(67g) Sibling-Collagen Scaffolds As a Novel Bone Tissue Engineering Material

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 acellular bone tissue engineering scaffold can be developed. In this work, we show preliminary studies which suggest that both mineralization and cell growth can be enhanced by the addition of SIBLINGs to a collagen scaffold.

The mineralization induction 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. ¹²⁵I radiolabeled adsorption isotherms were constructed for each of the SIBLING proteins on the collagen substrate and were then used to ensure equivalent amounts of were bound to each substrate in subsequent experiments. Collagen substrates were then 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 with both atomic force microscopy and colorimetric assays for Ca²⁺ and PO₄^3-. 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. ¹²⁵I adsorption isotherms were developed for DPP adsorbing to both tissue culture polystyrene (TCPS) and collagen coated TCPS to ensure equal amounts of DPP were bound to each substrate. An MC3T3-E1 cell adhesion assay was performed on TCPS and collagen-coated TCPS in the presence of identical amounts of adsorbed DPP. 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. A cell inhibition assay was performed to ensure that the results seen were due to interactions between the RGD domain of DPP and the MC3T3-E1 cell integrins.

The effects of SIBLINGs on the early stages of collagen fibrillogenesis were also investigated. Fibrillogenesis assays were performed in a 96-well plate using a solution of collagen, a fibrillogenesis buffer, and one of three SIBLINGs. Absorbance measurements were performed at 400 nm to quantify both the rate and extent of collagen fibrillogenesis in the presence of either BSP, DPP, or OPN.

This 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 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.