-890 (2003).
  20. Mauck, R. L., et al. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J. Biomech. Eng. 122, 252-260 (2000).
  21. Demarteau, O., Jakob, M., Schafer, D., Heberer, M., Martin, I. Development and validation of a bioreactor for physical stimulation of engineered cartilage. Biorheology. 40, 331-336 (2003).
  22. Grad, S., et al. Surface motion upregulates superficial zone protein and hyaluronan production in chondrocyte-seeded three-dimensional scaffolds. Tissue Eng. 11, 249-256 (2005).
  23. Schatti, O., et al. A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells. Eur. Cell Mater. 22, 214-225 (2011).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved