The Role of Cell Communication and 3d Cell-Matrix Environment in a Stem Cell-Based Tissue Engineering Strategy for Bone Repair

The use of stem cells for the repair of bone and cartilage is a primary research interest in modern tissue engineering strategies. The goal of this doctoral thesis was to investigate the role of cell communication and mechanical stimulation in a 3D tissue engineered construct, and to determine whether this construct could be optimized for bone healing. It is well established that there is an interconnected relationship between connexin-containing gap junctions, cadherins, and integrins in cell communication and the ability of cells to respond to mechanical stimulation. The specific aim of this project was to determine the role of connexin containing gap junctions and confined compression on the biosynthetic activity of embryonic stem cells stimulated to form osteoblasts in a 3D collagen-I scaffold. Results from this project showed that this construct could be optimized for bone repair and cell differentiation could be accelerated by applying mechanical stimulation to cells at an early state of differentiation. This could be further enhanced with the application of a nitric oxide donor. As well, cell communication and the presence of functional connexin-43 gap junctions was required for lineage determination of these cells and to initiate mineralization within the construct. The necessity for cell communication was such that cells were not able to recover their function following communication inhibition. It was shown that dissociation between connexin-43 and integrin α5β1 occurred under communication impairment and could be slightly restored with the application of nitric oxide. Thus, in order for osteoblasts to perceive a mechanical stimulus and initiate mineralization of the matrix in response, functional gap junctions and their association to integrins are required to guide cell fate and function. Under impairment of cell function, nitric oxide can be used as an exogenous factor to refocus differentiation and rescue cell function. Overall, this project helps to bridge in-vivo fracture research with basic stem cell biology, and provides an understanding of the importance of cell communication and 3-dimensional cell-matrix environment in the healing process of bone.

Year	Entry
2009	Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, Chen CS. Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell. July 2, 2009;5(1):17-26.
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.
2005	Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/β-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell. May 2005;8(5):739-750.
2005	Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater. July 2005;4(7):518-524.
2000	Knothe Tate M, Knothe U. An ex vivo model to study transport processes and fluid flow in loaded bone. J Biomech. February 2000;33(2):247-254.
2003	Cherian PP, Cheng B, Gu S, Sprague E, Bonewald LF, Jiang JX. Effects of mechanical strain on the function of gap junctions in osteocytes are mediated through the prostaglandin EP₂ receptor. J Biol Chem. October 31, 2003;278(44):43146-43156.
1999	Burger EH, Klein-Nulend J. Mechanotransduction in bone: role of the lacunocanalicular network. FASEB J. May 1999;12(9001):S101-S112.
2001	Bakker AD, Soejima K, Klein-Nulend J, Burger EH. The production of nitric oxide and prostaglandin E₂ by primary bone cells is shear stress dependent. J Biomech. May 2001;34(5):671-677.
2006	Rubin J, Rubin C, Jacobs CR. Molecular pathways mediating mechanical signaling in bone. Gene. February 15, 2006;367:1-16.
2000	Teitelbaum SL. Bone resorption by osteoclasts. Science. September 1, 2000;289(5484):1504-1508.
2008	Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C_T method. Nat Protoc. 2008;3(6):1101-1108.
2013	Hazenbiller O. Micro-Environmental Factors Directing Differentiation of Murine Embryonic Stem Cells Down Osteogenic and Chondrogenic Lineages [Master's thesis]. Calgary, AB: University of Calgary; February 2013.
1978	McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg. May 1978;60B(2):150-162.
2001	Cheng B, Zhao S, Luo J, Sprague E, Bonewald LF, Jiang JX. Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells. J Bone Miner Res. February 2001;16(2):249-259.
2005	Wang L, Wang Y, Han Y, Henderson SC, Majeska RJ, Weinbaum S, Schaffler MB. In situ measurement of solute transport in the bone lacunar-canalicular system. Proc Natl Acad Sci USA. August 16, 2005;102(46):11911-11916.
2004	Awad HA, Wickham MQ, Leddy HA, Gimble JM, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials. July 2004;25(16):3211-3222.
2011	Marsell R, Einhorn TA. The biology of fracture healing. Injury. June 2011;42(6):551-555.
2006	Krishnan V, Bryant HU, MacDougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest. May 2006;116(5):1202-1209.
2011	Price C, Zhou X, Li W, Wang L. Real‐time measurement of solute transport within the lacunar‐canalicular system of mechanically loaded bone: direct evidence for load‐induced fluid flow. J Bone Miner Res. February 2011;26(2):277-285.
2007	Taylor AF, Saunders MM, Shingle DL, Cimbala JM, Zhou Z, Donahue HJ. Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions. Am J Physiol Cell Physiol. January 2007;292(1):C545-C552.
2000	Donahue HJ. Gap junctions and biophysical regulation of bone cell differentiation. Bone. May 2000;26(5):417-422.
2005	Hill TP, Später D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell. May 2005;8(5):727-738.
1999	Zaman G, Pitsillides AA, Rawlinson SCF, Suswillo RFL, Mosley JR, Cheng MZ, Platts LAM, Hukkanen M, Polak JM, Lanyon LE. Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes. J Bone Miner Res. July 1999;14(7):1123-1131.
2003	Srinivasan S, Agans SC, King KA, Moy NY, Poliachik SL, Gross TS. Enabling bone formation in the aged skeleton via rest-inserted mechanical loading. Bone. December 2003;33(6):946-955.
1985	Goodship AE, Kenwright J. The influence of induced micromovement upon the healing of experimental tibial fractures. J Bone Joint Surg. 1985;67B(4):650-655.
2002	Srinivasan S, Weimer DA, Agans SC, Bain SD, Gross TS. Low‐magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle. J Bone Miner Res. September 2002;17(9):1613-1620.
2006	Hadjidakis DJ, Androulakis II. Bone remodeling. Annals NY Acad Sci. December 2006;1092(1):385-396.
2004	You L, Weinbaum S, Cowin SC, Schaffler MB. Ultrastructure of the osteocyte process and its pericellular matrix. Anat Rec. June 2004;278A(2):505-513.
2014	Sims NA, Martin TJ. Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit. BoneKEy Rep. January 2014;3:481.
1996	Johnson DL, McAllister TN, Frangos JA. Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts. Am J Physiol Endocrinol Metab. July 1996;271(1):E205-E208.
2007	Genetos DC, Kephart CJ, Zhang Y, Yellowley CE, Donahue HJ. Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO‐Y4 osteocytes. J Cell Physiol. July 2007;212(1):207-214.
2005	Genetos DC, Geist DJ, Liu D, Donahue HJ, Duncan RL. Fluid shear‐induced ATP secretion mediates prostaglandin release in MC3T3‐E1 osteoblasts. J Bone Miner Res. January 2005;20(1):41-49.

Year

Entry

2009

Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, Chen CS. Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell. July 2, 2009;5(1):17-26.

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.

2005

Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/β-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell. May 2005;8(5):739-750.

2005

Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater. July 2005;4(7):518-524.

2000

Knothe Tate M, Knothe U. An ex vivo model to study transport processes and fluid flow in loaded bone. J Biomech. February 2000;33(2):247-254.

2003

Cherian PP, Cheng B, Gu S, Sprague E, Bonewald LF, Jiang JX. Effects of mechanical strain on the function of gap junctions in osteocytes are mediated through the prostaglandin EP₂ receptor. J Biol Chem. October 31, 2003;278(44):43146-43156.

1999

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

2001

Bakker AD, Soejima K, Klein-Nulend J, Burger EH. The production of nitric oxide and prostaglandin E₂ by primary bone cells is shear stress dependent. J Biomech. May 2001;34(5):671-677.

2006

Rubin J, Rubin C, Jacobs CR. Molecular pathways mediating mechanical signaling in bone. Gene. February 15, 2006;367:1-16.

2000

Teitelbaum SL. Bone resorption by osteoclasts. Science. September 1, 2000;289(5484):1504-1508.

2008

Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C_T method. Nat Protoc. 2008;3(6):1101-1108.

2013

Hazenbiller O. Micro-Environmental Factors Directing Differentiation of Murine Embryonic Stem Cells Down Osteogenic and Chondrogenic Lineages [Master's thesis]. Calgary, AB: University of Calgary; February 2013.

1978

McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg. May 1978;60B(2):150-162.

2001

Cheng B, Zhao S, Luo J, Sprague E, Bonewald LF, Jiang JX. Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells. J Bone Miner Res. February 2001;16(2):249-259.

2005

Wang L, Wang Y, Han Y, Henderson SC, Majeska RJ, Weinbaum S, Schaffler MB. In situ measurement of solute transport in the bone lacunar-canalicular system. Proc Natl Acad Sci USA. August 16, 2005;102(46):11911-11916.

2004

Awad HA, Wickham MQ, Leddy HA, Gimble JM, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials. July 2004;25(16):3211-3222.

2011

Marsell R, Einhorn TA. The biology of fracture healing. Injury. June 2011;42(6):551-555.

2006

Krishnan V, Bryant HU, MacDougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest. May 2006;116(5):1202-1209.

2011

Price C, Zhou X, Li W, Wang L. Real‐time measurement of solute transport within the lacunar‐canalicular system of mechanically loaded bone: direct evidence for load‐induced fluid flow. J Bone Miner Res. February 2011;26(2):277-285.

2007

Taylor AF, Saunders MM, Shingle DL, Cimbala JM, Zhou Z, Donahue HJ. Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions. Am J Physiol Cell Physiol. January 2007;292(1):C545-C552.

2000

Donahue HJ. Gap junctions and biophysical regulation of bone cell differentiation. Bone. May 2000;26(5):417-422.

2005

Hill TP, Später D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell. May 2005;8(5):727-738.

1999

Zaman G, Pitsillides AA, Rawlinson SCF, Suswillo RFL, Mosley JR, Cheng MZ, Platts LAM, Hukkanen M, Polak JM, Lanyon LE. Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes. J Bone Miner Res. July 1999;14(7):1123-1131.

2003

Srinivasan S, Agans SC, King KA, Moy NY, Poliachik SL, Gross TS. Enabling bone formation in the aged skeleton via rest-inserted mechanical loading. Bone. December 2003;33(6):946-955.

1985

Goodship AE, Kenwright J. The influence of induced micromovement upon the healing of experimental tibial fractures. J Bone Joint Surg. 1985;67B(4):650-655.

2002

Srinivasan S, Weimer DA, Agans SC, Bain SD, Gross TS. Low‐magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle. J Bone Miner Res. September 2002;17(9):1613-1620.

2006

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

2004

You L, Weinbaum S, Cowin SC, Schaffler MB. Ultrastructure of the osteocyte process and its pericellular matrix. Anat Rec. June 2004;278A(2):505-513.

2014

Sims NA, Martin TJ. Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit. BoneKEy Rep. January 2014;3:481.

1996

Johnson DL, McAllister TN, Frangos JA. Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts. Am J Physiol Endocrinol Metab. July 1996;271(1):E205-E208.

2007

Genetos DC, Kephart CJ, Zhang Y, Yellowley CE, Donahue HJ. Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO‐Y4 osteocytes. J Cell Physiol. July 2007;212(1):207-214.

2005

Genetos DC, Geist DJ, Liu D, Donahue HJ, Duncan RL. Fluid shear‐induced ATP secretion mediates prostaglandin release in MC3T3‐E1 osteoblasts. J Bone Miner Res. January 2005;20(1):41-49.

Year	Entry
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.
2019	Ferrie LE. The Contribution of Endogenous and Exogenous Stem Cells in Fracture Repair [Master's thesis]. Calgary, AB: University of Calgary; December 2019.

Year

Entry

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.

2019

Ferrie LE. The Contribution of Endogenous and Exogenous Stem Cells in Fracture Repair [Master's thesis]. Calgary, AB: University of Calgary; December 2019.