A Vitronectin-Derived Bioactive Peptide Improves Bone Healing Capacity of SLA Titanium Surfaces

Abstract

In this study, we evaluated early bone responses to a vitronectin-derived, minimal core bioactive peptide, RVYFFKGKQYWE motif (VnP-16), both in vitro and in vivo, when the peptide was treated on sandblasted, large-grit, acid-etched (SLA) titanium surfaces. Four surface types of titanium discs and of titanium screw-shaped implants were prepared: control, SLA, scrambled peptide-treated, and VnP-16-treated surfaces. Cellular responses, such as attachment, spreading, migration, and viability of human osteoblast-like HOS and MG63 cells were evaluated in vitro on the titanium discs. Using the rabbit tibia model with the split plot design, the implants were inserted into the tibiae of four New Zealand white rabbits. After two weeks of implant insertion, the rabbits were sacrificed, the undecalcified specimens were prepared for light microscopy, and the histomorphometric data were measured. Analysis of variance tests were used for the quantitative evaluations in this study. VnP-16 was non-cytotoxic and promoted attachment and spreading of the human osteoblast-like cells. The VnP-16-treated SLA implants showed no antigenic activities at the interfaces between the bones and the implants and indicated excellent bone-to-implant contact ratios, the means of which were significantly higher than those in the SP-treated implants. VnP-16 reinforces the osteogenic potential of the SLA titanium dental implant.

Keywords: RVYFFKGKQYWE motif; cellular responses; dental implants; osseointegration; vitronectin.

Figure 1

Surface characteristics of the titanium specimens investigated in this study. (A) Field emission scanning electron microscopy definitely shows different topographical features between the polished and sandblasted, large-grit, acid-etched (SLA) surfaces. (B) The mean values of the measured surface parameters indicated that the peptide treatment did not change the surfaces physically at the micro level. Note the significant differences in the surface parameters between the polished and the other SLA surfaces. ^** p < 0.01 vs. the polished surface. (C) Electron spectroscopy for chemical analysis detected high nitrogen content on the peptide-treated surfaces. Almost no nitrogen was found in the other groups. ^** p < 0.01 vs. the polished and SLA surfaces (significant differences are marked only for the nitrogen content).

Figure 2

Cell attachment, spreading, and migration of osteoblast-like HOS cells seeded on culture plates treated with vitronectin and synthetic peptides. (A) Photographs of osteoblast-like HOS cells adhering (upper panel) and spreading (lower panel) to culture plates treated with 1% bovine serum albumin (BSA), vitronectin (0.26 μg/cm²), scrambled peptide (SP), and VnP-16 peptide (10.5 μg/cm²). Bar = 100 μm. (B,C) Cell attachment (B) and spreading (C) to immobilized synthetic peptides. HOS cells were allowed to adhere to peptide-treated plates for 1 h (B) or 3 h (C) in serum-free medium. (D) Migration of osteoblast-like HOS cells induced by vitronectin and synthetic peptides. HOS cells were seeded into the upper chambers of transwell filters coated with vitronectin (0.26 μg/cm²), SP, or VnP-16 (10.5 μg/cm²) and were incubated for 24 h. ND, not detected. (E) The viabilities of osteoblast-like HOS cells treated with VnP-16 for 24 or 48 h. ^** p < 0.01 vs. the SP-treated control group. Data in (B–E) (n = 4) represent the mean ± SD.

Figure 3

Cell attachment and spreading of osteoblast-like MG-63 cells seeded on culture plates treated with vitronectin and synthetic peptides. (A) Photographs of osteoblast-like MG-63 cells adhering (upper panel) and spreading (lower panel) to culture plates treated with 1% bovine serum albumin (BSA), vitronectin (0.26 μg/cm²), scrambled peptide (SP), and VnP-16 peptide (10.5 μg/cm²). Bar = 100 μm. (B–C) Cell attachment (B) and spreading (C) to immobilized synthetic peptides. MG-63 cells were allowed to adhere to peptide-treated plates for 1 h (B) or 3 h (C) in serum-free medium. (D) The viabilities of osteoblast-like MG-63 cells treated with VnP-16 for 24 or 48 h. ^** p < 0.01 vs. the SP-treated control group. Data in (B–D) (n = 4) represent the mean ± SD.

Figure 4

The histologic views and histomorphometric data for bone responses to the turned, SLA, SP-treated SLA, and VnP-16-treated SLA titanium implant surfaces. (A) The demarcation lines (white arrowheads), difference in stained colors and maturity of the bone (cancellous or cortical) differentiate the new bone from the existing old bone. Here, the new bone is stained more reddish while the old bone is stained more blueish. (B) Bone-to-implant contact ratios were measured, which are defined as the percentage of the implant surface in contact with bone to the total implant surface at the region of interest, which was the area ranging from the bone crest to 2 mm in depth in this study (green edged rectangle in (A)). (C) The ratio of the area filled with bone to the total area of the region of interest (bone area, or BA ratio) was also measured for each implant.