Adsorption of fibronectin 9-10 on pure titanium in a magnetic field ; A pilot study
Abstract
HJ Kim*, JY Koak*, SJ Heo*, IT Chang*, JB Lee, Yim SH**, JH Jang***
* Department of Prothodontic Dentistry, Seoul National University
** Sungkyunkwan University, School of Medicine
*** IBEC & BK 21, Seoul National University
이 논문의 목적은 fibronectin 이 machined 타이타늄 디스크에 흡착되는데 약한 자기장이 어떠한 영향을 미치는지를 평 가하는 것이다. 0 (대조군), 10, 20, 50, 100 gauss 의 자기장이 각각 가해진 상태에서 1 M fibronectin 용액에 타이타늄 디스크를 1일간 위치시킨후 흡착된 fibronectin 의 optical density를 측정하였다. 대조군과 비교할 때 20, 100 gauss 에서 통계적으로 유의하게 높은 값을 보였고 50 gauss 에서는 농도가 감소하였다. 자기장의 세기에 따라 fibronectin 흡착이 증가된다기 보다는 특정 세기에서 흡착이 증가하였다.
Key word ; fibronectin, machined titanium, magnetic field
This work was supported by a grant from the Korea Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (02- PJ3-PG6-EV11-0002).
Introduction
U
pon implantation, the surfaces of implants become coated with a thin proteinacious film.1) Protein adsorption will be dependent on the particular binding surface and various conditions may modify their biologic activity in relation to cell attachment.2) Titanium forms an oxide layer,3)and the interaction of cells with this layer is mediated by the extracellular matrix proteins produced by the cell as well as by cell surface proteins.Application of magnetic field is a useful method to promote bone formation and several studies have demonstrated its clinical usefulness for treatment of bone injuries. Degan et al4) reported the promoting effect of static magnetic field (SMF)on fracture repair. Bruce et al.5) reported that the mechanical strength of fractured bone of rabbits was increased by SMF exposure. However, in in vivo studies, the effect of magnetic field on bone formation can't be demonstrated owing to various factors participating in osteogenesis.
In dental field, since Freedman used Alnico magnet for enhancing retention of lower denture in 1941,6) magnet has been widely used in dentistry, such as attachment in overdenture7-8) and orthodontic devices.9-10) But the effect of magnetic field on neighboring tissue has not been studied enough.
In this pilot study, the correlation between the intensity of magnetic field and adsorption of fibronectin 9-10 on titanium was investigated.
Materials and methods
1. Preparation of Ti disks
Commercially pure titanium disks (Grade 4) were provided by Daidon steel co., Korea. No polishing procedures were performed on the surfaces of disks. The disks were 25mm in diameter and 1mm in thickness. All disks were placed in trichloroethylen and sonicated for 15 minutes followed by sonication with 70% ethanol three times. The machined samples were dried, and rinsed in sterile phosphate buffered saline (PBS, pH 7.4)
2. Static magnetic field
Various magnetic fields - 10, 20, 50, 100 gauss - were estabilished by controlling the distance from Nd-Fe- B magnet (Magtopia, Gumi, Korea) to the disks (Fig.1). The intensities of magnetic field on each designatied plate site in the acrylic boxes were measured by Gauss meter (Kanetec, Japan) in a triplicate manner. Control disks were placed in an acrylic box without magnet. Three sets of Nd-Fe-B magnets along with four titanium disks in each designated gauss were prepared..
3. Fibonectin adsorption
Each titanium disk in a single plate was put in the acrylic box and was incubated in 1500µl 1µM
II
I
▲ Fig. 1, 2.
Schematic diagrams of experiment apparatus and magnetic field of Nd-Fe-B magnet.
▲ Fig. 3.
PowerWave X340-1
▲ Fig. 4.
Substrates in 96 well plate.
G 20 19.8 19.6
19.4 19.2
-1 -0.5 0
0 cm
cm
0.5
1 -1
1
-0.5 0.5
After rinsing in PBS, the samples were incubated in His probe (H-3) antibody (Santa Cruz, biotechnology) for 90 minutes at room temperature and rinsed in TBST once. One step Turbo TMB ELISA (Pierce) was added to each plate and incubation was done for 30 minutes at room temperature. 100µl substrates in each plate were transferred to the well plate. Optical density (OD) was measured at 450nm in ELISA reader (PowerWave X340-1) after adding reaction stopping solution consisting of 2M H2SO4. The adsorption of fibronectin on titanium plates were measured in a triplicate manner.
4. Statistical analysis
SPSS ver. 10.0 package for Windows was used.
Three samples were used for each experimental
used to determine the statistical significance of the differences observed groups. p- values smaller than 0.05 were considered significant.
Results
Compared with control group, optical densities of adsorbed fibronectin were higher in 20 and 100 gauss groups (p= 0.006, 0.003). In 50 gauss, OD values decreased and showed no significant difference compared with the values showed in 0 gauss.
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Table. 1.
Effect of magnetic field on adsorption of fibronectin 9-10
Intensity of magnetic field (gauss) Optical density (OD)
Control ( 0 ) 0.040 +- 0.0017
10 0.063 +- 0.0060
20 0.075 +- 0.0150*
50 0.053 +- 0.0050**
100 0.079 +- 0.0071*
Measurements are mean +- standard deviation.
* Significant difference from control (0gauss) (p <0.05)
** Significant difference from 100 gauss (p < 0.05)
Discussion
In this study, we investigated the correlation between the intensity of static magnetic field and fibronectin adsorption. In 20 and 100 gauss, the amount of adsorbed fibronectin was higher than those in 0 gauss. Highest OD value was observed in 100 gauss, which had insignificant difference with those in 20 gauss. In 0-100 gauss, weak magnetic field influenced fibronectin adsorption in titanium disks. However, the effect did not demonstrate dose dependent correlation. Masumoto et al.11)reported
similar results. In his experiment, 0.2 mT and 0.3 mT promoted a greater bone formation than 0.8mT which he concluded that minimal essential magnetic intensity stimulates more effective bone formation than higher magnetic intensity.
Pulsated electromagnetic fields (PEMF) have been reported to affect bone formation in animals and humans,12-14)and it is clinically used to treat the bone fractures. PEMF osteogenic action is believed to occur through several mechanisms. It changes the permeability of ions across the cell membrane and affects the activities of intercellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine
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▲ Fig. 2.
Mean and SD of OD value of adsorbed fibronectin 9-10.
OD
gauss
0.100
0.075
0.050
0.025
0.000
0 10 20 50 100
electric and magnetic vectors periodically.
However, as the static magnetic field (SMF) doesn't induce the electric currents and the vectorial changes, the mechanism of osteogenic actions of PEMF and SMF may be different.
SMF of gauss order were reported to induce bone formation in in vivo model.5, 17-20)Camilleri and McDonald studied the effect of the SMF using a rat model with a neodymiumion-boron magnet placed over a skull suture and showed that the SMF affected the cell mitotic activity.19) Bruce et al.
reported that the SMF stimulation enhanced the strength of the fractured radii of rabbits.5) It is assumed that the SMF initiated an increase in localized calcium deposits, which neutralized the net negative charge of tissues and allowed the subsequent vascularization of osteogenisis.20)
Compared with in vivo study, in vitro study is easy to evaluate the magnetic field effect on each step of bone formation. This test model has been used to compare biocompatibility and bone forming capacity among different materials and materials with various surface treatment.21-23)To see the pure effect of the magnetic field, unpolished machined titanium discs were used and fibronectins were diluted in PBS. Magnetic field intensity was controlled only through a distance between titanium discs and magnet. This experiment showed that the magnetic field can modulate the precondition of osteoblast adhesion.
Fibronectin is one of the earliest cell-binding proteins produced by odontoblasts and
chains, each 60nm in length and 2.5nm in diameter, connected by flexible disulfide bonds near one each of each chain. Fibronectin protein binding involves the interaction of charged groups at the surface of the molecule. Garcia et al.26) demonstrated that precoating of substrates with fibronectin resulted in higher cell detachment strength. Apart from an initial attachment enhancement, fibronectin has been found to be a survival factor for differentiated osteoblasts. 27)
Fibronectin has been reported to have globular and filamentous forms depending on substrate and solution conditions.28)The globular, compact form of a particle may represent collapsed fibronectin molecules, which is the form found in solution under normal physiologic conditions. At temperature above 40。C, extreme pH levels, or increased ionic strength, fibronectin assumes a more extended form. From the study of Wolff and Lai,29) the energy transfer of fluorescein-labeled amino terminus groups and coumarin-labeled sulfhydryl groups was completely reduced, suggesting that interchain separation occurs during protein adsorption. Of the two protein chain sulfhydryl groups, one (SH-1) becomes exposed while the other (SH-2) remains buried when plasma fibronectin binds to polystyrene beads. These conformational changes can be expected to influence further the protein's exposed potential binding sites.
Magnetic field may be one of variables influencing the conformational change of fibronectin. Further
investigations are needed to know more information about interaction and its mechanism of magnetic field and serum protein.
Conclusion
1. A weak magnetic field increased fibronectin adsorption on titanium disks.
2. The effect of magnetic field was significantly higher in 20 and 100 gauss than 0 gauss.
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