Osteoblast response to titanium surfaces functionalized with extracellular matrix peptide biomimetics

Abstract

Objective: Functionalizing surfaces with specific peptides may aid osteointegration of orthopedic implants by favoring attachment of osteoprogenitor cells and promoting osteoblastic differentiation. This study addressed the hypothesis that implant surfaces functionalized with peptides targeting multiple ligands will enhance osteoblast attachment and/or differentiation. To test this hypothesis, we used titanium (Ti) surfaces coated with poly-l-lysine-grafted polyethylene glycol (PLL-g-PEG) and functionalized with two peptides found in extracellular matrix proteins, arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), which have been shown to increase osteoblast attachment. KSSR, which does not promote osteoblast attachment, was used as a control.

Materials and methods: Sandblasted acid-etched titanium surfaces were coated with PLL-g-PEG functionalized with varying combinations of RGD and KRSR, as well as KSSR. Effects of these surfaces on osteoblasts were assessed by measuring cell number, alkaline phosphatase-specific activity, and levels of osteocalcin, transforming growth factor beta-1 (TGF-β1), and PGE(2).

Results: RGD increased cell number, but decreased markers for osteoblast differentiation. KRSR alone had no effect on cell number, but decreased levels of TGF-β1 and PGE(2). KRSR and RGD/KRSR coatings inhibited osteoblast differentiation vs. PLL-g-PEG. KSSR decreased cell number and increased osteoblast differentiation, indicated by increased levels of osteocalcin and PGE(2).

Conclusions: The RGD and KRSR functionalized surfaces supported attachment but did not enhance osteoblast differentiation, whereas KSSR increased differentiation. RGD decreased this effect, suggesting that multifunctional peptide surfaces can be designed that improve peri-implant healing by optimizing attachment and proliferation as well as differentiation of osteoblasts, but peptide combination, dose and presentation are critical variables.

Fig. 1

(a) Schematic of peptide-functionalized poly-L-Lysine grafted polyethylene glycol (PLL-g-PEG) following self-assembly on the titanium SLA surface. (b) Array showing the experimental groups used in this study to evaluate the interaction of RGD and KRSR. The large gray dots represent the previous study by Tosatti et al. (Tosatti et al. 2004) while the black dots represent groups used in the present study. Values are measured in pmol/cm². The dose-dependent response to RGD was measured with the following experimental groups coated on titanium SLA: (4) PLL-g-PEG/PEGRGD(0.05) and (5) PLL-g-PEG/PEG-RGD(1.26). The response to KSSR was measured with the following experimental groups coated on titanium SLA: (6) PLL-g-PEG/PEG-KSSR(10), (7) PLL-gPEG/PEG-RGD(0.05)/PEG-KSSR(10), and (8) PLL-g-PEG/PEG-RGD(1.26)/PEG-KSSR(10). The dose-dependent response to KRSR was measured with the following experimental groups coated on titanium SLA: (9) PLL-g-PEG/PEG-KRSR(5), (10) PLL-g-PEG/PEG-KRSR(10), (11) PLL-g-PEG/PEGKRSR(15), and (12) PLL-g-PEG/PEG-KRSR(20). Multifunctional peptide experimental groups included: (13) PLL-g-PEG/PEG-RGD(0.05)/PEGKRSR(5), (14) PLL-g-PEG/PEG-RGD(0.05)/PEGKRSR(10), (15) PLL-g-PEG/PEG-RGD(0.05)/PEGKRSR(15), (16) PLL-g-PEG/PEG-RGD(1.26)/PEGKRSR(5), (17) PLL-g-PEG/PEG-RGD(1.26)/PEGKRSR(10), and (18) PLL-g-PEG/PEG-RGD(1.26)/ PEG-KRSR(15). KRSR at a surface peptide density of 20 pmol/cm² was near the saturating surface peptide density and therefore could not be combined with RGD.

Fig. 2

Effects of RGD, KRSR, and KSSR on cell number in osteoblast-like MG63 cells. (a) Cell number decreased on PLL-g-PEG surfaces compared with TCPS and SLA surfaces. Addition of KSSR further decreased cell number vs. PLL-g-PEG controls. (b) KRSR alone had no discernable effect on cell number. The combination of KRSR and RGO at a high peptide surface density increased cell number. (a) *P <0.05. Ti surfaces v. plastic; ^**P<0.05, PEG surfaces vs. SLA; ^•P<0.05, KSSR vs. PEG. (b) +P<0.05. RGD vs. KRSR alone (0 RGO); ‡P<0.05, KRSR vs. RGD alone (0 KRSR).

Fig. 3

Effects of RGD, KRSR, and KSSR on alkaline phosphatase specific activity in osteoblast-like MG63 cells. (a) Alkaline phosphatase activity increased on PLL-g-PEG surfaces compared with TCPS and SLA surfaces . There was no effect of KSSR on alkaline phosphatase activity. (b) Addition of either RGD or KRSR decreased alkaline phosphatase activity. The effect of combining RGD and KRSR caused a further decrease in alkaline phosphatase activity at high peptide densities. (a) ^**P<0.05, PEG surfaces vs. SLA. (b) +P<0.05, RGD vs. KRSR alone (0 RGD) ; ‡P<0.05, KRSR vs. RGD alone (0 KRSR).

Fig. 4

Effects of RGD, KRSR, and KSSR on osteocalcin levels in osteoblast-like MG63 cells. (a) Levels of osteocalcin increased on PLL-g-PEG surfaces compared with TCPS and SLA surfaces. The addition of 1.26 pmol/cm² RGD decreased levels of osteocalcin. Addition of KSSR increased osteocalcin levels. (b) KRSR alone did not affect osteocalcin levels, although the combination of RGD and KRSR at high peptide density decreased osteocalcin levels. (a) ^*P<0 .05, Ti surfaces vs. plastic; ^**P<0.05, PEG surfaces vs. SLA; ^•P<0. 05. KSSR vs. PEG. (b) +P<0.05, RGD vs. KRSR alone (0 RGD) ; ‡P<0.0 5, KRSR vs. RGD alone (0 KRSR) .

Fig. 5

Effects of RGD, KRSR, and KSSR on TGF-b1 levels in osteoblast-likeMG63 cells. Levels of latent and active TGF-β1 were increased on PLL-g-PEG surfaces vs. TCPS and SLA surfaces (a,c). KSSR did not affect active or latent TGF-β1 levels. Addition of 1.26 pmol/cm² RGD reduced latent and active TGF-β1 levels, as did addition of 15 pmol/ cm² of KRSR (b,d). (a,c) ^*P<0.05, Ti surfaces vs. plastic; ^**P<0.05, PEG surfaces vs. SLA. (b,d) +P<0.05 , RGD vs. KRSR alone (0 RGD); ‡P<0.05, KRSR vs. RGO alone (0 KRSR).

Fig. 6

Effects of RGD, KRSR, and KSSR on PGE2 levels in osteoblast-like MG63 cells. (a) PGE2 levels were increased on PLL-g-PEG surfaces vs. TCPS and SLA surfaces. Addition of KSSR further increased levels of PGE2. (b) Addition of 1.26 pmol/cm² RGD reduced levels of PGE2, as did addition of KRSR. Combining RGD and KRSR caused further inhibition of PGE2, especially at high surface peptide densities. (a) ^*<P0.05, Ti surfaces vs. plastic;^**P<0.05, PEG surfaces vs. SLA. ^•P< 0.05, KSSR vs. PEG. (b) +P< 0.05, RGD vs. KRSR alone (0 RGD) ; ‡P<0.05, KRSR vs. RGD alone (0 KRSR).