J Korean Orthop Assoc.  2007 Jun;42(3):386-394. 10.4055/jkoa.2007.42.3.386.

Osteoinduction using by Human BMP-2-physio- functional Bioactive Ceramics

• Kim H
• Moon SH
• Lee JW
• Kim HM
• Park M
• Lee HM

• KMID: 2186565
• DOI: http://doi.org/10.4055/jkoa.2007.42.3.386

Abstract

PURPOSE: To assess the properties and the osteogenic potency of the calcium phosphate-recombinant human morphogenetic protein-2 (CaP-rhBMP-2 composite) on glass-ceramics.
MATERIALS AND METHODS
Bioactive glass-ceramics,as a scaffold, and a calcium phosphate (CaP) solution (pH7.4) were prepared. Recombinant human bone morphogenetic protein-2 (rhBMP-2) was purified from CHO-K1 cells by transfecting the cells with BMP-2 cDNA. The glass-ceramics were soaked for 3 days at room temperature in saline, a CaP only solution, and a CaP solution containing rhBMP-2. Scanning electron microscopy (SEM), Fourier transform infrared reflection spectroscopy (FT-IR), thin film X-ray diffraction (TF-XRD) and immunofluorescent staining (IF) of the anti-human BMP-2 to composite-coated scaffold were performed to verify the characterization of the scaffold surface. In addition, RT-PCR of osteogenic marker gene and SEM photography were performed after adhering the mouse preosteoblast MC3T3-E1 cells in order to assess the osteoinductivity.
RESULTS
CaP-rhBMP-2 composite was coated on the surface of glass-ceramics, as confirmed by SEM, FT-IR, TF-XRD spectrum, and IF. The CaP-rhBMP-2 composite on the glass-ceramic showed a globular shape covered with fine spikes while the CaP on the glass-ceramic showed a fine spike structure on the flat glass surface. The expression of collagen type I and alkaline phosphatase mRNAs had increased 4 hours after cell seeding. In addition, the level of osteocalcin mRNA expression had increased significantly by 3 days in the CaP-rhBMP-2 composite compared with the control and CaP group. The SEM photographs showed more active filopodia formation in the CaP-rhBMP-2 composite than the other groups. There was extensive newly synthesized extracellular matrix around the osteoblasts and CaP-rhBMP-2 composite nodule.
CONCLUSION
The application of CaP-rhBMP-2 composite-surface coating technique on bioactive glass-ceramic is a powerful tool for osteoinduction.

Keyword

Calcium phosphate-rhBMP-2 composite; Surface coating technique; Osteoinductivity

MeSH Terms

Alkaline Phosphatase
Animals
Calcium
Ceramics*
Collagen Type I
DNA, Complementary
Extracellular Matrix
Fourier Analysis
Glass
Humans*
Mice
Microscopy, Electron, Scanning
Osteoblasts
Osteocalcin
Photography
Pseudopodia
RNA, Messenger
Spectrum Analysis
X-Ray Diffraction
Alkaline Phosphatase
Calcium
Ceramics
Collagen Type I
DNA, Complementary
Osteocalcin
RNA, Messenger

Figure

• 

  Fig. 1 Diagram of CaP-rhBMP-2 composite generation and surface coating technique (A) standardized group, (B) control group with CaP coating, (C) experimental group with CaP and rhBMP-2 coating.

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  Fig. 2 Scanning electronic microscopic finding of glass ceramic only, glass ceramic with CaP coating, and glass ceramic with CaP and rhBMP-2 coating.

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  Fig. 3 TF-XRD and FT-IRRS analysis after glass ceramic coating.

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  Fig. 4 Immuno-fluorescent stain with antibody to rhBMP-2 (magnification ×100).

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  Fig. 5 mRNA expression of osteogenic marker gene mRNA (collagen type I, osteocalcin, alkaline phosphatase) in mouse osteoblasts (MC3T3-E1) on glass ceramic, treated with saline, CaP, CaP+rhBMP-2.

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  Fig. 6 Scanning electronic microscopic finding of mouse osteoblasts (MC3T3-E1) on glass ceramic only, glass ceramic with CaP coating, and glass ceramic with CaP and rhBMP-2 coating with 3 days' incubation.

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