J Periodontal Implant Sci.  2011 Oct;41(5):227-233. 10.5051/jpis.2011.41.5.227.

Response of osteoblast-like cells cultured on zirconia to bone morphogenetic protein-2

• Han SH
• Kim KH
• Han JS
• Koo KT
• Kim TI
• Seol YJ
• Lee YM
• Ku Y
• Rhyu IC

• KMID: 1783617
• DOI: http://doi.org/10.5051/jpis.2011.41.5.227

Abstract

PURPOSE
The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2.
METHODS
MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days.
RESULTS
At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7.
CONCLUSIONS
The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium.

Keyword

Bone morphogenetic protein-2; Cell differentiation; Cell proliferation; Zirconium oxide

MeSH Terms

Alkaline Phosphatase
Bone Matrix
Cell Differentiation
Cell Proliferation
Collagen Type I
Gene Expression
Integrin-Binding Sialoprotein
Microscopy, Confocal
Osteoblasts
Osteocalcin
Real-Time Polymerase Chain Reaction
RNA, Messenger
Seeds
Titanium
Transcription Factors
Zirconium
Alkaline Phosphatase
Collagen Type I
Integrin-Binding Sialoprotein
Osteocalcin
RNA, Messenger
Titanium
Transcription Factors
Zirconium

Figure

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  Figure 1 Cumulative release of bone morphogenetic protein-2 (BMP-2) from the demineralized bone matrix gel with BMP-2. The amount of BMP-2 released from the gel was determined by enzyme-linked immunosorbent assay.

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  Figure 2 Fluorescence staining of MC3T3-E1 cells 24 hours after gel loading. Typical images are shown for ziconia-demineralized bone matrix (DBM) gel (left), titanium-DBM gel (right) (A), zirconia-bone morphogenetic protein (BMP) gel (left), titanium-BMP gel (right) (B), zirconia alone (left), titanium alone (right) (C), and cover glass (D).

• 

  Figure 3 Methyl tetrazole sulfate (MTS) assay in MC3T3-E1 cells cultured on zirconia and titanium discs at day 1, day 4, and day 7 after gel loading. Data are expressed as the mean values±standard deviation of three independent experiments. DBM, demineralized bone matrix; BMP, bone morphogenetic protein. a)P<0.05.

• 

  Figure 4 ALPase activity of MC3T3-E1 cells at 7 days after gel loading. 3×104 cells/cm2 were plated on the different samples. The released 4-nitrophenylate was determined photometrically at 405 nm and related to protein concentration of the lysates. The results are presented as mean±standard deviation (N=3). a) or b) indicate the significant difference with other groups (P<0.05). However, in cells cultured with bone morphogenetic protein (BMP)-2, there was no difference between zirconia and titanium. DBM, demineralized bone matrix.

• 

  Figure 5 Expression levels of the osteogenic genes were measured by real-time reverse transcription-polymerase chain reaction and normalized to that of the 18s rRNA gene. The relative gene expression level was calculated by the 2-ΔΔCt formula and that may be interpreted as 'the expression of the gene of interest relative to the internal control (18r RNA) in a treated sample compared with the untreated control (in this study, titanium disc)'. (A) ALPase; (B) BSP; (C) Type I collagen; (D) Osteocalcin; (E) Osterix; (F) Runx-2. DBM, demineralized bone matrix; BMP, bone morphogenetic protein.

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