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Abstract
Objective
This study examined the potential of the in vitro osteogenesis of microtopographically modified surfaces, RBM (resorbable blasting media) surfaces, which generate hydroxyapatite grit-blasting.
Methods
RBM surfaces were modified hydroxyapatite grit-blasting to produce microtopographically modified surfaces and the surface morphology, roughness or elements were examined. To investigate the potential of the in vitro osteogenesis, the osteoblastic cell adhesion, proliferation, and differentiation were examined using the human osteoblast-like cell line, MG-63 cells. Osteoblastic cell proliferation was examined as a function of time. In addition, osteoblastic cell differentiation was verified using four different methods of an ALP activity assay, a mineralization assay using alizarin red-s staining, and gene expression of osteoblastic differentiation marker using RT-PCR or ELISA.
Results
Osteoblastic cell adhesion, proliferation and ALP activity was elevated on the RBM surfaces compared to the machined group. The cells exhibited a high level of gene expression of the osteoblastic differentiation makers (osteonectin, type I collagen, Runx-2, osterix). imilar data was represented in the ELISA produced similar results in that the RBM surface increased the level of osteocalcin, osteopontin, TGF-beta1 and PGE2 secretion, which was known to stimulate the osteogenesis. Moreover, alizarin red-s staining revealed significantly more mineralized nodules on the RBM surfaces than the machined discs.
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Fig. 1.
Surface morphologhy of titanium discs for osteoblastic cell culture. SEM images of machined titanium (A) and RBM (B). Bar = 10 μm. EDA Data of machined titanium (C) and RBM (D). Surfaces were examined using the JSM-6480LV SEM at working distance of 10 mm and accelerating voltage of 20 kV.
Fig. 2.
Effect of surface roughness on the adhesion of MG-63 cells. MG-63 cells were stained with cresyl violet dye and counted using spectrophotometer on surfaces of differing roughness. Data are represented as the average ± standard deviation. RBM surface, the rough surface, were promoted the cell adhesion compared to smooth machined surface. *: Statistically significant compared with cells cultured on machined surfaces.(P<0.05)
Fig. 3.
Results of MG-63 cell proliferation experiments. Data are represented as the average±standard deviation. The differences between the number of cells on RBM and machined surfaces during 5 days is statistically significant. *: Statistically significant compared with cells cultured on machined surfaces.(P<0.05)
Fig. 4.
Effect of surface roughness on the osteoblastic differentiation of MG-63 cells. Data are represented as the average± standard deviation. (A) ALP (alkaline phosphatase) activity of MG-63 cells cultured either on machined Ti and RBM surfaces. Culture plate without osteogenic differentiation was served as a internal negative control. And a culture plate with osteogenic differentiation was a internal positive control. On RBM surface, the ALP activity enhanced about 10% than machined surface (B). Mineralization properties of the machined Ti and RBM surface. MG-63 cells were cultured as before for 14 days with osteogenic differentiation medium. Total calcium deposition measured by alizarin red-s stain via extraction with acetic acid. (C-F) Effect of surface roughness on osteogenesis-related local factor levels determined by using an ELISA kit. Osteocalcin levels were measure in the conditioned media using an ELISA kit specific for human osteoclacin (C). Osteopontin levels were measured in the conditioned media using an ELISA kit specific for human osteopontin (D). PGE2 levels were measured in the conditioned media using an ELISA kit specific for humans PGE2 (E). And Active of latent TGF-β1 (transforming growth factor-β1) levels were measure in the conditioned media using an ELISA kit specific for human TGF-β1 (F). (G) RT-PCR analysis of roughness-influenced osteogenic marker gene. Type I collagen, osteonectin, osterix and Runx-2 RNA levels in MG-63 cells exposure for 72hrs on a machined Ti or RBM surface. RT-PCR products were subjected to electrophoresis on 2% agarose gel and visualized UV exposure. A representative analysis is shown inside of the each figure. And the level of specific bands was normalized for the β-actin cDNA content. Statistically significant compared with cells cultured on machined surfaces by unpaired t-tests.(*P<0.01, **P<0.05, ***P<0.001)
Table 1.
Rrimer sequences of osteogenic marker and conditions for polymerase chain reaction
Table 2.
Roughbess values of a machined Ti and a RBM surfaces measured by profilometer
| Machied | RBM | |
|---|---|---|
| Ra, [um] | 0.222 (±0.039) | 1.523 (±0.156) |
| Ra2max, [um] | 0.244 (±0.032) | 1.663 (±0.163) |
| Rz, [um] | 1.566 (±0.199) | 10.514 (±1.177) |
| Rt, [um] | 1.727 (±0.278) | 11.648 (±1.378) |
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