Beta 1 Integrins in Bone Formation During Development and Engineering Integrin-Specific Hydrogels for Enhanced Bone Healing

Links

Abstract

Bone formation is a complex physiological process which is orchestrated by multiple microenvironmental cues such as soluble factors, cell-cell interactions and the extracellular matrix. Integrins are heterodimeric transmembrane receptors consisting of α and β subunits which mediate cell interactions with the extracellular matrix. Beta 1 integrins encompass the majority of integrins and represent the main integrin binding partners of collagen I, the most abundant extracellular matrix component of bone. The central goals of this dissertation project were to elucidate the role of β1 integrins on bone development and healing in vivo, and to design biomimetic α2β1 integrin-specific polyethylene glycol hydrogels to enhance bone healing within segmental bone defects.

Because global β1 knockout mice are embryonically lethal, in order to study the role of β1 integrins in vivo, we used the Cre-Lox system to generate mice with conditional beta 1 integrin deletions in osteolineage cells at three stages: (1) mesodermal cells [under Twist 2/Dermo 1], (2) osteoprogenitors [under the osterix promoter] and (3) mature osteoblasts and osteocytes [under the osteocalcin promoter]. We found that β1 integrin deletion in mesodermal cells severely impaired pre-natal skeletal mineralization, particularly in the calvarium, and also resulted in late-stage embryonic lethality. In contrast, β1 integrin deletion in pre-osteoblasts resulted in viable but runted mice with decreased cranial mineralization, tooth defects, impaired femur development and some perinatal mortality. Finally, mice with β1 integrin null osteoblasts and osteocytes displayed very mild bone phenotypes with no change in femur fracture healing capacity. Taken together, these results suggest that β1 integrins play an important role in the early bone formation process but are not essential for the function of mature osteoblasts and osteocytes.

We also sought to engineer a biomimetic bone graft substitute by incorporating the following two bioactive components into a matrix metalloproteinase (MMP)-sensitive synthetic polyethylene glycol (PEG) hydrogel: (1) the collagen I-mimetic triple-helical synthetic ligand GFOGER, which specifically binds to the pro-osteogenic α2β1 integrin, and (2) recombinant human bone morphogenetic protein 2 (rhBMP-2). We synthesized PEG hydrogels incorporating GFOGER or the commonly used non-integrin selective adhesive peptide, RGD, in equimolar densities and studied hMSC differentiation responses to each of these surfaces. We then examined the effects of treating murine radial segmental defects with either GFOGER functionalized PEG-MAL hydrogels or GFOGER gels which also incorporated a low dose of rhBMP-2. Our data indicated that GFOGER hydrogels enhanced bone healing compared to empty defects and that incorporating low dose rhBMP-2 in GFOGER gels further improved bone formation. We evaluated the roles of the GFOGER ligand and the MMP-sensitive crosslinker, GCRDVPMSMRGGDRCG (VPM), in this response by comparing bone formation in defects treated with non-degradable hydrogels, degradable hydrogels lacking the GFOGER ligand, and in defects treated with degradable GFOGER hydrogels. Minimal bone formation occurred in response to PEG hydrogels which were not functionalized with any adhesive ligand and there was no bone formation in non-degradable PEG hydrogels, indicating that adhesive function and degradability are essential to bone regeneration in response to GFOGER hydrogels. Our examination of rhBMP-2 dose response within GFOGER hydrogels suggested that low 0.02mg/ml (0.03 μg) dose was sufficient for robust healing, but that the medium 0.04 mg/ml (0.06 μg) dose increased bone volume and mineral density within the defect compared to the low dose. The high 0.2 mg/ml (0.3 μg) BMP-2 dose induced less bone formation within the defect than the medium dose and altered the structure of the ulna so that it encircled the radius and fused with the radius. FMT analysis and in vitro BMP release assays revealed that GFOGER hydrogels provided sustained release of rhBMP-2. Finally, we evaluated the bone regeneration capacity of low dose rhBMP-2 delivery from GFOGER functionalized PEG hydrogels in comparison with collagen sponges, the clinical standard for BMP-2 delivery. We observed superior bone healing in response to GFOGER hydrogel treatment. In conclusion, our bioengineered integrin-specific hydrogel may be a promising bone graft substitute for the treatment of large bone defects.

Cited Works (17)

Year	Entry
2002	Cho T-J, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor β superfamily during murine fracture healing. J Bone Miner Res. March 2002;17(3):513-520.
2002	Zhang M, Xuan S, Bouxsein ML, von Stechow D, Akeno N, Faugere MC, Malluche H, Zhao G, Rosen CJ, Efstratiadis A, Clemens TL. Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem. November 15, 2002;277(46):44005-44012.
1997	Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsent G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. May 30, 1997;89(5):747-754.
2002	Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell. September 20, 2002;110(6):673-687.
2002	Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell. April 2002;2(4):389-406.
2007	Buie HR, Campbell GM, Klinck RJ, MacNeil JA, Boyd SK. Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis. Bone. October 2007;41(4):505-515.
2003	Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as a post‐natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem. April 2003;88(5):873-884.
1999	Giancotti FG, Ruoslahti E. Integrin signaling. Science. August 13, 1999;285(5430):1028-1033.
2010	Litzenberger JB, Kim J-B, Tummala P, Jacobs CR. Β₁ integrins mediate mechanosensitive signaling pathways in osteocytes. Calcif Tiss Int. April 2010;86(4):325-332.
2006	Tsuji K, Bandyopadhyay A, Harfe BD, Cox K, Kakar S, Gerstenfeld L, Einhorn T, Tabin CJ, Rosen V. BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet. December 2006;38(12):1424-1429.
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.
2002	Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. January 11, 2002;108(1):17-29.
1997	Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y-H, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. May 30, 1997;89(5):755-764.
1997	Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science. May 30, 1997;276(5317):1425.
1997	Salter DM, Robb JE, Wright MO. Electrophysiological responses of human bone cells to mechanical stimulation: evidence for specific integrin function in mechanotransduction. J Bone Miner Res. July 1997;12(7):1133-1141.
2007	Duvall CL, Taylor WR, Weiss D, Wojtowicz AM, Guldberg RE. Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin‐deficient mice. J Bone Miner Res. February 2007;22(2):286-297.
2003	Lutolf MP, Lauer-Fields JL, Schmoekel HG, Metters AT, Weber FE, Fields GB, Hubbell JA. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. Proc Natl Acad Sci USA. April 29, 2003;100(9):5413-5418.