RGD‐containing peptide GCRGYGRGDSPG reduces enhancement of osteoblast differentiation by poly(L‐lysine)‐graft‐poly(ethylene glycol)‐coated titanium surfaces

Abstract

Osteoblasts exhibit a more differentiated morphology on surfaces with rough microtopographies. Surface effects are often mediated through integrins that bind the RGD motif in cell attachment proteins. Here, we tested the hypothesis that modulating access to RGD binding sites can modify the response of osteoblasts to surface microtopography. MG63 immature osteoblast-like cells were cultured on smooth (Ti sputter-coated Si wafers) and rough (grit blasted/acid etched) Ti surfaces that were modified with adsorbed monomolecular layers of a comb-like graft copolymer, poly-(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), to limit nonspecific protein adsorption. PLL-g-PEG coatings were functionalized with varying amounts of an integrin-receptor-binding RGD peptide GCRGYGRGDSPG (PLL-g-PEG/PEG-RGD) or a nonbinding RDG control sequence GCRGYGRDGSPG (PLL-g-PEG/PEG-RDG). Response to PLL-g-PEG alone was compared with response to surfaces on which 2-18% of the polymer sidechains were functionalized with the RGD peptide or the RDG peptide. To examine RGD dose-response, peptide surface concentration was varied between 0 and 6.4 pmol/cm(2). In addition, cells were cultured on uncoated Ti or Ti coated with PLL-g-PEG or PLL-g-PEG/PEG-RGD at an RGD surface concentration of 0.7 pmol/cm(2), and free RGDS was added to the media to block integrin binding. Analyses were performed 24 h after cultures had achieved confluence on the tissue culture plastic surface. Cell number was reduced on smooth Ti compared to plastic or glass and further decreased on surfaces coated with PLL-g-PEG or PLL-g-PEG/PEG-RDG, but was restored to control levels when PLL-g-PEG/PEG-RGD was present. Alkaline phosphatase specific activity and osteocalcin levels were increased on PLL-g-PEG alone or PLL-g-PEG/PEG-RDG, but PLL-g-PEG/PEG-RGD reduced the parameters to control levels. On rough Ti surfaces, cell number was reduced to a greater extent than on smooth Ti. PLL-g-PEG coatings reduced alkaline phosphatase and increased osteocalcin in a manner that was synergistic with surface roughness. The RDG peptide did not alter the PLL-g-PEG effect but the RGD peptide restored these markers to their control levels. PLL-g-PEG coatings also increased TGF-beta1 and PGE(2) in conditioned media of cells cultured on smooth or rough Ti; there was a 20x increase on rough Ti coated with PLL-g-PEG. PLL-g-PEG effects were inhibited dose dependently by addition of the RGD peptide to the surface. Free RGDS did not decrease the effect elicited by PLL-g-PEG surfaces. These unexpected results suggest that PLL-g-PEG may have osteogenic properties, perhaps correlated with effects that alter cell attachment and spreading, and promote a more differentiated morphology.

Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

“…Related studies in our lab using PT and SLA substrates coated with polyethylene glycol and functionalized with an RGD-containing peptide demonstrated suggested that greater numbers of attachment sites might actually result in a less differentiated cell [18]. The behavior of MG63 cells on coated SLA could be restored to the behavior of these cells on PT and even tissue culture plastic simply by increasing the number of RGD peptides on the surface.…”

An RGD-restricted substrate interface is sufficient for the adhesion, growth and cartilage forming capacity of human chondrocytes

“…The uncertainties introduced by working with natural protein layers has driven the development and synthesis of materials which provide full control over the ligandreceptor interactions (Hubbell, 1999), not only for optimizing cell culture labware but also for the development of smart materials in regenerative medicine. To this end, RGD grafted poly-L-lysine-graftpoly(ethylene glycol) (PLL-g-PEG) copolymers offer an elegant solution to block non-specific protein adsorption from serum through their PEG compartment, while introducing biofunctionality via PEG chains grafted with a peptide that contains the cell adhesion sequence RGD (VandeVondele et al, 2003, for a schematic depiction see Fig. 1).…”

“…The biology of chondrocytes is highly influenced by interactions with specific extracellular matrix (ECM) molecules (Scully et al, 2001;van der Kraan et al, 2002). Thus, targeted modification of cell culture substrates with biological motives has the potential to improve HAC expansion strategies.…”

Osseointegration and Bioscience of Implant Surfaces - Current Concepts at Bone-Implant Interface

“…In particular, the RGD motif, as discussed before, is one of the most studied protein sequence capable of promoting cell adhesion and thereby initiating intracellular signalling cascades through multiple integrins including  v  3 and  5  1 (Ruoslahti, 1996). This motif is usually covalently attached to titanium using silanization (Bagno et al, 2007) or functionalized using polymer chemistry (Tosatti et al, 2004), and has been reported to increase osteoblast attachment and proliferation (Schuler et al, 2006). While several in vivo studies (Elmengaard et al, 2005;Kroese-Deutman et al, 2005) demonstrated better osseointegration results, others did not find any significant enhancement for the RGD functionalization (Petrie et al, 2008;Schliephake et al, 2009).…”