RESUMO
Bone loss occurs as a consequence of a variety of diseases as well as from traumatic injuries, and often requires therapeutic intervention. Strategies for repairing and replacing damaged and/or lost bone tissue include the use of biomaterials and medical implant devices with and without osteoinductive coatings. The soluble growth factor angiopoietin-1 (Ang-1) has been found to promote cell adhesion and survival in a range of cell types including cardiac myocytes, endothelial cells and fibroblasts through an integrin-dependent mechanism. Furthermore, the short sequence QHREDGS has been identified as the integrin-binding sequence of Ang-1 and as a synthetic peptide has been found to possess similar integrin-dependent effects as Ang-1 in the aforementioned cell types. Integrins have been implicated in osteoblast differentiation and bone mineralization, processes critical to bone regeneration. By binding integrins on the osteoblast surface, QHREDGS could promote cell survival and adhesion, as well as conceivably osteoblast differentiation and bone mineralization. Here we immobilized QHREDGS onto polyacrylate (PA)-coated titanium (Ti) plates and polyethylene glycol (PEG) hydrogels. The osteoblast differentiation marker, alkaline phosphatase, peaked in activity 4-12 days earlier on the QHREDGS-immobilized PA-coated Ti plates than on the unimmobilized, DGQESHR (scrambled)- and RGDS-immobilized surfaces. Significantly more bone matrix was deposited on the QHREDGS-immobilized Ti surface than on the other surfaces as determined by atomic force microscopy. The QHREDGS-immobilized hydrogels also had a significantly higher mineral-to-matrix (M/M) ratio determined by Fourier transform infrared spectroscopy. Alizarin Red S and von Kossa staining and quantification, and environmental scanning electron microscopy showed that while both the QHREDGS- and RGDS-immobilized surfaces had extensive mineralization relative to the unimmobilized and DGQESHR-immobilized surfaces, the mineralization was more considerable on the QHREDGS-immobilized surface, both with and without the induction of osteoblast differentiation. Finally, treatment of cell monolayers with soluble QHREDGS was demonstrated to upregulate osteogenic gene expression. Taken together, these results demonstrate that the QHREDGS peptide is osteoinductive, inducing osteoblast differentiation, bone matrix deposition and mineralization.
RESUMO
Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.
