Design and Validation of an Open-Source 3D Printable Bioreactor System for Ex Vivo Bone Culture

In Canada, osteoporosis is a prevailing skeletal disease that is underdiagnosed and undertreated, with an annual economic cost of $4.6 billion. Trabecular bone is widely understood to undergo modelling and remodelling in response to biochemical and mechanical loads during physical activity. Further insights into the adaptation process of bone could help clinicians improve prevention and treatment methods, including physical activity recommendations, and lead to increased bone health. Previous studies have successfully implemented a polycarbonate (PC) bioreactor system to study trabecular bone adaptation in response to biochemical and mechanical stimulation in long-term ex vivo bone organ culture. However, the PC bioreactors are expensive and difficult to fabricate and have been limited to testing bone cores with maximum dimensions of 5 mm x 10 mm (height x diameter), which is below recommended standards for bone compression testing. Recent advancements in additive manufacturing can reduce fabrication cost and difficulty and allow for high dimensional precision in 3D printed designs with biocompatible material options. Thus, the first objective of the presented research was to develop an open-source 3D printable bioreactor with the photopolymer MED610™ that addresses the PC bioreactors fabrication and bone core height limitations. The second objective was to test the role of the MED610™ material on cell viability and determine a cleaning and sterilization protocol for MED610™ in cell and tissue culture applications. Finally, the third objective was to validate the 3D printed bioreactor in an ex vivo bovine trabecular bone study with mechanical stimulation by measuring the change in apparent elastic modulus over 21-days. Collectively, this thesis demonstrated that 3D printed MED610™ bioreactors cleaned and sterilized with a sonication and autoclave protocol are suitable for ex vivo bone organ culture and can replicate trends in trabecular bone apparent elastic modulus found in previous studies. Recommendations for next steps are provided including adjustments to the bioreactor design and continued biocompatibility and validation testing

Year	Entry
2009	Rincón-Kohli L, Zysset PK. Multi-axial mechanical properties of human trabecular bone. Biomech Model Mechanobiol. June 2009;8(3):195-208.
2002	Currey JD. Bones: Structure and Mechanics. Princeton, NJ: Princeton University Press; 2002.
2011	Cartner JL, Hartsell ZM, Ricci WM, Tornetta P III. Can we trust ex vivo mechanical testing of fresh‒frozen cadaveric specimens? the effect of postfreezing delays. J Orthop Trauma. 2011;25(8):459-461.
2006	Davies CM, Jones DB, Stoddart MJ, Koller K, Smith E, Archer CW, Richards RG. Mechanically loaded ex vivo bone culture system “Zetos”: systems and culture preparation. Eur Cell Mater. January–June 2006;11:57-75.
2020	Xie S, Wallace RJ, Pankaj P. Time-dependent behaviour of demineralised trabecular bone: experimental investigation and development of a constitutive model. J Mech Behav Biomed Mater. September 2020;109:103751.
1997	Keaveny TM, Pinilla TP, Crawford RP, Kopperdahl DL, Lou A. Systematic and random errors in compression testing of trabecular bone [published correction appears in J Orthop Res. 1995;17(1):151]. J Orthop Res. 1997;15(1):101-110.
2008	Sharir A, Barak MM, Shahar R. Whole bone mechanics and mechanical testing. Vet J. July 2008;177(1):8-17.
2012	Al Nazer R, Lanovaz J, Kawalilak C, Johnston JD, Kontulainen S. Direct in vivo strain measurements in human bone: a systematic literature review. J Biomech. January 3, 2012;45(1):27-40.
1998	Hou FJ, Lang SM, Hoshaw SJ, Reimann DA, Fyhrie DP. Human vertebral body apparent and hard tissue stiffness. J Biomech. November 1998;31(11):1009-1015.
2001	Keaveny TM, Morgan EF, Niebur GL, Yeh OC. Biomechanics of trabecular bone. Annu Rev Biomed Eng. 2001;3:307-333.
1998	Martin RB, Burr DB, Sharkey NA. Skeletal Tissue Mechanics. New York, NY: Springer-Verlag; 1998.
2008	Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. November 2008;3(suppl 3):S131-S139.
2003	Frost HM. Bone's mechanostat: a 2003 update. Anat Rec. 2003;275A(2):1081-1101.
2015	Oftadeh R, Perez-Viloria M, Villa-Camacho JC, Vaziri A, Nazarian A. Biomechanics and mechanobiology of trabecular bone: a review. J Biomech Eng. January 2015;137(1):010802.
1998	Weiner S, Wagner HD. The material bone: structure-mechanical function relations. Ann Rev Mater Sci. August 1998;28:271-298.
1997	Kalidindi SR, Abusafieh A, El-Danaf E. Accurate characterization of machine compliance for simple compression testing. Exp Mech. June 1997;37(2):210-215.
2018	Wu D, Isaksson P, Ferguson SJ, Persson C. Young’s modulus of trabecular bone at the tissue level: a review. Acta Biomater. September 15, 2018;78:1.
2011	Donnelly E. Methods for assessing bone quality: a review. Clin Orthop Relat Res. August 2011;469:2128-2138.
2018	Zhao S, Arnold M, Ma S, Abel RL, Cobb JP, Hansen U, Boughton O. Standardizing compression testing for measuring the stiffness of human bone. Bone Joint Res. August 2018;7(8):524-538.
2006	Xie L, Jacobson JM, Choi ES, Busa B, Donahue LR, Miller LM, Rubin CT, Judex S. Low-level mechanical vibrations can influence bone resorption and bone formation in the growing skeleton. Bone. November 2006;39(5):1059-1066.
2004	Rubin C, Recker R, Cullen D, Ryaby J, McCabe J, McLeod K. Prevention of postmenopausal bone loss by a low‐magnitude, high‐frequency mechanical stimuli: a clinical trial assessing compliance, efficacy, and safety. J Bone Miner Res. March 2004;19(3):343-351.
1993	Fung YC. Biomechanics: Mechanical Properties of Living Tissues. 2nd ed. New York, NY: Springer-Verlag; 1993.
2003	Jones DB, Broeckmann E, Pohl T, Smith EL. Development of a mechanical testing and loading system for trabecular bone studies for long term culture. Eur Cell Mater. January–June 2003;5:48-60.
2004	Pearson OM, Lieberman DE. The aging of Wolff's “law”: ontogeny and responses to mechanical loading in cortical bone. Yearb Phys Anthropol. 2004;47:63-99.
1987	Frost HM. Bone “mass” and the “mechanostat”: a proposal. Anat Rec. September 1987;219(1):1-9.
2021	Kistler-Fischbacher M, Weeks BK, Beck BR. The effect of exercise intensity on bone in postmenopausal women (part 2): a meta-analysis. Bone. February 2021;143:115697.
2019	Bravo AE, Osnaya LC, Ramírez EI, Jacobo VH, Ortiz A. The effect of bone marrow on the mechanical behavior of porcine trabecular bone. Biomed Phys Eng Express. 2019;5(6):065023.
2016	Meyer LA, Johnson MG, Cullen DM, Vivanco JF, Blank RD, Ploeg H-L, Smith EL. Combined exposure to big endothelin-1 and mechanical loading in bovine sternal cores promotes osteogenesis. Bone. April 2016;85:115-122.
2010	Lievers WB, Petryshyn AC, Poljsak AS, Waldman SD, Pilkey AK. Specimen diameter and “side artifacts” in cancellous bone evaluated using end-constrained elastic tension. Bone. August 2010;47(2):371-377.
2019	Wescott DJ. Postmortem change in bone biomechanical properties: loss of plasticity. Forensic Sci Int. July 2019;300:164-169.
2012	Currey JD. The structure and mechanics of bone. J Mater Sci. January 2012;47(1):41-54.
1994	Deligianni DD, Maris A, Missirlis YF. Stress relaxation behaviour of trabecular bone specimens. J Biomech. December 1994;27(12):1469-1476.
2006	Adharapurapu RR, Jiang F, Vecchio KS. Dynamic fracture of bovine bone. Mater Sci Eng C Mater Biol Appl. September 2006;26(8):1325-1332.
2018	Watson SL, Weeks BK, Weis LJ, Harding AT, Horan SA, Beck BR. High-intensity resistance and impact training improves bone mineral density and physical function in postmenopausal women with osteopenia and osteoporosis: the LIFTMOR randomized controlled trial. J Bone Miner Res. February 2018;33(2):211-220.
2021	Anam M, Ponnusamy Va, Hussain M, Nadeem MW, Javed M, Goh HG, Qadeer S. Osteoporosis prediction for trabecular bone using machine learning: a review. CMC-Comput Mater Con. 2021;67(1):89-105.
2021	Bennison MBL, Pilkey AK, Lievers WB. Evaluating a theoretical and an empirical model of “side effects” in cancellous bone. Med Eng Phys. August 2021;94:8-15.
2021	Kendler DL, Adachi JD, Brown JP, Juby AG, Kovacs CS, Duperrouzel C, McTavish RK, Cameron C, Slatkovska L, Burke N. A scorecard for osteoporosis in Canada and seven Canadian provinces. Osteoporos Int. January 2021;32(1):123-132.
2005	Ruimerman R, Hilbers P, van Rietbergen B, Huiskes R. A theoretical framework for strain-related trabecular bone maintenance and adaptation. J Biomech. April 2005;38(4):931-941.
1996	Burr DB, Milgrom C, Fyhrie D, Forwood M, Nyska M, Finestone A, Hoshaw S, Saiag E, Simkin A. In vivo measurement of human tibial strains during vigorous activity. Bone. May 1996;18(5):405-410.
2013	Vivanco J, Garcia S, Ploeg HL, Alvarez G, Cullen D, Smith EL. Apparent elastic modulus of ex vivo trabecular bovine bone increases with dynamic loading. Proc Inst Mech Eng Part H-J Eng Med. August 2013;227(8):904-912.
1998	Rho J-Y, Kuhn-Spearing L, Zioupos P. Mechanical properties and the hierarchical structure of bone. Med Eng Phys. 1998;20(2):92-102.
1993	Keaveny TM, Borchers RE, Gibson L, Hayes WC. Trabecular bone modulus and strength can depend on specimen geometry. J Biomech. August 1993;26(8):991-995.
1993	Linde F, Sørensen HCF. The effect of different storage methods on the mechanical properties of trabecular bone. J Biomech. 1993;26(10):1249-1252.
2010	Papaioannou A, Morin S, Cheung AM, Atkinson S, Brown JP, Feldman S, Hanley DA, Hodsman A, Jamal SA, Kaiser SM, Kvern B, Siminoski K, Leslie WD; Scientific Advisory Council of Osteoporosis Canada. 2010 clinical practice guidelines for the diagnosis and management of osteoporosis in Canada: summary. Can Med Assoc J. November 23, 2010;182(17):1864-1873.
2006	Mann V, Huber C, Kogianni G, Jones D, Noble B. The influence of mechanical stimulation on osteocyte apoptosis and bone viability in human trabecular bone. J Musculoskel Neuron Interact. December 2006;6(4):408-417.
2021	Bennison MBL, Pilkey AK, Lievers WB. Misalignment error in cancellous bone apparent elastic modulus depends on bone volume fraction and degree of anisotropy. J Biomech Eng. February 2021;143(2):021005.
2000	Lucchinetti E, Thomann D, Danuser G. Micromechanical testing of bone trabeculae: potentials and limitations. J Mater Sci. December 2000;35(24):6057-6064.

Year

Entry

2009

Rincón-Kohli L, Zysset PK. Multi-axial mechanical properties of human trabecular bone. Biomech Model Mechanobiol. June 2009;8(3):195-208.

2002

Currey JD. Bones: Structure and Mechanics. Princeton, NJ: Princeton University Press; 2002.

2011

Cartner JL, Hartsell ZM, Ricci WM, Tornetta P III. Can we trust ex vivo mechanical testing of fresh‒frozen cadaveric specimens? the effect of postfreezing delays. J Orthop Trauma. 2011;25(8):459-461.

2006

Davies CM, Jones DB, Stoddart MJ, Koller K, Smith E, Archer CW, Richards RG. Mechanically loaded ex vivo bone culture system “Zetos”: systems and culture preparation. Eur Cell Mater. January–June 2006;11:57-75.

2020

Xie S, Wallace RJ, Pankaj P. Time-dependent behaviour of demineralised trabecular bone: experimental investigation and development of a constitutive model. J Mech Behav Biomed Mater. September 2020;109:103751.

1997

Keaveny TM, Pinilla TP, Crawford RP, Kopperdahl DL, Lou A. Systematic and random errors in compression testing of trabecular bone [published correction appears in J Orthop Res. 1995;17(1):151]. J Orthop Res. 1997;15(1):101-110.

2008

Sharir A, Barak MM, Shahar R. Whole bone mechanics and mechanical testing. Vet J. July 2008;177(1):8-17.

2012

Al Nazer R, Lanovaz J, Kawalilak C, Johnston JD, Kontulainen S. Direct in vivo strain measurements in human bone: a systematic literature review. J Biomech. January 3, 2012;45(1):27-40.

1998

Hou FJ, Lang SM, Hoshaw SJ, Reimann DA, Fyhrie DP. Human vertebral body apparent and hard tissue stiffness. J Biomech. November 1998;31(11):1009-1015.

2001

Keaveny TM, Morgan EF, Niebur GL, Yeh OC. Biomechanics of trabecular bone. Annu Rev Biomed Eng. 2001;3:307-333.

1998

Martin RB, Burr DB, Sharkey NA. Skeletal Tissue Mechanics. New York, NY: Springer-Verlag; 1998.

2008

Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. November 2008;3(suppl 3):S131-S139.

2003

Frost HM. Bone's mechanostat: a 2003 update. Anat Rec. 2003;275A(2):1081-1101.

2015

Oftadeh R, Perez-Viloria M, Villa-Camacho JC, Vaziri A, Nazarian A. Biomechanics and mechanobiology of trabecular bone: a review. J Biomech Eng. January 2015;137(1):010802.

1998

Weiner S, Wagner HD. The material bone: structure-mechanical function relations. Ann Rev Mater Sci. August 1998;28:271-298.

1997

Kalidindi SR, Abusafieh A, El-Danaf E. Accurate characterization of machine compliance for simple compression testing. Exp Mech. June 1997;37(2):210-215.

2018

Wu D, Isaksson P, Ferguson SJ, Persson C. Young’s modulus of trabecular bone at the tissue level: a review. Acta Biomater. September 15, 2018;78:1.

2011

Donnelly E. Methods for assessing bone quality: a review. Clin Orthop Relat Res. August 2011;469:2128-2138.

2018

Zhao S, Arnold M, Ma S, Abel RL, Cobb JP, Hansen U, Boughton O. Standardizing compression testing for measuring the stiffness of human bone. Bone Joint Res. August 2018;7(8):524-538.

2006

Xie L, Jacobson JM, Choi ES, Busa B, Donahue LR, Miller LM, Rubin CT, Judex S. Low-level mechanical vibrations can influence bone resorption and bone formation in the growing skeleton. Bone. November 2006;39(5):1059-1066.

2004

Rubin C, Recker R, Cullen D, Ryaby J, McCabe J, McLeod K. Prevention of postmenopausal bone loss by a low‐magnitude, high‐frequency mechanical stimuli: a clinical trial assessing compliance, efficacy, and safety. J Bone Miner Res. March 2004;19(3):343-351.

1993

Fung YC. Biomechanics: Mechanical Properties of Living Tissues. 2nd ed. New York, NY: Springer-Verlag; 1993.

2003

Jones DB, Broeckmann E, Pohl T, Smith EL. Development of a mechanical testing and loading system for trabecular bone studies for long term culture. Eur Cell Mater. January–June 2003;5:48-60.

2004

Pearson OM, Lieberman DE. The aging of Wolff's “law”: ontogeny and responses to mechanical loading in cortical bone. Yearb Phys Anthropol. 2004;47:63-99.

1987

Frost HM. Bone “mass” and the “mechanostat”: a proposal. Anat Rec. September 1987;219(1):1-9.

2021

Kistler-Fischbacher M, Weeks BK, Beck BR. The effect of exercise intensity on bone in postmenopausal women (part 2): a meta-analysis. Bone. February 2021;143:115697.

2019

Bravo AE, Osnaya LC, Ramírez EI, Jacobo VH, Ortiz A. The effect of bone marrow on the mechanical behavior of porcine trabecular bone. Biomed Phys Eng Express. 2019;5(6):065023.

2016

Meyer LA, Johnson MG, Cullen DM, Vivanco JF, Blank RD, Ploeg H-L, Smith EL. Combined exposure to big endothelin-1 and mechanical loading in bovine sternal cores promotes osteogenesis. Bone. April 2016;85:115-122.

2010

Lievers WB, Petryshyn AC, Poljsak AS, Waldman SD, Pilkey AK. Specimen diameter and “side artifacts” in cancellous bone evaluated using end-constrained elastic tension. Bone. August 2010;47(2):371-377.

2019

Wescott DJ. Postmortem change in bone biomechanical properties: loss of plasticity. Forensic Sci Int. July 2019;300:164-169.

2012

Currey JD. The structure and mechanics of bone. J Mater Sci. January 2012;47(1):41-54.

1994

Deligianni DD, Maris A, Missirlis YF. Stress relaxation behaviour of trabecular bone specimens. J Biomech. December 1994;27(12):1469-1476.

2006

Adharapurapu RR, Jiang F, Vecchio KS. Dynamic fracture of bovine bone. Mater Sci Eng C Mater Biol Appl. September 2006;26(8):1325-1332.

2018

Watson SL, Weeks BK, Weis LJ, Harding AT, Horan SA, Beck BR. High-intensity resistance and impact training improves bone mineral density and physical function in postmenopausal women with osteopenia and osteoporosis: the LIFTMOR randomized controlled trial. J Bone Miner Res. February 2018;33(2):211-220.

2021

Anam M, Ponnusamy Va, Hussain M, Nadeem MW, Javed M, Goh HG, Qadeer S. Osteoporosis prediction for trabecular bone using machine learning: a review. CMC-Comput Mater Con. 2021;67(1):89-105.

2021

Bennison MBL, Pilkey AK, Lievers WB. Evaluating a theoretical and an empirical model of “side effects” in cancellous bone. Med Eng Phys. August 2021;94:8-15.

2021

Kendler DL, Adachi JD, Brown JP, Juby AG, Kovacs CS, Duperrouzel C, McTavish RK, Cameron C, Slatkovska L, Burke N. A scorecard for osteoporosis in Canada and seven Canadian provinces. Osteoporos Int. January 2021;32(1):123-132.

2005

Ruimerman R, Hilbers P, van Rietbergen B, Huiskes R. A theoretical framework for strain-related trabecular bone maintenance and adaptation. J Biomech. April 2005;38(4):931-941.

1996

Burr DB, Milgrom C, Fyhrie D, Forwood M, Nyska M, Finestone A, Hoshaw S, Saiag E, Simkin A. In vivo measurement of human tibial strains during vigorous activity. Bone. May 1996;18(5):405-410.

2013

Vivanco J, Garcia S, Ploeg HL, Alvarez G, Cullen D, Smith EL. Apparent elastic modulus of ex vivo trabecular bovine bone increases with dynamic loading. Proc Inst Mech Eng Part H-J Eng Med. August 2013;227(8):904-912.

1998

Rho J-Y, Kuhn-Spearing L, Zioupos P. Mechanical properties and the hierarchical structure of bone. Med Eng Phys. 1998;20(2):92-102.

1993

Keaveny TM, Borchers RE, Gibson L, Hayes WC. Trabecular bone modulus and strength can depend on specimen geometry. J Biomech. August 1993;26(8):991-995.

1993

Linde F, Sørensen HCF. The effect of different storage methods on the mechanical properties of trabecular bone. J Biomech. 1993;26(10):1249-1252.

2010

Papaioannou A, Morin S, Cheung AM, Atkinson S, Brown JP, Feldman S, Hanley DA, Hodsman A, Jamal SA, Kaiser SM, Kvern B, Siminoski K, Leslie WD; Scientific Advisory Council of Osteoporosis Canada. 2010 clinical practice guidelines for the diagnosis and management of osteoporosis in Canada: summary. Can Med Assoc J. November 23, 2010;182(17):1864-1873.

2006

Mann V, Huber C, Kogianni G, Jones D, Noble B. The influence of mechanical stimulation on osteocyte apoptosis and bone viability in human trabecular bone. J Musculoskel Neuron Interact. December 2006;6(4):408-417.

2021

Bennison MBL, Pilkey AK, Lievers WB. Misalignment error in cancellous bone apparent elastic modulus depends on bone volume fraction and degree of anisotropy. J Biomech Eng. February 2021;143(2):021005.

2000

Lucchinetti E, Thomann D, Danuser G. Micromechanical testing of bone trabeculae: potentials and limitations. J Mater Sci. December 2000;35(24):6057-6064.

Year	Entry
2024	Huitema E. Design and Validation of an Accessible and 3D-Printable 24-Channel Peristaltic Pump for Cell Culture Research [Master's thesis]. Queen's University; July 15, 2024.

Year

Entry

2024

Huitema E. Design and Validation of an Accessible and 3D-Printable 24-Channel Peristaltic Pump for Cell Culture Research [Master's thesis]. Queen's University; July 15, 2024.