J Adv Prosthodont.  2011 Sep;3(3):145-151. 10.4047/jap.2011.3.3.145.

The effect of immobilization of heparin and bone morphogenic protein-2 to bovine bone substitute on osteoblast-like cell's function

• Huh JB
• Kim SE
• Song SK
• Yun MJ
• Shim JS
• Lee JY
• Shin SW

• KMID: 2118124
• DOI: http://doi.org/10.4047/jap.2011.3.3.145

Abstract

PURPOSE
This study was performed to investigate the ability of recombinant human-bone morphogenic protein-2 immobilized on a heparin-grafted bone substrate to enhance the osteoblastic functions.
MATERIALS AND METHODS
The Bio-Oss(R), not coated with any material, was used as a control group. In rhBMP-2-Bio-Oss(R) group, rhBMP-2 was coated with Bio-Oss(R) using only deep and dry methods (50 ng/mL, 24 h). In heparinized rhBMP-2-Bio-Oss(R) group, dopamine was anchored to the surface of Bio-Oss(R), and coated with heparin. rhBMP-2 was immobilized onto the heparinized- Bio-Oss(R) surface. The release kinetics of the rhBMP-2-Bio-Oss(R) and heparinized rhBMP-2-Bio-Oss(R) were analyzed using an enzyme-linked immunosorbent assay. The biological activities of the MG63 cells on the three groups were investigated via cytotoxicity assay, cell proliferation assay, alkaline phosphatase (ALP) measurement, and calcium deposition determination. Statistical comparisons were carried out by one-way ANOVA test. Differences were considered statistically significant at *P<.05 and **P<.001.
RESULTS
The heparinized rhBMP-2-Bio-Oss(R) showed more sustained release compared to the rhBMP-2-Bio-Oss(R) over an extended time. In the measurement of the ALP activity, the heparinized group showed a significantly higher ALP activity when compared with the non-heparinized groups (P<.05). The MG63 cells cultivated in the group with rhBMP-2 showed increased calcium deposition, and the MG63 cells from the heparinized group increased more than those that were cultivated in the non-heparinized groups.
CONCLUSION
Heparin increased the rhBMP-2 release amount and made sustained release possible, and heparinized Bio-Oss(R) with rhBMP-2 successfully improved the osteoblastic functions.

Keyword

Heparin; rhBMP-2; Osteoblast-like cell; Bovine bone

MeSH Terms

Alkaline Phosphatase
Bone Substitutes
Calcium
Cell Proliferation
Dopamine
Enzyme-Linked Immunosorbent Assay
Heparin
Immobilization
Kinetics
Osteoblasts
Alkaline Phosphatase
Bone Substitutes
Calcium
Dopamine
Heparin

Figure

• 

  Fig. 1 SEM images of the (A) Bio-Oss®, (B) rhBMP-2 (50 ng/ml)-Bio-Oss®, and (C) heparinized rhBMP-2 (50 ng/ml)-Bio-Oss®.

• 

  Fig. 2 Release kinetics of the rhBMP-2-(50 ng)-Bio-Oss® and heparinized rhBMP-2-(50 ng)-Bio-Oss®.

• 

  Fig. 3 Cytotoxicity of the osteoblast-like cells (MG63 cells) grown on the Bio-Oss®, rhBMP-2-(50 ng)-Bio-Oss®, and heparinized rhBMP-2-(50 ng)-Bio-Oss®.

• 

  Fig. 4 Proliferation of the osteoblast-like cells (MG63 cells) grown on the Bio-Oss®, rhBMP-2-(50 ng)-Bio-Oss®, and heparinized rhBmp-2-(50 ng)-Bio-Oss®.

• 

  Fig. 5 ALP activity of the osteoblast-like cells (MG63 cells) grown on the Bio-Oss®, rhBMP-2-(50 ng)-Bio-Oss®, and heparinized rhBMP-2-(50 ng)-Bio-Oss® after 7-, 14-, and 21-day incubation.

• 

  Fig. 6 Alizarin-red-S staining of the osteoblast-like cells (MG63 cells) grown on the Bio-Oss® (A), rhBMP-2-(50 ng)-Bio-Oss® (B), and heparinized rhBMP-2 (50 ng)-Bio-Oss® (C) after 21-day incubation (scale bar = 20 µm).

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