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