KR20150092662A - Method for Manufacturing Surface- Treated Implant - Google Patents

Abstract

The present invention relates to a manufacturing method of a surface-treated implant. According to an embodiment of the present invention, provided is a manufacturing method of a surface-treated implant, which comprises the steps of: preparing an implant; anodic oxidation treating on the surface of the implant; and immersing the anodic oxidation treated implant in modified simulated body fluid (SBF).

Description

TECHNICAL FIELD [0001] The present invention relates to a method for manufacturing a surface-treated implant,

The present invention relates to a method of manufacturing a surface-treated implant.

Clinical success of dental implants depends on various factors such as the biocompatibility of the implant materials, the macroscopic and microscopic properties of the implant surface, the condition of the implant bed, the surgical technique itself, the complete healing phase, the subsequent prosthetic design and the long term loading stage. Among these factors, the characteristics of the implant surface are crucial to the initial healing stage for successful osseointegration. This is because the physical and chemical properties of the implant surface affect the interaction between the implant surface and neighboring biological systems, which in turn affects the rate of bone adhesion and the amount and quality. The chemical composition and the degree of hydrophilicity and roughness of implant surface are important properties in the interaction of implant and tissue and osseointegration.

Therefore, various attempts have been made in order to achieve rapid healing and early loading by deforming the implant surface. Implant surface deformation is mainly related to surface shape and surface material. There are many in vitro and in vivo studies showing that implants with moderate surface roughness have many advantages such as clinically early fixation and long term stability. Currently, tooth dental implants with micro-level roughness are widely used in the dental field. In addition, it is known that properties such as surface energy and hydrophilicity of the implant surface may be important during the initial state of protein regulation and during the initial cell adhesion period. If the hydrophilicity is sufficient, the blood component increases the initial ability to regulate the surface of the implant and affects subsequent cellular responses. Therefore, hydrophilic surfaces with high surface energies have been accepted as ideal for bone implants. As part of an attempt to increase the surface energy and hydrophilicity of the titanium implant surface, an SLActive surface treatment method has been introduced which is treated in a nitrogen atmosphere to prevent air exposure and stores it in a sealed tube containing an isotonic NaCl solution. In addition, UV treatment of titanium surfaces is another attempt to increase the surface energy and surface hydrophilicity of titanium implants.

On the other hand, osteoclasts are responsible for the absorption of mineral tissue and are multinucleated giant cells derived from mononuclear / macrophage hematopoietic system. The osteoclast formation depends on the microenvironment, which is composed of both cell-binding factors and soluble factors provided by the bone marrow stromal cells or special osteoblasts lined up along the bone surface. There are currently two cytokine receptor activator of nuclear factor kappa B ligand (RANKL) and colony-stimulating factor-1 (CSF-1) , And osteoclast differentiation is achieved by subsequent activation of receptor activator of nuclear factor kappa B (RANK) on the surface of hematopoietic progenitor cells. The mature polynuclear osteoclasts are activated by signaling, thereby initiating bone remodeling. The mature osteoclasts can move along the bone surface and locate and attach to the area to be absorbed, establish a seal zone, proton to absorb minerals and organic matter in the bone, and form a wrinkle membrane that specializes in hydrolytic secretion It is a cell with excellent mobility and forms an absorption space.

For the successful fixation of the implant, it is desirable to activate bone formation but inhibit bone resorption in order to enable bone formation around the implant as early as possible in the initial stage. It is also required that the bone resorption around the implant be minimized for the long-term stability of the implant during the maintenance phase. That is, in order to minimize the bone absorption around the implant, a technique for inhibiting the formation of osteoclasts around the implant is needed. In view of this point, it is questionable whether osteoclast formation can be inhibited through the surface treatment of the implant.

An object of the present invention is to improve the success rate of implant treatment by inhibiting osteoclast formation around the implant through surface treatment of the titanium implant.

In order to accomplish the above-mentioned object, a representative configuration of the present invention is as follows.

According to one aspect of the present invention, there is provided a method of manufacturing a surface-treated implant, comprising the steps of preparing an implant, performing anodic oxidation on the surface of the implant, and implanting the anodized implant with a modified SBF body fluid, comprising the step of immersing the implant in a body fluid.

According to the present invention, osteoclast formation around the implant can be suppressed through the surface treatment of the titanium implant, and the success rate of the implant treatment can be improved.

1 is a view illustrating a method of manufacturing a surface-treated titanium implant according to an embodiment of the present invention.
FIG. 2 is a graph showing TRAP activation on days 3 and 6 of culture of cells cultured on the surface of four groups of titanium samples. FIG.
Figure 3 is an FE-SEM image of RAW cells differentiated on the surface of four groups of titanium samples.
Figure 4 is a CLMS photograph of RAW cells differentiated on the surface of four groups of titanium samples.
5 is a graph showing osteoclast gene transcription factor expression levels of cells cultured on the surface of four groups of titanium samples.
Figure 6 is a photograph showing Western blots of NFATc1, c-Fos and beta-actin of cells cultured on four groups of titanium sample surfaces.

DETAILED DESCRIPTION OF THE INVENTION The following detailed description of the invention refers to the accompanying drawings that illustrate specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. It should be understood that the various embodiments of the present invention are mutually exclusive, but need not be mutually exclusive. For example, certain features, specific structures, and specific features described herein may be implemented in other embodiments without departing from the spirit and scope of the invention in connection with one embodiment. It is also to be understood that the position or arrangement of the individual components within each disclosed embodiment may be varied without departing from the spirit and scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is to be limited only by the appended claims, along with the full scope of equivalents to which such claims are entitled. The length, area, thickness, and shape of the embodiments shown in the drawings may be exaggerated for convenience.

[Preferred Embodiment of the Present Invention]

Hereinafter, a method of manufacturing a surface-treated titanium implant for improving osteogenesis on the titanium implant surface as well as inhibiting osteoclast and inhibiting osteoclast-induced bone resorption will be described.

1 is a view illustrating a method of manufacturing a surface-treated titanium implant according to an embodiment of the present invention.

Referring to FIG. 1, as a first step, a titanium implant for surface treatment can be prepared (S1).

As a second step, the surface of the titanium implant can be anodized (S2). Anodic oxidation refers to a treatment method in which a metal is immersed in an electrolytic solution, and an electrolytic solution is used as a cathode as a cathode and electrochemically forms an oxide film on the surface of the metal. According to one embodiment of the present invention, the anodizing treatment on the surface of the titanium implant can proceed with a final voltage of 300 V in a constant current mode of 70 A / m < 2 >. The electrolyte solution used for the anodic oxidation may be 0.15 M calcium acetate monohydrate and 0.02 M calcium glycerophosphate. After anodizing, titanium implants can be washed with distilled water and anhydrous alcohol, dried, and then sterilized with ethylene oxide.

The surface roughness of the anodized titanium implant surface was measured by an optical interferometer (Accura 2000), and the average surface roughness was found to be 0.873 ± 0.162 μm.

As a third step, anodized titanium implants can be immersed in a modified simulated body fluid (SBF). In order to prepare the modified SBF, (1) the two thermodynamically stable stock solutions (solution A and solution B) were filtered with a filter, (2) solution A and solution B were mixed at a ratio of 1: 1, Lt; / RTI > The specific components contained in the solution A and the solution B are as shown in Table 1. Each of the components of the solutions A and B described in Table 1 is dissolved in a 1.00 M HCl solution to constitute the solutions A and B .

Further, in order to control the carbon content in the modified SBF, a 5% CO2 atmosphere can be maintained. Table 2 shows the ionic composition of modified SBF and actual plasma prepared by this method.

Anodized titanium implants can be immersed in the modified SBF produced by this method. Titanium implants can be immersed in modified SBF at 37 ° C, pH 7.4, p (CO2) 0.05 atm for 2 weeks.

Once the immersion in the modified SBF is complete, it can be rinsed with distilled water and deionized water and dried.

[Experimental Example]

The inventor of the present invention conducted the following experiment to demonstrate the effect of the titanium implant manufactured according to one embodiment of the present invention.

1. Titanium sample preparation

First, after preparing a plurality of titanium samples each having a thickness of 1 mm and a diameter of 25 mm, the respective titanium samples were classified into the following four groups.

Group I: Processed and modified SBF non-soaked group

Group II: Processed and modified groups immersed in SBF

Group III: Anodic oxidation treatment and modification Groups not immersed in SBF

Group IV: Anodized and modified SBF group

Here, the processing means that the surface of the titanium implant is cleaned using an ultrasonic cleaning apparatus and ethanol and stored in distilled water for 24 hours, which means that only the mechanical cleaning treatment is performed without the anodizing treatment.

2. Cell culture

Rat macrophages RAW 264.7 macrophages / mononuclear cells (TIB-71; ATCC) were cultured on four groups of titanium sample surfaces. RANKL RAW 264.7, an extra-differentiated mouse bone marrow macrophage line, is a widely accepted model and is commonly used for in vitro studies of osteoclastogenesis. Gibco, NY, USA) containing 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA), penicillin 100 U / ml and streptomycin 100 μg / And macrophages of rats were cultured under the condition of 5% CO 2 atmosphere and 37 ° C. Cells were grown at a density of 0.5 × 10 4 cells / well and the next day, 100 ng / ml of RANKL was supplied to the rats. Median alpha and RANKL were changed every 2 days. Cells were cultured for up to 11 days.

3. Analysis of tartaric acid-resistant phosphoric acid degradation (TRAP) activation

Tartaric acid-resistant phosphorylation (TRAP) activation of the titanium surface is measured to quantitatively assess osteoclast differentiation activity.

TRAP activation was measured using a TRACP assay kit (Takara, Kyoto, Japan) after 3 and 6 days of culture according to the analysis protocol. TRAP activation of cultured cells in each of the four groups of titanium samples was investigated by colorimetric analysis utilizing the conversion of colorless p-nitrophenol phosphate (pNPP, Takara) to colored p-nitrophenol. The sample absorbance was measured spectrophotometrically at 405 nm (Bio-Rad, Hercules, Calif., USA). Measurements were made in triplicate and repeated in 6 cultures (n = 6).

FIG. 2 is a graph showing TRAP activation on days 3 and 6 of culture of cells cultured on the surface of four groups of titanium samples. FIG. Referring to FIG. 2, there was a significant difference in TRAP activities of cells grown on the titanium surface as a result of immersion in modified SBF on the 6th day of culture. However, there was no significant difference between the groups on the third day of culture. On day 6, TRAP activation was significantly higher in the cells grown on the anodized titanium surface (Group IV) immersed in the modified SBF than in the cells grown on the anodized titanium surface (Group III) without being immersed in the modified SBF Respectively. Similarly, TRAP activation of cultured cells on the treated titanium surface (Group II) immersed in the modified SBF was significantly lower than that of the treated surface (Group I) without immersion in modified SBF.

That is, there was no statistically significant difference between the groups on the third day of culture, but on the sixth day, group II and group IV showed lower values compared with the respective groups I and III.

4. Field emission scanning electron microscopy (FE-SEM)

Field emission scanning electron microscopy (FE-SEM) is used to predict and compare the differentiation of osteoclasts on titanium surfaces.

Cells cultured on each of the four groups of titanium surfaces were fixed with 2% glutaraldehyde in phosphate buffered saline (PBS) for 10 min at room temperature after 11 days of incubation, It was colored by hate and gradually dehydrated by increasing ethanol. Titanium samples containing the cultured cells were sputter coated with 6 nm platinum after critical point drying and examined on Field Emission Scanning Electron Microscope (FE-SEM).

Titanium surfaces were observed at low 15 kV accelerating voltage using FE-SEM, and high magnification (Χ 100, Χ 500) FE-SEM observations were performed in five different parts.

Figure 3 is an FE-SEM image of RAW cells differentiated on the surface of four groups of titanium samples. (magnification 100 times), (b) is a surface of a titanium sample of group II (magnification 100 times), (c) is a surface of a titanium sample of group III (magnification of 100 times) (d) is the surface of the titanium sample of group IV (100-fold magnification), (e) osteoclast differentiated in (a) (2,000 magnification), (f) osteoclast differentiated in (c) 2,000 times), and arrows in (a) and (c) represent differentiated osteoclasts.

3, it can be seen that the titanium sample immersed in the deformed SBF does not progress to osteoclast differentiation in comparison with the surface of the titanium sample immersed in the deformed SBF.

5. Confocal Laser Scanning Microscope (CLMS)

To determine the degree and type of adhesion of differentiated osteoclasts on the surface of the four groups of titanium samples, RAW cells cultured on titanium surface for F-action (cytoskeleton) and DNA (nucleus) were immunostained and observed with CLMS .

After 11 days of culture, the cells were washed twice with PBS, fixed in formaldehyde 4% solution (Sigma-Aldrich, BUchs, Switzerland) for 10 minutes at room temperature, and washed three times with PBS. Thereafter, it was allowed to permeate in PBST (containing 0.25% Triton X-100 in PBS) for 10 minutes, washed 3 times in PBS, and then blocked with 1% BSA for 1 hour at room temperature. The actin cytoskeleton was stained with 1: 100 phalloidin in PBS for 1 hour, and the nuclei were stained for 15 minutes with DAPI 1: 1000 in PBS. Four groups of titanium samples, including cells using confocal laser scanning microscopy (CLSM), were photographed at three different points. 100 times magnification was used to observe the overall differentiation activation of the cells in the osteoclasts on each titanium surface. A 400 fold magnification was used to identify polymorphonical osteoclasts.

Figure 4 is a CLMS photograph of RAW cells differentiated on the surface of four groups of titanium samples. (a) is a group I, (b) is a group II, (c) is a group III, and (d) is a confocal photograph (100 times magnification) of a RAW cell on the titanium sample surface of the group IV. There was no significant difference between CLMS images of Group I and Group II. In both groups, the cells showed a mixed distribution similar to the fusiform and round shaped cells. Fully differentiated or partially differentiated osteoclasts with podosomes, Actin rings, and three or more nuclei were rarely found in the images of the two groups. In contrast, there was a difference in the differentiated osteoclasts found between Group III and Group IV. In group III, differentiated osteoclasts were observed, but in group IV, the number of differentiated osteoclasts found was much lower than in group III.

6. Real-time RT-PCR (RT-PCR)

To compare the level of c-Fos mRNA expressed at the early differentiation stage, real-time PCR of c-Fos on the first and third day after RANKL and NFATAc1 supplementation performed by c-Fos is performed.

Real-time RT-PCR (RT-PCR) was performed to determine the amount of osteoclast gene mRNA expression for the reference gene HPRT and the following two target genes.

a. c-Fos (1 day after RANKL supplementation)

b. NFATc1 (3 days after RANKL replacement)

Total RNA was isolated from cell samples after 1 and 3 days of RANKL supplementation. Cells were isolated by adding 1 mL of TRIzol® reagent (Invitrogen, Carlsbad, Calif., USA) per 5 × 10 6 cells. The homogenized sample was incubated at room temperature for 5 minutes to allow complete dissociation of the nuclear protein complex. 0.2 mL of chloroform was added to each sample, and the samples were mixed well and incubated again at room temperature for 3 minutes. The samples were centrifuged at 12,000 G for 20 minutes at 4 ° C. After centrifugation, the mixture was separated into low red phenol chloroform phase and interphase and upper aqueous phase. The aqueous phase was mixed with 0.5 mL of isopropyl alcohol to precipitate RNA. Samples were incubated at RT for 20 min and centrifuged at 12,000 G for 15 min at 4 ° C. The RNA precipitate forms a gel-like pellet on the side and bottom of the tube. After removing the supernatant, the RNA pellet was washed with 1 mL of 75% ethanol and centrifuged at 12,000 G for 10 minutes at 4 ° C. Finally, after drying the RNA pellet for 10 minutes, the RNA was dissolved in RNase-free water for 10 minutes and cultured at 55 ° C. Total RNA was quantified using a spectrophotometer (ThermoScientific Nanodrop Technologies, Wilmington, DE, USA).

1 μg of total RNA was reverse transcribed to cDNA at 42 ° C. for 50 minutes using Superscript II reverse transcriptase (Invitrogen) containing oligo (dT) 2.5 mM dNTP, 5 × FS buffer, 0.1 M DTT, SSII enzyme and RNase.

5'-CTTTCA GCAGATTGGCAATCTC-3 'and c-fos [(F) 5'-CCACAGGGACTAGAACACCTGCTAA-3, (R) 5'-CTTGTGGACTGTGTGACT- Mouse primers such as NFATc1 [(F) 5'-CGGCTGCCTTCCGTCTCA TAG-3 ', (R) 5'-CGGCTGCCTTCCGTCTCATAG-3'

Real-time PT-PCR was performed in iCycler (Bio-Rad, Hercules, Calif., USA) using SYBR Green (Invitrogen) detection. For each reaction, a final volume of 20 μl contains 5 μl of cDNA, 0.4 μl of forward and reverse specific primers and 0.4 μl of Rox. The amplification program consists of a pre-incubation step for template cDNA (3 min, 95 ° C) denaturation, followed by 40 cycles of denaturation (15 sec, 95 ° C), heating and cooling (15 sec, 60 ° C) 30 seconds, 72 ° C). The correlation level of c-fos and NFAcT1 was normalized to HPRT.

5 is a graph showing osteoclast gene transcription factor expression levels of cells cultured on the surface of four groups of titanium samples. (a) shows the level of c-Fos mRNA on the first day, and (b) shows the level of NFATc1 mRNA on the third day.

The c-Fos mRNA was anodically oxidized and significantly lower in the cells grown on the surface of the titanium samples immersed in the modified SBF compared to the surface of the titanium samples immersed in the modified SBF. However, there was no statistically significant difference between the surfaces of the treated titanium samples depending on whether they were immersed in the modified SBF.

In addition, NFATc1 mRNA expression was anodically oxidized compared to the titanium surface immersed in the modified SBF and significantly lower in the cells grown on the titanium surface immersed in the modified SBF. However, the treated surface showed a similar tendency.

In other words, c-Fos mRNA expression was significantly lower on the anodized surface when immersed in modified SBF, and there was a significant difference in the expression of NFATc1 mRNA between the immobilized group and the non-immobilized group in modified SBF.

7. Western Blot

RANKL supplementation Western blots were performed on the following two proteins in RAW cells growing around the titanium samples on days 0, 1, 2, and 3. β-Actin was used for loading control.

a. c- Fos

b. NFATc1

Total cell lysates were washed with phosphate buffered saline (PBS) and then washed with cold RIPA buffer (10 mM Tris pH 7.2, 150 mM NaCl, 5 mM EDTA, 1 mM NaF, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate , 1 mM phenylmethylsulfonyl fluoride, 1 μg / ml leupeptin, 1 μg / ml aprotinin, 1% Triton X-100, 0.1% SDS and 1% deoxycholate). Total cell lysates were incubated for 20 minutes and centrifuged at 14,000 rpm for 20 minutes at 4 ° C. The collected proteins were separated by 8% or 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane (Whatman GmbH, Dassel, Germany). This membrane was probed with a specific antibody and immunocomplex reactivity was detected using an ECL (enhanced chemiluminescence) reagent.

Figure 6 is a photograph showing Western blots of NFATc1, c-Fos and beta-actin of cells cultured on four groups of titanium sample surfaces.

c-Fos protein levels peaked at 1 day after RANKL supplementation, decreased over time, and thereafter NFATc1 was induced. NFATc1 protein levels peaked after 3 days of RANKL supplementation. This tendency was observed in all experimental groups.

No significant differences in c-Fos protein were observed on the treated surface after immersion in modified SBF. However, in the case of the anodized surface, the group immersed in the modified SBF lowered the c-Fos protein expression compared to the group not immersed in the modified SBF. On the third day, NFATc1 protein was significantly reduced by immersion in modified SBF on both surfaces. β-Actin was used as a load control device.

In other words, on both treated and anodized surfaces, the NFATc1 protein levels were significantly different in the modified SBF, while the c-Fos protein was significantly different only in the anodized surface.

8. Conclusion

A prominent feature of osteoclast differentiation is the increased amount of TRAP. TRAP enzyme activation was anodized after 6 days of RANKL supplementation and lowest in cells cultured on titanium surface immersed in modified SBF.

As a result of FE-SEM observation and CLMS observation, the number of osteoclasts differentiated from the titanium surface immersed in the anodized and deformed SBF was the smallest when compared with the control group.

To explain the effect of titanium surface immersed in anodic oxidation and modified SBF on the osteoclast differentiation activity at the molecular level, the effect of two major transcription factors related to osteoclast formation, NFATcl and c-Fos, on gene expression . Hereinafter, the relationship between gene expression of NFATc1 and c-Fos and osteoclast differentiation will be described.

FIG. 7 is a diagram illustrating the osteoclast formation process. 7, osteoclasts are derived from bone marrow mononuclear cells / macrophage lineage progenitor cells, and two cytokines, macrophage colony stimulating factor (M-CSF) and RANKL, are basic for osteoclast differentiation into progenitor cells . RANKL is mainly expressed in a membrane-bound form for osteoblasts. M-CSF secreted by osteoblasts leads to cell proliferation and osteoclast formation Responsible for survival signals.

The RANKL signal, coupled with the receptor RANK expressed on the precursor cells, activates TRAF-6 and c-Fos and the calcium signaling pathway, both of which are activated T cells (NFATc1), the master transcription factor for bone formation, Is essential for the induction and activation of nuclear factor. NFATc1 is the most strongly inducible transcription factor after RANKL stimulation. It binds to its own promoter and automatically amplifies its own gene, whose induction depends on c-Fos. This signaling pathway induced by RANKL also induces and activates two transcription factors, active protein 1 (AP-1) through c-Fos and nuclear factor kappa B (NF-kB) through TRAF-6. The AP-1 transcription factor consisting of c-Fos, Jun and ATF members is crucial for NFATc1 induction in the early stages of differentiation and is an important transcriptional partner of NFATc1. At the end of differentiation, NFATc1 cooperates with AP-1 to induce osteoclast-specific genes such as TRAP, calcitonin receptor and cathepsin K.

NFATc1 mRNA levels were found to be significantly lower in RAW cells cultured on titanium surfaces immersed in modified SBF compared to non-immersed in modified SBF. The c-Fos mRNA levels were also anodically oxidized compared to the other groups and significantly lower on the titanium surface immersed in the modified SBF.

Thus, NFATc1 protein expression and c-Fos protein expression levels were anodized and lowest on the titanium surface immersed in the modified SBF.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken in conjunction with the present invention. Variations and changes are possible. Such variations and modifications are to be considered as falling within the scope of the invention and the appended claims.

Claims (4)

A method for producing a surface-treated implant,
Preparing an implant,
Anodic oxidation of the surface of the implant, and
Immersing the anodized implant in a modified SBF (simulated body fluid)
≪ / RTI > The method according to claim 1,
Wherein the implant is formed of titanium (Ti). The method according to claim 1,
Wherein the modified SBF comprises at least one of Na ⁺ , Cl ^- , HCO ^{3 -} , K ⁺ , Mg ^{2 +} , Ca ^{2 +} , HPO ₄ ^{2 -} , and SO ₄ ^{2 -} Way. The method according to claim 1,
Wherein the anodizing treatment is performed with a final voltage of 300 V in a current mode of 70 A / m < ² >.

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