Stable biofunctionalization of hydroxyapatite (HA) surfaces by HA-binding/osteogenic modular peptides for inducing osteogenic differentiation of mesenchymal stem cells

Abstract

Hydroxyapatite (HA), the principal component of bone mineral, shows osteoconductive properties when employed for coating metal implants as well as scaffold materials in synthetic bone grafts. With the goal of providing this material with osteoinductive capabilities to promote faster bone regeneration, we show an easy approach to functionalize HA implant surfaces and enrich them with osteoinductive properties by the use of HA-binding modular peptides. The modular peptides are designed as a combination of two domains, an HA-binding peptide motif and an osteogenic peptide motif derived from the osteogenic growth peptide (OGP) or bone morphometric protein 7 (BMP-7). To identify the best HA-binding peptide, several nature-inspired peptides derived from natural bone extracellular matrix proteins (bone sialoprotein, osteonectin, osteocalcin, and salivarin statherin) were compared for HA-binding activity, revealing concentration-dependent and incubation-time-dependent behaviours. We discovered that a Poly-E heptamer (E7) is the best HA-binding peptide, and thus combined it with a second osteogenic peptidic domain to create an osteoinductive modular peptide. After binding/release characterization, we found that the addition of the second osteogenic peptide domain did not change the binding profile of the modular peptides and caused only a slight change in their release kinetics. Mesenchymal stem cells (MSCs) were cultured on the HA substrates functionalized with modular peptides, and cell adhesion, proliferation, and differentiation in a basal medium (i.e., without any osteogenic supplements) were investigated. Gene expression data clearly showed that MSCs were committed to differentiate into osteoblasts in the presence of the modular peptides. HA discs functionalized with the E7 BMP-7 modular peptide showed the best capability in inducing the osteogenic differentiation of MSCs among all modular peptides studied. The modular peptides can easily be used to functionalize the HA implants through its constituent HA-binding motif, leaving the osteogenic peptide motif protruding from the surface for inducing osteogenesis. Our work opens up a new approach to the formulation of new bioactive HA coatings and implants for bone and dental repair.

Adding Functions to Biomaterial Surfaces through Protein Incorporation

“…It has also been shown to direct neurite sprouting and growth on material supports, [296] which has been widely investigated to guide axonal regeneration in bioengineering of nerve graft substitutes. [297] Additional applicationspecific sequences include osteogenic growth peptide or peptides derived from bone morphometric proteins that are used to generate bioactive surfaces with improved osteoinductive properties, [298] hyaluronic acid-binding motifs used to increase cartilage lubrication in joints [299] and coat medical devices, a complement linked factor H-binding peptide for regulation of inflammation on glass-and polystyrene-based surfaces, [300] collagen mimetic peptides such as GPP 7 , [301] and the angiogenic VEGF receptor-binding QK peptide. [302] A multitude of cellspecific peptide sequences have also been identified for tagging of drug carriers or imaging vehicles for delivery to specific cell types in vivo of course, [303] with increasing numbers of these motifs likely to continue to become available due to technological advances in library synthesis and screening methods that ease their identification.…”

Peptide-biofunctionalization of biomaterials for osteochondral tissue regeneration in early stage osteoarthritis: challenges and opportunities

“…The (Table 2). 73,[87][88][89][90][91][92][93][94][95] Besides their success, GFs present some limitations regarding their low protein stability, short circulating half-life, the rapid rate of cellular internalization and side effects. Therefore, alternatives to the use of the whole proteins have been investigated due to the evidence that using smaller GF fragments or mimetic peptides motivates receptor-mediated signal transduction.…”

Osteogenic growth peptide and its use as a bio‐conjugate in regenerative medicine applications

“…Along with titanium, hydroxyapatite (HA) is one of the most widely utilized implants for clinical bone repair because of both its compositional and structural similarity to natural bone . Recently, HA‐based materials were functionalized with modular peptides which contained both an HA‐binding domain and a cell‐responsive OGP motif . Compared to a non‐osteogenic control, modular OGP‐peptide functionalized HA displayed an upregulation of OSC, osteopontin (OPN), and RUNX2 mRNA expression for r MSCs after 2 weeks in vitro .…”

“…Recently, HA‐based materials were functionalized with modular peptides which contained both an HA‐binding domain and a cell‐responsive OGP motif . Compared to a non‐osteogenic control, modular OGP‐peptide functionalized HA displayed an upregulation of OSC, osteopontin (OPN), and RUNX2 mRNA expression for r MSCs after 2 weeks in vitro . Additionally, proliferation of r MSCs on the OGP‐functionalized surfaces decreased within the first 7 days of in vitro culture, suggesting that these cells had entered the differentiation phase earlier than those on the non‐osteogenic control surfaces …”