Analysis of attachment, proliferation and differentiation response of human mesenchymal stem cell to various implant surfaces coated with rhBMP-2

In-Ku Lee; In-Ho Han; Sun-Wook Hwang; Jae-Jun Ryu

doi:10.4047/jkap.2012.50.1.44

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Lee, Han, Hwang, and Ryu: Analysis of attachment, proliferation and differentiation response of human mesenchymal stem cell to various implant surfaces coated with rhBMP-2

Original Article

The Journal of Korean Academy of Prosthodontics

The Journal of Korean Academy of Prosthodontics 2012; 50(1): 44-52.

Published online: 31 January 2012

DOI: https://doi.org/10.4047/jkap.2012.50.1.44

Analysis of attachment, proliferation and differentiation response of human mesenchymal stem cell to various implant surfaces coated with rhBMP-2

In-Ku Lee, DDS, MSD, PhD¹, In-Ho Han, PhD², Sun-Wook Hwang, PhD³, Jae-Jun Ryu, DDS, MSD, PhD⁴

¹Department of Medical Science, Major in Dentistry, Korea University, Seoul, Korea.

²Department of Chemical and Biomolecular Engineering, Patent and Tradement office, Daejeon, Korea.

³Department of Neuroscience, Medical research institute, Ansan Hospital, Korea University, Ansan, Korea.

⁴Department of Prosthodontics, Ansan Hospital, Korea University, Seoul, Ansan, Korea.

Corresponding Author: Jae-Jun Ryu. Department of Prosthodontics, Ansan Hospital, Korea University, Gojan 1-dong, Danwon-gu, Ansan, 425-707, Korea. +82 31 412 5370, koprosth@unitel.co.kr

Received 26 December 2011 Revised 6 January 2011 Accepted 11 January 2012

Abstract

Purpose

In this paper we tried to evaluate the most appropriate surface for rhBMP-2 coating among 4 rough titanium surfaces.

Materials and methods

We used machined surface as a control group and anodized, RBM and SLA surfaces as test groups. We coated rhBMP-2 on the 4 surfaces and with uncoated surfaces for each case, we cultured human mesenchymal stem cells on all 8 surfaces. 24 hours after we measured the stem cell'attachment with SEM, and on 3rd, 7th, and 14th days, we checked the cell proliferation and differentiation by using MTT and ALP activity assay. And on the 7th day after the culture, we performed RT-PCR assay to determine whether the expression levels of Type I collagen, osteocalcin, osteopontin were changed.

Results

We observed with SEM that 4 rhBMP-2 coated surfaces exhibited wider and tighter cell attachment and more cell process spreading than uncoated surfaces. The anodized rhBMP-2 surface caused robustest effects. In MTT assay we could not find any meaningful difference. In ALP assay there was a significant increase (P<.05) in the ALP activity of anodized rhBMP-2 coated surface compared with that of the control (3rd and 14th days) and with that of the RBM rhBMP-2 coated surface (14th day). In RT-PCR assay there was increased expressions in the anodized rhBMP-2 coated surface for osteocalcin, and osteopontin.

Conclusion

We found that the anodized rhBMP-2 coated surface were most prominent stem cell attachment and differentiation in compared to control and Machined rhBMP-2 coated, RBM rhBMP-2 coated surface.

Keywords: Stem cell, rhBMP-2, Anodized, RBM, SLA surface

Figures and Tables

Fig. 1

SEM of treated surfaces after 24 hours culture.

M: machined surface (×50), M-1: (×500), MB: machined rhBMP-2 coated surface (×50), MB-1: (×500), A: anodized surface (×50), A-1: (×500), AB: anodized rhBMP-2 coated surface (×50), AB-1: (×500) R: RBM surface (×50), R-1: (×500), RB: RBM rhBMP-2 coated surface (×50), RB-1: (×500), S: SLA surface (× 50), S-1: (×500), SB: SLA rhBMP-2 coated surface (×50), SB-1: (×500).

Fig. 2

MTT assay measured as cell proliferation..

^* significant differences (P<.05) compared with control group, ^v significant differences (P<.05) compared with no BMP-2 coating group. Non: culture plastic. Error bars represent the standard errors of each group.

Fig. 3

ALP activity in the cells attached to each type of titanium surfaces indicates osteoblast differentiation.

^* significant differences (P<.05) compared with control group, ^v significant differences (P<.05) compared with no BMP-2 coating group. Non: culture plastic.

Fig. 4

RT-PCR of Type I collagen, osteocalcin, and osteopontin of eight surface treated samples.

Table 1

4 different surfaces and rhBMP-2 treated surfaces

Table 2

Surface roughness (Ra) of each surface treated titanium discs

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