In vitro osteogenic properties of two dental implant surfaces

Abstract

Current dental implant research aims at understanding the biological basis for successful implant therapy. The aim of the study was to perform a full characterization of the effect of two commercial titanium (Ti) surfaces, OsseoSpeed and TiOblast, on the behaviour of mouse preosteoblast MC3T3-E1 cells. The effect of these Ti surfaces was compared with tissue culture plastic (TCP). In vitro experiments were performed to evaluate cytotoxicity, cell morphology and proliferation, alkaline phosphatase activity, gene expression, and release of a wide array of osteoblast markers. No differences were observed on cell viability and cell proliferation. However, changes were observed in cell shape after 2 days, with a more branched morphology on OsseoSpeed compared to TiOblast. Moreover, OsseoSpeed surface increased BMP-2 secretion after 2 days, and this was followed by increased IGF-I, BSP, and osterix gene expression and mineralization compared to TiOblast after 14 days. As compared to the gold standard TCP, both Ti surfaces induced higher osteocalcin and OPG release than TCP and differential temporal gene expression of osteogenic markers. The results demonstrate that the gain of using OsseoSpeed surface is an improved osteoblast differentiation and mineralization, without additional effects on cell viability or proliferation.

Figure 1

Scanning electron microscope images of MC3T3-E1 cells attached to each coin after 2, 7, and 14 days of cell culture. 400x magnification images are shown.

Figure 2

Detail of surface morphology and cell monolayer of MC3T3-E1 cells after 2 days by scanning electron microscope. 2000x magnification images are shown.

Figure 3

DAPI staining of the nuclei (20x) of the cell monolayer attached to each coin after 2, 7, and 14 days of cell culture.

Figure 4

Cell viability, proliferation and RNA content of MC3T3-E1 cells (a) LDH activity measured from culture media collected after 2, 7 and 14 days of culture. Data expressed as a relative to the mean value of the TCP group at day 2, which was set as 100%. (b) Number of nuclei were counted with the ImageJ software and referred to the number of cells/mm². (c) RNA content of cells attached after 2, 7 and 14 days of culture. Values represent the mean ± SEM. Student's t-test: *P ≤ 0.05 versus TCP; ^# P ≤ 0.05 versus TiOblast.

Figure 5

BMP-2 released to culture media after 2 days of culture. Values represent the mean ± SEM. Student's t-test: ^# P ≤ 0.05.

Figure 6

Relative mRNA levels of osteogenic markers (Coll-I, BSP, ALP, CD44) after 2, 7, and 14 days of culture. Ratios of target genes relative to housekeeping genes (GAPDH, B-actin) were expressed relative to the mean value of the TCP group at day 2, which was set as 100%. Values represent the mean ± SEM. Differences between groups (n = 6) were assessed by Student's t-test. ^# P ≤ 0.05 versus TiOblast; *P ≤ 0.05 versus TCP.

Figure 7

Relative mRNA levels of transcriptional factors and regulators (Hes1, Dlx2, Osx, Smad1, Smad5) after 2, 7 and 14 days of culture. Ratios of target genes relative to housekeeping genes (GAPDH, B-actin) were expressed relative to the mean value of the TCP group at day 2, which was set as 100%. Values represent the mean ± SEM. Differences between groups (n = 6) were assessed by Student's t-test. ^# P ≤ 0.05 versus TiOblast; *P ≤ 0.05 versus TCP.

Figure 8

Relative mRNA levels of cytokines and growth factors (IGF-I, IL-6, OPG, RANKL) after 2, 7, and 14 days of culture. Ratios of target genes relative to housekeeping genes (GAPDH, B-actin) were expressed relative to the mean value of the TCP group at day 2, which was set as 100%. Values represent the mean ± SEM. Differences between groups (n = 6) were assessed by Student's t-test. ^# P ≤ 0.05 versus TiOblast; *P ≤ 0.05 versus TCP.

Figure 9

OC, OPG, and IL-6 released to culture media after 2, 7, and 14 days of culture. Values represent the mean ± SEM. Student t-test: *P ≤ 0.05 versus TCP; ^# P ≤ 0.05 versus TiOblast.

Figure 10

ALP activity measured from culture media collected after 2, 7, and 14 days of culture. Data expressed as a relative to the mean value of the TCP group at day 2, which was set as 100%. Values represent the mean ± SEM. Student t-test: *P ≤ 0.05 versus TCP; ^# P ≤ 0.05 versus TiOblast.