High affinity binding of an engineered, modular peptide to bone tissue

Abstract

Bone grafting procedures have become common due in part to a global trend of population aging. Native bone graft is a popular choice when compared to various synthetic bone graft substitutes, owing to superior biological activity. Nonetheless, the insufficient ability of bone allograft to induce new bone formation and the insufficient remodeling of native bone grafts call for osteoinductive factors during bone repair, exemplified by recombinant human bone morphogenetic protein 2 (rhBMP2). We previously developed a modular bone morphogenetic peptide (mBMP) to address complications associated with the clinical use of rhBMP2 as a bone graft substitute. The mBMP is designed to strongly bind to hydroxyapatite, the main inorganic component of bone and teeth, and to provide pro-osteogenic properties analogous to rhBMP2. Our previous in vivo animal studies showed that mBMP bound to hydroxyapatite-coated orthopedic implants with high affinity and stimulated new bone formation. In this study, we demonstrate specific binding of mBMP to native bone grafts. The results show that mBMP binds with high affinity to both cortical and trabecular bones, and that the binding is dependent on the mBMP concentration and incubation time. Importantly, efficient mBMP binding is also achieved in an ex vivo bone bioreactor where bone tissue is maintained viable for several weeks. In addition, mBMP binding can be localized with spatial control on native bone tissue via simple methods, such as dip-coating, spotting, and direct writing. Taken together with the pro-osteogenic activity of mBMP established in previous bone repair models, these results suggest that mBMP may promote bone healing when coated on native bone grafts in a clinically compatible manner.

Figure 1

Fluorescence images of cortical bones after incubating in rhodamine-labeled modular peptide solution with different concentrations for different time periods. Green and red fluorescence were emitted from native bone and rhodamine, respectively.

Figure 2

(a) Amount of rhodamine-labeled mBMP bond to cortical bone after incubating in various conditions. Quantification of fluorescence intensity of (b) rhodamine-labeled mBMP bound to cortical bone by incubating in various conditions, and (c) rhodamine-labeled mBMP and mBMP-mut bound to cortical bone by incubating in 100 µg/mL peptide solution for different time periods. Data are shown as mean ± standard deviation. * p < 0.01 and ** p < 0.05.

Figure 3

(a) Fluorescence images and (b) fluorescence intensity of modular peptide-bound cortical bones after incubating in simulated body fluid for different time periods. Data are shown as mean ± standard deviation.

Figure 4

(a) Fluorescence images and (b) fluorescence intensity of trabecular bone cores after incubating in rhodamine-labeled mBMP solution for different time periods in a bone bioreactor. Data are shown as mean ± standard deviation. ** p < 0.05.

Figure 5

Fluorescence images of (a) cortical bone and (b) trabecular bone dip-coated in rhodamine-labeled mBMP solution. (c) Fluorescence image of cortical bone spotted with rhodamine-labeled mBMP solution with different concentrations: (i) 6.25, (ii) 12.5, (iii) 25, (iv) 50, (v) 100 and (vi) 200 µg/mL. (d) Fluorescence image of “UW” written with rhodamine-labeled mBMP solution on cortical bone.