Modular Peptide Growth Factors for Substrate‐Mediated Stem Cell Differentiation

Abstract

Natural proteins are often multifunctional, and therefore capable of activating cell surface receptors, and also binding with high affinity and specificity to natural extracellular matrices (ECMs). To achieve these diverse functions, a strategy commonly employed by nature involves creating modular proteins, in which distinct domains within a single protein are designed to enable either cell signaling or ECM binding. For example, modular proteins such as osteocalcin (OCN) and bone sialoprotein (BSP) contain a domain that binds to hydroxyapatite (HA) -the major biomineral component in the ECM of bony tissues -and a distinct domain that interacts with integrin receptors to mediate cell adhesion.[1] Therefore, these proteins are capable of influencing cell behavior in particular locations within an organism by virtue of their non-covalent linkage to a specific ECM material.The mechanisms that enable binding of signaling molecules to ECM in nature can potentially be extended to synthetic biomaterials as well. For example, a recent study indicates that it is possible to mimic nature's modular cell adhesion proteins (e.g. OCN, BSP) by engineering synthetic modular peptide molecules that bind to synthetic HA, yet remain capable of affecting cell adhesion. [2] This modular design approach has been used to promote cell adhesion to HA materials, which are now used in a wide range of common clinical orthopedic applications. However, previous studies have not yet extended this type of modular design strategy in order to non-covalently immobilize growth factors, which are capable of actively regulating stem cell phenotype.We hypothesized that modular peptides inspired by portions of natural proteins could provide a mechanism to decorate common biomedical materials with growth factors. We reasoned that the surface-immobilized peptide growth factor could then promote stem cell differentiation on the material surface. Specifically, we synthesized modular peptide growth factors, which mimic the HA-binding ability of OCN and the ability of bone morphogenetic protein-2 (BMP-2) to promote stem cell differentiation. OCN is a 5.7 kDa protein that binds to calcium in the HA crystal lattice (600 nM dissociation constant).[3] HA-OCN binding can be largely attributed a 9-mer sequence on the N-terminus, which contains three γ-carboxylated glutamic acid (Gla) residues that coordinate with Ca 2+ ions in the HA crystal. [4] Therefore, we reasoned that the N-terminal helix derived from OCN could be used as a linker to attach BMP-2 to a HA surface. BMP-2 is a 26 kDa protein that exerts its effects by stimulating differentiation of progenitor cells toward an osteoblastic lineage. [5,6] Recently Tanihara and coworkers discovered that a 20 amino acid peptide sequence from the "knuckle" epitope of BMP-2 retains the biological activity of the full-length BMP-2 protein.[7-10] Therefore, we hypothesized that a modular peptide containing an OCN-inspired portion and the 20-mer derived from the BMP-2 knuckle epitope could be used to promote substrate-med...

Osteocalcin Biomimic Recognizes Bone Hydroxyapatite

“…All peptides investigated in this study were prepared following reported protocols with minor modifications. [13,14] Detail experimental information about materials and methods is described in the Supporting Information. Binding affinity and release kinetics of HABPs with HA were evaluated by incubating various amounts of peptides (0-20 mm) with HA (5 mg mL À1 ) solution.…”

“…CD spectra were collected to confirm conformational changes of HABPs in the absence or presence of HA. [7,13] For binding competition assay of HABP-19 and PAM, HA (5 mg mL À1 ) was incubated in mixtures that were composed of HABP-19 (10 mm) and PAM (0-200 mm). Cy-HABP-19 was engineered by conjugating Cy5.5 with Cys-containing HABP-19 peptide.…”

“…Even in the presence of 200 mm CaCl 2 solution, they did not form an a-helical structure (Figure 2 E and Figure S3 in the Supporting Information). [13] In the presence of HA (0.2 mg mL À1 ) HABP-19 showed the characteristic spectrum of an a-helical structure with negative mean ellipticity at 208 and 222 nm. As expected, HABP-10 and cHABP-19 did not exhibit any noticeable CD spectrum change.…”

“…In contrast, neither cHABP-10 nor cHABP-19 showed appreciable HA binding (Figure 2 A and B); this supports the critical role of Gla residues in HA binding, as previously reported. [7,13,14] To confirm the selectivity with HA, HABP-19 was validated with a panel of calcium salts: calcium carbonate (CC), calcium oxalate (CO), calcium phosphate (CP), calcium pyrophosphate (CPP) and HA, under the same experimental condition. HABP-19 exhibited exceptional binding affinity with HA but not to other tested calcium salts (P < 0.005; Figure 2 C and D).…”

Scaffold Translation: Barriers Between Concept and Clinic

“…Our recent experience with nanostructured mineral coatings that bind to biologics provides one example of the utility of a modular approach. [124][125][126][127][128] We have also developed molecular coatings that can induce bone formation, 108,[128][129][130][131] and the interaction between the osteoinductive molecule and the scaffold can be used to dictate the release rate. In each case, the biologics delivery is decoupled from device physical properties, thus allowing for independent optimization of each component in a modular scaffold.…”