The Role of Osteopontin in Postnatal Vascular Growth: Functional Effects in Ischemic Limb Collateral Vessel Formation and Long Bone Fracture Healing

Postnatal vascular growth is a complex process involving multiple cells types whose functionality is orchestrated by a variety of soluble extracellular growth factors, mechanical stimuli, and matrix derived cues. The central goal for this dissertation project was to elucidate the role of osteopontin, a non-collagenous extracellular matrix protein, in postnatal vascular growth.

At the onset, we concluded that the current methods for measurement of vascularity in small animal models were lacking. To address this shortcoming, we pursued micro-CT imaging for analysis of three-dimensional blood vessel architecture. We were able to demonstrate that micro-CT imaging provides an objective, quantitative, and three-dimensional methodology for evaluation of vascular networks that has broad applicability to preclinical studies.

Next, we sought to apply the developed imaging techniques along with other complementary methodologies to explore the role of osteopontin in postnatal vascular growth. Osteopontin was previously known to elicit survival, migration, and other relevant activities in multiple cell types involved in postnatal vascular growth. Therefore, we sought to determine the in vivo significance of osteopontin in this process. To do so, we compared wild type and Osteopontin^-/- mice for (1) their ability to form collateral vessels and functionally recover following acute induction of hind limb ischemia and (2) their capacity for neovascularization, mineralization, remodeling, and the restoration of mechanical properties during fracture healing. Data suggested that OPN is a critical regulator of collateral vessel formation and that this effect is driven by its role in mediating monocyte/macrophage migration and functionality. Secondly, we found that the presence of osteopontin was essential for normal early callus formation, neovascularization, late stage callus remodeling, and restoration of biomechanical strength. Abnormal collagen organization was observed within the remodeling fractures of Osteopontin^-/- mice, and we hypothesize that a unifying link between the vascular and bone defects may be related to deficient matrix organization and remodeling.

In conclusion, the imaging techniques developed in this thesis provide a novel methodology for quantitative analysis of vascular structures in small animal models. Secondly, this project has yielded an improved understanding of the basic pathophysiological mechanisms that control postnatal blood vessel growth and bone fracture healing.

Year	Entry
1998	Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res. October 1998;355(suppl):S7-S21.
1998	Yasuda H, Shima N, Akagawa NN, Yamaguchi K, Kinosaki M, Mochizuki S-I, Omoyasu AT, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Igashio KH, Udagawa N, Tkahashi N, Suda T. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA. March 31, 1998;95(7):3597-3602.
2003	Amblard D, Lafage-Proust M-H, Laib A, Thomas T, Rüegsegger P, Alexandre C, Vico L. Tail suspension induces bone loss in skeletally mature mice in the C57BL/6J strain but not in the C3H/HeJ strain. J Bone Miner Res. March 2003;18(3):561-569.
1984	Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Res. 1984;2(1):97-101.
2001	Le AX, Miclau T, Hu D, Helms JA. Molecular aspects of healing in stabilized and non-stabilized fractures. J Orthop Res. January 2001;19(1):78-84.
2001	Jepsen KJ, Pennington DE, Lee Y, Warman M, Nadeau J. Bone brittleness varies with genetic background in A/J and C57BL/6J inbred mice. J Bone Miner Res. October 2001;16(10):1854-1862.
1961	Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys. May 1961;93(2):440-447.
1999	Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulate embryonic skeletal formation? Mech Dev. September 1999;87(1-2):57-66.
1978	McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg. May 1978;60B(2):150-162.
2002	Deckers MML, van Bezooijen RL, van der Horst G, Hoogendam J, van der Bent C, Papapoulos SE, Löwik CWGM. Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology. April 2002;143(4):1545-1553.
1979	Junqueira LCU, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histchem J. 1979;11:447-455.
2002	Boskey AL, Spevak L, Paschalis E, Doty SB, McKee MD. Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone. Calcif Tiss Int. August 2002;71(2):145-154.
1997	Hildebrand T, Rüegsegger P. A new method for the model-independent assessment of thickness in three-dimensional images. J Microsc (Oxford). 1997;185(1):67-75.
1984	Feldkamp LA, Davis LC, Kress JW. Practical cone-beam algorithm. J Opt Soc Am. 1984;A1(6):612-619.
2001	Thompson JB, Kindt JH, Drake B, Hansma HG, Morse DE, Hansma PK. Bone indentation recovery time correlates with bond reforming time. Nature. December 13, 2001;414(6865):773-776.
1989	Feldkamp LA, Goldstein SA, Parfitt MA, Jesion G, Kleerekoper M. The direct examination of three‐dimensional bone architecture in vitro by computed tomography. J Bone Miner Res. 1989;4(1):3-11.
2001	Kon T, Cho T-J, Aizawa T, Yamazaki M, Nooh N, Graves D, Gerstenfeld LC, Einhorn TA. Expression of osteoprotegerin, receptor activator of NF-κB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res. June 2001;16(6):1004-1014.
2004	Duvall CL, Taylor WR, Weiss D, Guldberg RE. Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury. Am J Physiol Heart Circ Physiol. July 2004;287(1):H302-H310.
1999	Hildebrand T, Laib A, Müller R, Dequeker J, Rüegsegger P. Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res. July 1999;14(7):1167-1174.
2002	Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, van Bruggen N, Redmond HP, Carano RAD, Filvaroff EH. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA. July 23, 2002;99(15):9656-9661.
1993	Odgaard A, Gundersen HJG. Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions. Bone. March–April 1993;14(2):173-182.
2001	Hausman MR, Schaffler MB, Majeska RJ. Prevention of fracture healing in rats by an inhibitor of angiogenesis. Bone. December 2001;29(6):560-564.
2005	Fantner GE, Hassenkam T, Kindt JH, Weaver JC, Birkedal H, Pechenik L, Cutroni JA, Cidade GAG, Stucky GD, Morse DE, Hansma PK. Sacrificial bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture. Nat Mater. August 2005;4(8):612-616.
1973	Hounsfield GN. Computerized transverse axial scanning (tomography), I: description of system. Br J Radiol. 1973;46:1016-1022.
2005	Hansma PK, Fantner GE, Kindt JH, Thurner PJ, Schitter G, Turner PJ, Udwin SF, Finch MM. Sacrificial bonds in the interfibrillar matrix of bone. J Musculoskel Neuron Interact. October–December 2005;5(4):313-315.

Year

Entry

1998

Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res. October 1998;355(suppl):S7-S21.

1998

Yasuda H, Shima N, Akagawa NN, Yamaguchi K, Kinosaki M, Mochizuki S-I, Omoyasu AT, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Igashio KH, Udagawa N, Tkahashi N, Suda T. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA. March 31, 1998;95(7):3597-3602.

2003

Amblard D, Lafage-Proust M-H, Laib A, Thomas T, Rüegsegger P, Alexandre C, Vico L. Tail suspension induces bone loss in skeletally mature mice in the C57BL/6J strain but not in the C3H/HeJ strain. J Bone Miner Res. March 2003;18(3):561-569.

1984

Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Res. 1984;2(1):97-101.

2001

Le AX, Miclau T, Hu D, Helms JA. Molecular aspects of healing in stabilized and non-stabilized fractures. J Orthop Res. January 2001;19(1):78-84.

2001

Jepsen KJ, Pennington DE, Lee Y, Warman M, Nadeau J. Bone brittleness varies with genetic background in A/J and C57BL/6J inbred mice. J Bone Miner Res. October 2001;16(10):1854-1862.

1961

Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys. May 1961;93(2):440-447.

1999

Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulate embryonic skeletal formation? Mech Dev. September 1999;87(1-2):57-66.

1978

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

2002

Deckers MML, van Bezooijen RL, van der Horst G, Hoogendam J, van der Bent C, Papapoulos SE, Löwik CWGM. Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology. April 2002;143(4):1545-1553.

1979

Junqueira LCU, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histchem J. 1979;11:447-455.

2002

Boskey AL, Spevak L, Paschalis E, Doty SB, McKee MD. Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone. Calcif Tiss Int. August 2002;71(2):145-154.

1997

Hildebrand T, Rüegsegger P. A new method for the model-independent assessment of thickness in three-dimensional images. J Microsc (Oxford). 1997;185(1):67-75.

1984

Feldkamp LA, Davis LC, Kress JW. Practical cone-beam algorithm. J Opt Soc Am. 1984;A1(6):612-619.

2001

Thompson JB, Kindt JH, Drake B, Hansma HG, Morse DE, Hansma PK. Bone indentation recovery time correlates with bond reforming time. Nature. December 13, 2001;414(6865):773-776.

1989

Feldkamp LA, Goldstein SA, Parfitt MA, Jesion G, Kleerekoper M. The direct examination of three‐dimensional bone architecture in vitro by computed tomography. J Bone Miner Res. 1989;4(1):3-11.

2001

Kon T, Cho T-J, Aizawa T, Yamazaki M, Nooh N, Graves D, Gerstenfeld LC, Einhorn TA. Expression of osteoprotegerin, receptor activator of NF-κB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res. June 2001;16(6):1004-1014.

2004

Duvall CL, Taylor WR, Weiss D, Guldberg RE. Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury. Am J Physiol Heart Circ Physiol. July 2004;287(1):H302-H310.

1999

Hildebrand T, Laib A, Müller R, Dequeker J, Rüegsegger P. Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res. July 1999;14(7):1167-1174.

2002

Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, van Bruggen N, Redmond HP, Carano RAD, Filvaroff EH. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA. July 23, 2002;99(15):9656-9661.

1993

Odgaard A, Gundersen HJG. Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions. Bone. March–April 1993;14(2):173-182.

2001

Hausman MR, Schaffler MB, Majeska RJ. Prevention of fracture healing in rats by an inhibitor of angiogenesis. Bone. December 2001;29(6):560-564.

2005

Fantner GE, Hassenkam T, Kindt JH, Weaver JC, Birkedal H, Pechenik L, Cutroni JA, Cidade GAG, Stucky GD, Morse DE, Hansma PK. Sacrificial bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture. Nat Mater. August 2005;4(8):612-616.

1973

Hounsfield GN. Computerized transverse axial scanning (tomography), I: description of system. Br J Radiol. 1973;46:1016-1022.

2005

Hansma PK, Fantner GE, Kindt JH, Thurner PJ, Schitter G, Turner PJ, Udwin SF, Finch MM. Sacrificial bonds in the interfibrillar matrix of bone. J Musculoskel Neuron Interact. October–December 2005;5(4):313-315.

Year	Entry
2009	Kolambkar YM. Electrospun Nanofiber Meshes for the Functional Repair of Bone Defects [PhD thesis]. Atlanta, GA: Georgia Institute of Technology; December 2009.

Year

Entry

2009

Kolambkar YM. Electrospun Nanofiber Meshes for the Functional Repair of Bone Defects [PhD thesis]. Atlanta, GA: Georgia Institute of Technology; December 2009.