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

摘要

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 RGP 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 RGP peptide GCRGYGRGDSPG (PLL-g-PEG/PEG-RGP) 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 percent of the polymer sidechains were functionalized with the RGD peptide or the RDG peptide. To examine RGP 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-RDG at an RGP 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 in-creased on PLL-g-PEG clone 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 RGP peptide restored these markers to their control levels. PLL-g-PEG coatings also in-creased 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.