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

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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.

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