Micro-Environmental Factors Directing Differentiation of Murine Embryonic Stem Cells Down Osteogenic and Chondrogenic Lineages

The field of bone tissue engineering (BTE) aims to develop graft substitutes for diseased or difficult to heal fractures. We used a BTE construct made of collagen type I and murine embryonic stem cells (mESCs) which has been shown to trigger mESCs differentiation into osteoblasts, and successfully contribute to fracture repair in vivo within a mouse model system.

Bone healing is a complex process involving the interplay of biochemical and biomechanical cues. Therefore, this project aimed to systematically emulate the roles of chemical and mechanical cues present during fracture repair on the differentiation of mESCs in vitro in order to optimize treatment strategies for BTE.

Further characterization of this cell/gel construct revealed that mESCs differentiate into a heterogeneous cell population of chondrocytes and osteoblasts, replicating the process of endochondral ossification that normally occurs during fracture repair. To study the effect of biomechanical cues, a loading system was specifically designed and characterized to apply confined compressive load to a soft, viscoelastic cell/gel construct. Mechanical stimuli enhanced chondrogenic differentiation but had no effect on osteogenic differentiation. Moreover, the role of integrins in directing mESCs differentiation and transducing mechanical signals was evaluated. Finally, the synergistic effect between extracellular matrix mediated differentiation, mechanical stimulation and BMP-2 delivery to the system using nano-particles was studied.

Year	Entry
2003	Simmons CA, Matlis S, Thornton AJ, Chen S, Wang C-Y, Mooney DJ. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J Biomech. August 2003;36(8):1087-1096.
2004	Mullender M, El Haj AJ, Yang Y, van Duin MA, Burger EH, Klein-Nulend J. Mechanotransduction of bone cells in vitro: mechanobiology of bone tissue. Med Biol Eng Comput. January 2004;42(1):14-21.
2003	Kapur S, Baylink DJ, William Lau K-H. Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. Bone. March 2003;32(3):241-251.
2006	Ingber DE. Cellular mechanotransduction: putting all the pieces together again. FASEB J. May 2006;20(7):811-827.
2008	Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. November 2008;3(suppl 3):S131-S139.
1997	Owan I, Burr DB, Turner CH, Qiu J, Tu Y, Onyia JE, Duncan RL. Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain. Am J Physiol Cell Physiol. September 1997;273(3):C810-C815.
2002	Akiyama H, Chaboissier M-C, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. November 1, 2002;16(21):2813-2828.
1995	Turner CH, Owan I, Takano Y. Mechanotransduction in bone: role of strain rate. Am J Physiol Endocrinol Metab. September 1995;269(3):E438-E442.
1998	Jacobs CR, Yellowley CE, Davis BR, Zhou Z, Cimbala JM, Donahue HJ. Differential effect of steady versus oscillating flow on bone cells. J Biomech. November 1998;31(11):969-976.
2001	Hsieh Y, Turner CH. Effects of loading frequency on mechanically induced bone formation. J Bone Miner Res. May 2001;16(5):918-924.
1994	Urban JPG. The chondrocyte: a cell under pressure. Br J Rheumatol. October 1994;33(10):901-908.
2004	Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the art and future trends. Macromol Biosci. August 9, 2004;4(8):743-765.
2003	Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials. November 2003;24(24):4385-4415.
2003	Wong M, Siegrist M, Goodwin K. Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes. Bone. October 2003;33(4):685-693.
2007	Wang Y, McNamara LM, Schaffler MB, Weinbaum S. A model for the role of integrins in flow induced mechanotransduction in osteocytes. Proc Natl Acad Sci USA. October 2, 2007;104(40):15941-15946.
1993	Langer R, Vacanti JP. Tissue engineering. Science. May 14, 1993;260(5110):920-926.
1998	Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res. January 10, 1998;238(1):265-272.
2001	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods. December 2001;25(4):402-408.
1998	Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. November 6, 1998;282(5391):1145-1147.
1997	Lee DA, Bader DL. Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose. J Orthop Res. March 1997;15(2):181-188.
2006	Hadjidakis DJ, Androulakis II. Bone remodeling. Annals NY Acad Sci. December 2006;1092(1):385-396.
1999	Burger EH, Klein-Nulend J. Mechanotransduction in bone: role of the lacunocanalicular network. FASEB J. May 1999;12(9001):S101-S112.
2004	Huang CC, Hagar KL, Frost LE, Sun Y, Cheung HS. Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells. Stem Cells. May 2004;22(3):313-323.
2002	Hunter CJ, Imler SM, Malaviya P, Nerem RM, Levenston ME. Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels. Biomaterials. February 15, 2002;23(4):1249-1259.
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.

Year

Entry

2003

Simmons CA, Matlis S, Thornton AJ, Chen S, Wang C-Y, Mooney DJ. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J Biomech. August 2003;36(8):1087-1096.

2004

Mullender M, El Haj AJ, Yang Y, van Duin MA, Burger EH, Klein-Nulend J. Mechanotransduction of bone cells in vitro: mechanobiology of bone tissue. Med Biol Eng Comput. January 2004;42(1):14-21.

2003

Kapur S, Baylink DJ, William Lau K-H. Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. Bone. March 2003;32(3):241-251.

2006

Ingber DE. Cellular mechanotransduction: putting all the pieces together again. FASEB J. May 2006;20(7):811-827.

2008

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

1997

Owan I, Burr DB, Turner CH, Qiu J, Tu Y, Onyia JE, Duncan RL. Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain. Am J Physiol Cell Physiol. September 1997;273(3):C810-C815.

2002

Akiyama H, Chaboissier M-C, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. November 1, 2002;16(21):2813-2828.

1995

Turner CH, Owan I, Takano Y. Mechanotransduction in bone: role of strain rate. Am J Physiol Endocrinol Metab. September 1995;269(3):E438-E442.

1998

Jacobs CR, Yellowley CE, Davis BR, Zhou Z, Cimbala JM, Donahue HJ. Differential effect of steady versus oscillating flow on bone cells. J Biomech. November 1998;31(11):969-976.

2001

Hsieh Y, Turner CH. Effects of loading frequency on mechanically induced bone formation. J Bone Miner Res. May 2001;16(5):918-924.

1994

Urban JPG. The chondrocyte: a cell under pressure. Br J Rheumatol. October 1994;33(10):901-908.

2004

Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the art and future trends. Macromol Biosci. August 9, 2004;4(8):743-765.

2003

Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials. November 2003;24(24):4385-4415.

2003

Wong M, Siegrist M, Goodwin K. Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes. Bone. October 2003;33(4):685-693.

2007

Wang Y, McNamara LM, Schaffler MB, Weinbaum S. A model for the role of integrins in flow induced mechanotransduction in osteocytes. Proc Natl Acad Sci USA. October 2, 2007;104(40):15941-15946.

1993

Langer R, Vacanti JP. Tissue engineering. Science. May 14, 1993;260(5110):920-926.

1998

Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res. January 10, 1998;238(1):265-272.

2001

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods. December 2001;25(4):402-408.

1998

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. November 6, 1998;282(5391):1145-1147.

1997

Lee DA, Bader DL. Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose. J Orthop Res. March 1997;15(2):181-188.

2006

Hadjidakis DJ, Androulakis II. Bone remodeling. Annals NY Acad Sci. December 2006;1092(1):385-396.

1999

Burger EH, Klein-Nulend J. Mechanotransduction in bone: role of the lacunocanalicular network. FASEB J. May 1999;12(9001):S101-S112.

2004

Huang CC, Hagar KL, Frost LE, Sun Y, Cheung HS. Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells. Stem Cells. May 2004;22(3):313-323.

2002

Hunter CJ, Imler SM, Malaviya P, Nerem RM, Levenston ME. Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels. Biomaterials. February 15, 2002;23(4):1249-1259.

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.

Year	Entry
2014	Damaraju S. The Role of Cell Communication and 3d Cell-Matrix Environment in a Stem Cell-Based Tissue Engineering Strategy for Bone Repair [PhD thesis]. Calgary, AB: University of Calgary; July 2014.
2017	Nasr S. The Role of Mechanical Interactions Between Cells and the Extracellular Matrix on Mineralization in a Stem Cell-Seeded Scaffold: A Multi-Scale Approach [PhD thesis]. Calgary, AB: University of Calgary; June 2017.

Year

Entry

2014

Damaraju S. The Role of Cell Communication and 3d Cell-Matrix Environment in a Stem Cell-Based Tissue Engineering Strategy for Bone Repair [PhD thesis]. Calgary, AB: University of Calgary; July 2014.

2017

Nasr S. The Role of Mechanical Interactions Between Cells and the Extracellular Matrix on Mineralization in a Stem Cell-Seeded Scaffold: A Multi-Scale Approach [PhD thesis]. Calgary, AB: University of Calgary; June 2017.