Effect of titanium surface microgrooves and thermal oxidation on in vitro osteoblast responses

Jin-Ho Seo; Richard Leesungbok; Su-Jin Ahn; Su-Jung Park; Myung-Hyun Lee; Suk Won Lee

doi:10.4047/jkap.2015.53.3.198

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Seo, Leesungbok, Ahn, Park, Lee, and Lee: Effect of titanium surface microgrooves and thermal oxidation on in vitro osteoblast responses

Original Article

J Korean Acad Prosthodont 2015;53(3):198-206.

Published online: 9 February 2015

DOI: https://doi.org/10.4047/jkap.2015.53.3.198

Effect of titanium surface microgrooves and thermal oxidation on in vitro osteoblast responses

Jin-Ho Seo¹, Richard Leesungbok¹, Su-Jin Ahn¹, Su-Jung Park¹, Myung-Hyun Lee², Suk Won Lee^1,^∗

¹Department of Biomaterials & Prosthodontics, Kyung Hee University Hospital at Gangdong, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea

²Green Ceramics Division, Korea Institute of Ceramic Engineering and Technology, Seoul, Republic of Korea

^∗Corresponding Author: Suk Won Lee Department of Biomaterials & Prosthodontics, Kyung Hee University Hospital at Gangdong, Institute of Oral Biology, School of Dentistry, Kyung Hee University, 892 Dongnam-ro, Gangdong-gu, Seoul 134-727, Republic of Korea +82 2 440 7519: e-mail, ysprosth@hanmail.net

^※ This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-20100021268).

Received 18 June 201513 July 2015 Accepted 15 July 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

We aimed to investigate the effect of combined various microgrooves and thermal oxidation on the titanium (Ti) and to evaluate various in vitro responses of human periodontal ligament cells (PLCs).

Materials and methods

Grade II titanium disks were fabricated. Microgrooves were applied on titanium discs to have 0/0 ㎛, 15/3.5 ㎛,30/10 ㎛, and 60/10 ㎛ of respective width/depth by photolithography. Thermal oxidation was performed on the microgrooves of Ti substrata for 3 h at 700°C in air. The exper-iments were divided into 3 groups: control group (ST), thermal oxidation group (ST/TO), and combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO). Surface characterization was performed by field-emission scanning microscopy. Cell adhesion, osteoblastic differentiation, and mineralization were analyzed using the bromodeoxyurdine (BrdU), Alkaline phosphatase (ALP) activity, and extracellular calcium deposition assays, respectively. Statistical analysis was performed using the one-way analysis of variance and Pearson’ s bivariate correlation analysis (SPSS Version 17.0).

Results

In general, the combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO) showed significantly higher levels compared with the control (ST) or thermal oxidation (ST-TO) groups in the BrdU expression, ALP activity, and extracellular calcium deposition. Gr60-TO group induced highest levels of cell adhesion and osteoblastic differentiation.

Conclusion

Within the limitation of this study, we conclude that the Ti surface treatment using combined microgrooves and thermal oxidation is highly effective in inducing the cell adhesion andosteoblastic differentiation. The propose surface is also expected to be effective in inducing rapid and strong osseointegration of Ti oral implants.

Keywords: Titanium, Microgrooves, Thermal oxidation, Osteoblastic differentiation

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Fig. 1.

Field emission scanning electron microscopic images of (A) ST (×500),(B) Gr15 (×200), (C) Gr30 (×200) and (D) Gr60 (×200).

Fig. 2.

Field emission scanning electron microscopic images of the smooth titanium (ST) and thermally oxidized ST (ST-TO) groups at various magnifications.(A) ST (×1,000), (B) ST (×50,000), (C) ST-TO (×1,000), (D) ST-TO (×50,000). Note that polished texture at nano- to submicroscale widths appear in (A) and (B).

Fig. 3.

Comparison result of the cell adhesion of human periodontal ligament cells on ST, ST-TO, Gr15-TO, Gr30-TO and Gr60-TO titanium substrata after 16 h of culture using bromodeoxyuridine assay. One-way ANOVA (n = 4). ∗∗∗: significant difference (P<.001).

Fig. 4.

Comparison result of the alkaline phosphatase activity of human periodontal ligament cells on ST, ST-TO, Gr15-TO, Gr30-TO and Gr60-TO titanium substrata after 7 and 14 days of osteogenic culture. One-way ANOVA (n = 4). ∗∗∗: significant difference (P<.001).

Fig. 5.

Comparison result of the osteoblast differentiation of human periodontal ligament cells on ST, ST-TO, Gr15-TO, Gr30-TO and Gr60-TO titanium substrata after 24 days of osteogenic culture using extracellular calcium deposition assay. One-way ANOVA (n = 4). ∗∗∗: significant difference (P<.001).

Fig. 6.

Scatter-plot results from the Pearson' s correlation analysis. Significant correlations were present for (A), (B), (C), (D), (E) and (F) (P<.01) (n = 20).

Table 1.

Comparison of the cell adhesion of human periodontal ligament cells on titanium substrata with various surface topographies and chemistry after 16 h of culture by bromodeoxyuridine assay (optical density)

Titanium substrata with various surface topographies and chemistry
	ST	ST-TO	Gr15-TO	Gr30-TO	Gr60-TO	Sig.¹⁾
	n = 4	n = 4	n = 4	n = 4	n = 4
16 h	0.356 ± 0.005	0.376 ± 0.008	0.419 ± 0.009	0.389 ± 0.005	0.410 ± 0.010	<0.001
T²⁾	a	b	c	b	c

1) Statistical significances were tested by one-way analysis of variance among groups.

2) The same letters indicate non-significant difference between groups based on Tukey' s multiple comparison tests.

Table 2.

Comparison of the alkaline phosphatase activity of human periodontal ligament cells on titanium substrata with various surface topographies and chemistry after 7 and 14 days osteogenic culture (μ M/mg protein)

Titanium substrata with various surface topographies and chemistry
	ST	ST/TO	Gr15-TO	Gr30-TO	Gr60-TO	Sig.¹⁾
	n = 4	n = 4	n = 4	n = 4	n = 4
7 days	0.261 ± 0.006	0.337 ± 0.042	0.342 ± 0.018	0.493 ± 0.071	0.503 ± 0.027	<0.001
T²⁾	a	b	b	c	c
14 days	0.878 ± 0.073	1.033 ± 0.010	1.107 ± 0.044	1.264 ± 0.034	1.417 ± 0.145	<0.001
T²⁾	a	a,b	b,c	c,d	d

1) Statistical significances were tested by one-way analysis of variance among groups.

2) The same letters indicate non-significant difference between groups based on Tukey' s multiple comparison tests.

Table 3.

Comparison of the extracellular calcium deposition of human periodontal ligament cells on titanium substrata with various surface topographies and chemistry after 24 days of osteogenic culture by quantifying the calcium concentration per 24-well (μ g/well)

Titanium substrata with various surface topographies and chemistry
	ST n = 4	ST/TO n = 4	Gr15-TO n = 4	Gr30-TO n = 4	Gr60-TO n = 4	Sig.¹⁾
24 days	2.365 ± 0.127	2.881 ± 0.133	3.273 ± 0.168	3.375 ± 0.170	3.842 ± 0.105	<0.001
T²⁾	a	b	c	c	d

1) Statistical significances were tested by one-way analysis of variance among groups.

2) The same letters indicate non-significant difference between groups based on Tukey' s multiple comparison tests.

Table 4.

Pearson correlation coefficients between the results of the bromodeoxyuridine assay, alkaline phosphatase activity and extracellular calcium deposition of human periodontal ligament cells

	BrdU 16h	ALP 7 days	ALP 14 days
ALP 7 days	0.555^∗∗
ALP 14 days	0.713^∗∗	0.915^∗∗
Ca 24 days	0.844^∗∗	0.880^∗∗	0.941^∗∗

^∗∗ Correlation is significant at the 0.01 level (2-tailed).

N = 20.

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