Controlling SIBLING protein orientation and conformation via native binding interactions
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Native bone tissue is comprised of a mixture of collagen, non-collagenous proteins, and hydroxyapatite (HA). The SIBLING (small integrin binding, N-linked glycoprotein) family of proteins is the primary group of non-collagenous proteins found in bone tissue, several of which have been identified ahead of the mineralized front of developing bone. By replicating the native binding interactions between collagen and the SIBLING family of proteins, it is believed that a new type of bone tissue scaffold can be developed. In this work, preliminary studies are conducted which suggest that both mineralization and cell growth can be enhanced by the addition of SIBLINGs to a collagen scaffold. The mineralization forming capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), were directly compared on a biomimetic collagen substrate. Collagen substrates were prepared with either BSP, DPP, or OPN and exposed to a simulated body fluid for 5, 10, and 24 hours. The resulting mineral morphology was assessed and mineralization was seen in the presence of all three proteins, but only minerals formed in the presence of DPP were morphologically similar to those seen in developing bone tissue. The accessibility of the cell binding RGD domain of DPP when bound to collagen was explored and compared to previous studies with BSP and OPN. A statistically significant difference was seen between cell binding on collagen coated TCPS and TCPS with bound DPP, and the cells exhibited a preference for plain TCPS over collagen coated TCPS. This suggests that the RGD sequence of DPP is less accessible when it is bound to collagen. The effects of SIBLINGs on the early stages of collagen fibrillogenesis were also investigated using a solution of collagen, a fibrillogenesis buffer, and one of the three SIBLINGs mentioned earlier. It was shown that BSP and OPN will accelerate the rate of fibril formation, while DPP slowed down the fibril formation process significantly. This preliminary work has shown that a collagen-SIBLING scaffold is capable of both promoting cell growth and mineralization that is morphologically similar to that found in vivo developing bone tissue, allowing for an acellular bone tissue scaffold. By replicating the native binding interactions between collagen and the SIBLING family of proteins it is thought that a novel type of biomimetic bone scaffold can be developed without the need of cells.