J Korean Acad Prosthodont.  2011 Jul;49(3):245-253.

Effect of RGD peptide coating of implant titanium surface on human mesenchymal stem cell response

• Kim MS
• Jeong CM
• Jeon YC
• Ryu JJ
• Huh JB
• Yun MJ

Abstract

PURPOSE
The aim of this in vitro study was to estimate surface characteristic after peptide coating and investigate biological response of human mesenchymal stem cell to anodized titanium discs coated with RGD peptide by physical adhesion and chemical fixation.
MATERIALS AND METHODS
Fluorescence isothiocyanate (FITC) modified RGD-peptide was coated on the anodized titanium discs (diameter 12 mm, height 3 mm) using two methods. One was physical adhesion method and the other was chemical fixation method. Physical adhesion was performed by dip and dry procedure, chemical fixation was performed by covalent bond via silanization. In this study, human mesenchymal stem cell was used for experiments. The experiments consisted of surface characteristic evaluation after peptide coating, analysis about cell adhesion, proliferation, differentiation, and mineralization. Obtained data are statistically treated using Kruskal-Wallis test and Bonferroni test was performed as post hoc test (P=.05).
RESULTS
The evaluation of FE-SEM images revealed no diffenrence at micro-surfaces between each groups. Total coating dose was higher at physical adhesion experimental group than at chemical fixation experimental group. In cell adhesion and proliferation, RGD peptide coating did not show a statistical significance compared with control group (P>.05). In cell differentiation and mineralization, physical adhesion method displayed significantly increased levels compared with control group and chemical fixation method (P<.05).
CONCLUSION
RGD peptide coating seems to enhance osseointegration by effects on the response of human mesenchymal stem cell. Especially physical adhesion method showed more effective than chemical fixation method on response of human mesenchymal stem cell.

Keyword

Anodized titanium; RGD peptide; Human mesenchymal stem cell; Adhesion; Proliferation; Differentiation

MeSH Terms

Cell Adhesion
Cell Differentiation
Fluorescence
Humans
Isothiocyanates
Mesenchymal Stromal Cells
Oligopeptides
Osseointegration
Titanium
Isothiocyanates
Oligopeptides
Titanium

Figure

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  Fig. 1. Schematic diagram of the chemical fixation.

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  Fig. 2. FE-SEM images after peptide coating (×50,000 magnification). A: Control, B: Chemical fixation, C: Physical adhesion.

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  Fig. 3. Fluorescent microscope images after peptide coating. A: Control, B: Chemical fixation, C: Physical adhesion.

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  Fig. 4. Concentration curve by fluorescent spectroscopy.

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  Fig. 5. Confocal laser scanning microscopy images of cells grown on the surface of titanium specimen after incubation of 2 hours and 24 hours (×400 magnification). actin staining.

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  Fig. 6. Crystal violet assay of human mesenchymal stem cells grown on the surface of titanium specimen after incubation of 3 hours.

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  Fig. 7. Cell proliferation of human mesenchymal stem cells grown on the surface of titanium specimen after incubation of 3 days and 7 days.

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  Fig. 8. Real time-PCR analysis for mRNA of osteopontin grown on the surface of titanium specimen after incubation of 7 days (★:P<.05, Bonferroni test).

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  Fig. 9. Real time-PCR analysis for mRNA of collagen type I grown on the surface of titanium specimen after incubation of 7 days (★:P<.05, Bonferroni test).

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  Fig. 10. Real time-PCR analysis for mRNA of osteocalcin grown on the surface of titanium specimen after incubation of 7 days (★:P<.05, Bonferroni test).

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  Fig. 11. ALP activity of humen mesenchymal stem cells grown on the surface of titanium specimen after incubation of 7 days and 14 days (★:P<.05, Bonferroni test).

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  Fig. 12. Alizarin red assay of humen mesenchymal cells grown on the surface of titanium specimen after incubation of 21 days (★: P<.05, Bonferroni test).

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