J Adv Prosthodont.  2014 Jun;6(3):157-164. 10.4047/jap.2014.6.3.157.

Magnesium vs. machined surfaced titanium - osteoblast and osteoclast differentiation

• Kwon YD
• Lee DW
• Hong SO

• KMID: 2118225
• DOI: http://doi.org/10.4047/jap.2014.6.3.157

Abstract

PURPOSE
This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source.
MATERIALS AND METHODS
40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs).
RESULTS
MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM.
CONCLUSION
Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.

Keyword

Magnesium; Ion implantation; Titanium surface; MC3T3-E1; Mouse bone marrow-derived macrophages (BMMs)

MeSH Terms

Actins
Alkaline Phosphatase
Animals
Bone Remodeling
Cell Differentiation
Collagen
Gene Expression
Integrin-Binding Sialoprotein
Macrophages
Magnesium*
Mice
Microscopy, Electron, Scanning
Osteoblasts*
Osteocalcin
Osteoclasts*
Osteopontin
Phenotype
Plasma
Real-Time Polymerase Chain Reaction
RNA, Messenger
Surface Properties
Titanium*
Actins
Alkaline Phosphatase
Collagen
Integrin-Binding Sialoprotein
Magnesium
Osteocalcin
Osteopontin
RNA, Messenger
Titanium

Figure

• 

  Fig. 1 Titanium disc specimens (Shinheung, Co, Korea) of 10 mm diameter × 3 mm thickness (A). Machined surface (M) is more hydrophobic than the Mg-implanted on the machined surface (MM) contact angles are shown in Table 3 (B). SEM images of the different titanium surfaces are shown (C)(×100, ×500 and ×1,000).

• 

  Fig. 2 ALP activity of MC3T3-E1 cells on different Ti surfaces at 1st week, 2nd week, and 3rd week of culture. NC-negative control (machined Ti disc w/o dif.), PC-positive control (culture plate w/dif.), M-machined surface, MM-magnesium surface. Significant difference by Tukey's multiple comparison at * P<.01, and ** P<.05.

• 

  Fig. 3 Real Time PCR of Osteoblast cell. Quantitative real-time PCR analysis of the levels of mRNA for bone sialoprotein (BSP), and osteocalcin (OC) in cells grown on different surfaces at 7 and 14 days of culture. PC-positive control (culture plate with differentiation); M-machined surface; MM-magnesium surface.

• 

  Fig. 4 (A) OB-Alizarin red S Assay Alizarin red S staining for mineralized nodule formation in osteoblast cells, (B) Quantification of alizarin red S in osteoblasts. PT-positive control (culture plate with differentiation); M-machined surface; MM-magnesium surface.

• 

  Fig. 5 An effect on the ability of osteoclasts maturation and differentiation by actin ring formation assay. Mouse bone marrow-derived macrophages (BMMs) cells were plated on each disk; negative control (NC: machined Ti disc without differentiation)(A), Machined surface (M)(B), and Magnesium surface (MM)(C). Cells were stimulated with 100 µg/mL RANKL and 40 µg/mL M-CSF. In order to investigate the effect on actin ring formation, immunofluorescence analysis was performed. M and MM group cells showed many full actin rings and disrupted actin rings compared to NC group cells. Much fuller actin rings existed on MM compared with M.

• 

  Fig. 6 (A) OC - RT-PCR gene expression of TRAP of machined surface (M), Mg-implanted machined surface (MM). The mRNA expression levels of the indicated genes were determined by RT-PCR. Expression of the markers was normalised to the expression of the housekeeping gene GAPDH, (B) Trap density comparison between M and MM; lowest values were set at 0.

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