KR102265511B1 - Method for treating surface of Titanium Substrate - Google Patents

Abstract

_{The present invention comprises the steps of supplying nitrogen (N 2} ) gas to a substrate including titanium or a titanium alloy to form a nitrided surface; And it relates to a surface treatment method of a titanium substrate, comprising the step of forming a porous layer by anodizing the substrate on which the nitrided surface is formed in _{an electrolyte solution containing at least one of phosphoric acid (H 3} PO _{4 ) and hydrofluoric acid (HF).} .

Description

Method for treating surface of Titanium Substrate

The present invention relates to a method for surface treatment of a titanium substrate.

For therapeutic or cosmetic purposes, medical implants such as implants, dental implants, stents, screws, and bone substitutes are inserted. However, when this medical implant is inserted into the body, it is recognized as an external substance in the body, and a coagulation mechanism is activated or a foreign body reaction occurs in order to prevent bleeding and blood loss. In addition, since medical implants are in permanent contact with living tissues, they must have both surface compatibility and in vivo compatibility.

Recently, in order to improve the osseointegration of existing implants, one is a method to increase implant osseointegration by methods such as implant biomaterials, implant design, surface treatment, surface energy, surface chemistry, etc. In order to optimize osseointegration, low-level laser therapy or a method of photobiological control using light emitting diodes is being developed (Korea Registration Application No. 1845813).

In addition, when the implant is exposed in the oral cavity, a colony of bacteria causing periodontitis is formed within 2 weeks, and the subgingival flora is completed within 4 weeks, which may lead to implant failure.

Therefore, there is a need for an effective method for inhibiting bacterial growth in the oral cavity and immobilizing a bioactive material on the surface of a medical implant.

Accordingly, the present invention devised a surface-treated implant capable of inhibiting bacterial growth in the oral cavity and increasing the cell activity of periodontal tissue.

One object of the present invention is Streptococcus mutans ( Streptococcus mutans ) or Porphyromonas gingivalis ) While inhibiting the adhesion of harmful bacteria present in the oral cavity, such as, at the same time, periodontal cells such as gingival fibroblasts or osteoblasts An object of the present invention is to provide a method for treating a surface of a titanium substrate capable of increasing tissue adhesion and proliferation.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless specifically defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, " Streptococcus mutans " is a Gram-positive facultative bacteria, commonly found in the oral cavity, and has cilia, so it is easier to attach to the tooth surface or tissue than other bacteria, and mainly forms an initial bacterial film. The bacterial film formed by Streptococcus mutans accumulates down to the subgingival and facilitates late colonization by inducing adhesion of bacteria such as Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , and Fusobacterium nucleatum called 'red complex' through interaction.

In the present invention, " Porphyromonas gingivalis " is a Gram-negative bacterium, commonly found in the periodontal pockets of periodontitis patients, and can be observed in the osseointegration site where peri-implantitis has progressed. Since Porphyromonas gingivalis invades and proliferates in various tissues or cells of the host, adhesion of Porphyromonas gingivalis in the oral cavity may lead to pathogenicity. It is mainly found in the oral cavity and is known to be related to periodontal disease, but it is also found in the upper gastrointestinal tract, airways, and large intestine, and is also associated with bacterial vaginosis in women.

In the present invention, "peri-implantitis" is inflammation similar to periodontitis caused by a biofilm formed on the implant surface. The bacterial membrane is composed of various bacterial flora, and an acquired pellicle is formed as salivary proteins are attached to the surface. Unlike natural teeth, there is no periodontal ligament that protects the gum bone as the implant and the gum bone are agglutinated (ankylosis). For this reason, if food residues remain around the implant, plaque or tartar is not removed well, or if strong masticatory force is applied, inflammation may occur more easily than natural teeth. Unlike natural teeth, implants do not have blood vessels and nerve tissue, so there is a high probability that bone tissue damage will be severe without feeling any special symptoms. When this inflammation spreads to the bone tissue, bone loss occurs. Therefore, suppressing the formation of a bacterial film on the implant surface is very important for maintaining the implant.

In the present invention, "graphene" means one layer of carbons arranged in a honeycomb shape. It has a two-dimensional planar shape, and its thickness is 0.2 nm (1 nm is 1 billionth of a meter), that is, it is extremely thin, about 2 billionths of a meter, and has high physical and chemical stability. It conducts electricity 100 times better than copper and can move electrons 100 times faster than single crystal silicon, which is mainly used as a semiconductor. The strength is more than 200 times stronger than steel, and the thermal conductivity is more than twice that of diamond, which boasts the highest thermal conductivity. In addition, it has excellent elasticity and does not lose its electrical properties even when stretched or bent. It has more uniform metallic properties than carbon nanotubes. Graphene is relatively hydrophobic and is typically formed by exfoliation of graphite, which can occur using supercritical carbon dioxide, or by micromechanical cleavage, or by epitaxial growth on silicon carbide or certain metal substrates. do. Graphene can also be formed in the gas phase by passing droplets of ethanol into an argon plasma in an atmospheric pressure electromagnetic plasma reactor (Dato et al., "Substrate-Free Gas-Phase Synthesis of Graphene Sheets", Nano Lett., Vol. 8, pp. 2012-2016, 2008]).

In the present invention, "graphene derivative" includes a structure having a graphitic bond in which heteroatoms such as oxygen or structural defects in the carbon lattice are partially introduced. In general, the term graphene derivative also includes nanotubes, nanobuds, fullerenes, nano-pepods, endofullerenes, nano-onions, graphene oxide, lacy carbon, and others that may contain structural or chemical defects. It also contains structures such as graphitic carbon in the non-graphene form.

In the present invention, "gingival fibroblast" refers to a representative cell constituting the soft tissue surrounding the dental implant. As the degree of adhesion of gingival fibroblasts to the implant increases and proliferation increases, it helps the implant to be stably seated in the living body.

According to an embodiment of the present invention, _{the step of supplying nitrogen (N 2} ) gas to a substrate including titanium or a titanium alloy to form a nitrided surface; and anodizing the substrate on which the nitrided surface is formed in _{an electrolyte solution containing at least one of phosphoric acid (H 3} PO ₄ ) and hydrofluoric acid (HF) to form a porous layer. .

In the present invention, the forming of the nitrided surface may be performed while supplying nitrogen gas to the substrate including the titanium or titanium alloy under a vacuum atmosphere, but is not limited thereto.

In the present invention, the step of forming the nitrided surface may be performed at a temperature of 900 to 1100 °C. In the present invention, when the nitridation surface is formed, when the operating temperature is less than 900 ° C, there is a problem in that the internal temperature is low due to the nitriding treatment and thus the hardness is formed low, and when the execution temperature is 1100 ° C. There is a problem that the surface is deteriorated.

In the present invention, the step of forming the nitrided surface may be performed for 1 to 12 hours, preferably 2 to 6 hours, but is not limited thereto.

Forming the porous layer in the present invention may be performed for 40 to 80 minutes by applying a voltage of 10 to 30 V, preferably 20V. In the present invention, when the anodization is carried out below the range of the voltage or time, there may be a problem that it is formed in a thin non-porous form, or that a Mulberry-shaped coating layer cannot be obtained after applying graphene, and the voltage or If it is performed over the time range, there may be a problem in that a voltage drop occurs due to local stress and non-uniform anodization film thickness.

After the step of forming the porous layer in the present invention may further include the step of treating the plasma to the substrate on which the porous layer is formed, but is not limited thereto.

In the present invention, the plasma may be atmospheric pressure plasma.

In the present invention, the plasma may be generated under a carrier gas atmosphere of methane, argon, or a mixture thereof.

In the present invention, the carrier gas may be injected at a flow rate of 2 to 10 L/min, preferably 3 to 5 L/min.

In the present invention, the plasma may be treated for 2 to 10 minutes, preferably 4 to 8 minutes, but is not limited thereto.

In the present invention, after the plasma treatment, the step of coating graphene or a graphene derivative on the plasma-treated substrate may be further included, but the present invention is not limited thereto.

In the present invention, the surface of the porous layer is formed to exhibit a Mulberry shape, that is, a mulberry shape of a mulberry fruit. More specifically, the surface of the porous layer is formed by a phenomenon in which pores having a large diameter and pores having a small diameter are interspersed.

In the present invention, the porous layer is formed to include large pores and small pores of two types of sizes. The average size of the air pores in the porous layer may be 50-100㎛, the average size of the small pores may be 25-30nm.

In the present invention, the porous layer has 2 to 8, preferably 3 to 7, and more preferably 5 small pores per 10000 nm ^{2 unit area (that is, when the horizontal and vertical sizes are 100 nm).} can

As a surface treatment object of the present invention, it is preferable that the substrate including titanium or titanium alloy is for dental implants.

In the case of the surface treatment method of the titanium substrate according to the present invention, harmful bacteria present in the oral cavity on the surface of the titanium substrate that can be used for dental implants, for example, Streptococcus mutans or Porphyromonas Gingivalis ( Porphyromonas gingivalis ) It can prevent bacterial adhesion.

According to the surface treatment method of the titanium substrate according to the present invention, it is possible to increase the cell activity by increasing the adhesion and proliferation of gingival fibroblasts and/or osteoblasts to the surface of the titanium substrate that can be used for dental implants.

According to another embodiment of the present invention, there is provided a titanium substrate treated by the surface treatment method of the titanium substrate of the present invention.

In the present invention, the titanium substrate is preferably for dental implants.

According to another embodiment of the present invention, there is provided a dental implant comprising the surface-treated titanium substrate of the present invention.

The surface treatment method according to the present invention inhibits the adhesion of harmful bacteria present in the oral cavity, such as Streptococcus mutans or Porphyromonas gingivalis, to the surface of a titanium substrate, and at the same time, gingival fibers By increasing the adhesion and proliferation of periodontal tissues such as blasts or osteoblasts, when the treated titanium substrate is used as a dental implant, there is an effect of advancing an effective and stable healing process after periodontal surgery.

1 is a scanning electron microscope photograph in which an anodization treatment time is changed after nitridation according to the present invention.
2 is a graph showing the results of analysis using an X-ray photoelectron spectrometer.
Figure 3 is a graph showing the evaluation of the adhesion of live bacteria and dead bacteria to Streptococcus mutans, respectively.
Figure 4 is a graph showing the evaluation of the adhesion of live bacteria and dead bacteria to Porphyromonas gingivalis, respectively.
5 is a graph showing the evaluation results of adhesion and proliferation of gingival fibroblasts L-929 cells.
6 is a graph showing the evaluation results of adhesion and proliferation of osteoblast MC3T3-E1 cells.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited by the following examples.

Example

Material preparation and test method

Titanium Specimen Preparation

A titanium disk (ASTM Grade Ⅳ, Kobe Steel Co., Kobe, Japan) (T) was prepared with a diameter of 15 mm and a thickness of 3 mm. The titanium disk was polished with #600 SIC abrasive paper in a polishing machine (Labopol-5, Struers, Denmark), and was dried after ultrasonic cleaning for 20 minutes each with acetone, ethanol, and distilled water.

Nitrided surface formation

Using a vacuum nitriding furnace (Mirae Thermotec, Daegu, Korea) to the prepared specimen, N ₂ gas was supplied at a process temperature of 1020° C. in a vacuum atmosphere for 4 hours and then cooled to form a (N) nitrided surface.

Anodizing process

To form a porous surface, anodization was performed for 60 minutes by applying a voltage of 20V in _{1M H 3} PO _{4 + 1.5wt% hydrofluoric acid electrolyte.} A titanium specimen was connected to the positive electrode and a platinum plate was connected to the negative electrode, and the specimen and the platinum plate were spaced about 10 mm apart. The connected specimen and the platinum plate were immersed in the electrolyte, and after 1 minute, a voltage of 20V was applied and anodized for 60 minutes.

Plasma treatment

A mixed gas of methane and argon was applied to the porous surface treated with nitridation and anodization at a flow rate of 4 L/min. Atmospheric pressure plasma (PGS-300 Expantech Co. Suwon. Korea) was used for 6 minutes. (G) The graphene derivative was coated.

surface morphology analysis

The surface morphology was observed using a scanning electron microscope (FE-SEM S-4700, Hitachi horiba, Japan) in a vacuum state. The chemical composition and bonding state of the surface were analyzed using an X-ray photoelectron spectrometer (XPS, VG Mulrilab 2000, Thermo scientific, UK).

Assessment of bacterial attachment and proliferation

Streptococcus mutans ( Streptococcus mutans ) 24 hours and Porphyromonas gingivalis ( Porphyromonas gingivalis ) After 48 hours of bacterial culture, the culture medium on the specimen was removed. Thereafter, the non-adherent bacteria were removed by washing twice with a PBS (phosphate buffered saline) solution. After aliquoting 200 μl of a fluorescent reagent (SYTO9: dH ₂ O = 1.5 μl: 1 ml) to the specimen, incubated in an incubator for 15 minutes, followed by a fluorescence meter (ELISA reader: ELx 800UV, Bio-Tek Instrument. Ine, Winooski, VT, USA) was used for quantitative analysis. For cell adhesion and proliferation evaluation, mouse fibroblast L-929 cells and mouse osteoblast line MC3T3-E1 cells (MC3T3-E1 Subclone 4. ATCC CRL2593, Pockville, MD, USA) were mixed with 10% fetal bovine serum; FBS) and 100U/ml penicillin-containing α-MEM (α-Minimum Essential Medium, Dulbeco's modified Eagle's medium, Gibco-BRL, Grand Island, NY, USA) medium with 37℃, 5% CO ₂ incubator (Forma Series) II 3111 Water Jacketed CO2 Incubator, Thermo Scientific, USA). The specimens were placed in a 24-well plate and the prepared cells were aliquoted by 2 Х 10 ⁴ cell/ml. The well plate containing the specimen and cell solution was incubated _{for 24 hours and 72 hours in a 37°C, 5% CO 2} incubator, and then 100 μl of WST-8 reagent (EZ-Cytox Itsbio, Inc, Seoul, Korea) was added to each plate. was aliquoted and incubated again. In addition, 100 μl of each was transferred to a 96-well plate and absorbance was measured at 450 nm using an absorbance meter (ELISA reader: ELx 800UV, Bio-Tek Instrument. Ine, Winooski, VT, USA). Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). All results were tested for significance at the (p <0.05) level. The experimental group was divided into 4 groups according to conditions (refer to Table 1).

experimental group process T titanium N Nitride M Nitriding + Anodizing G Nitriding + Anodizing + Graphene

In the present invention, the experimental group performed with untreated titanium is indicated by a 'T' group, the experimental group performed with titanium with a nitrided surface is indicated by 'N', and the experimental group performed with titanium treated with an anodizing process after forming a nitrided surface is indicated by the 'M' group, and the experimental group performed with titanium coated with graphene derivatives by plasma treatment after formation of the nitrided surface and then anodizing process is indicated by the 'G' group.

result

Surface property evaluation

After nitriding, the anodization treatment time was varied, and observation was made using a scanning electron microscope (FE-SEM S-4700, Hitachi horiba, Japan). As a result, "Mulberry surface" was confirmed at 1 hour difference (60 minutes) of anodization (FIG. 1). Specifically, large pores with an average size of 50-100 μm and small pores with an average size of 25-30 nm were scattered and distributed, and in particular, it was confirmed that the small pores contained an average of 5 per ^{10000 nm 2 unit area.}

As a result of observation using a scanning electron microscope (FE-SEM S-4700, Hitachi horiba, Japan), porous surfaces having various sizes were confirmed. As a result of analysis using an X-ray photoelectron spectrometer (XPS, VG Mulrilab 2000, Thermo scientific, UK), it was possible to observe C, a characteristic peak of graphene, on the graphene-coated surface ( FIG. 2 ).

Bacterial adhesion assessment

As a result of quantitative analysis of the adhesion of Streptococcus mutans , it was confirmed that live bacteria relative fluorescence units were significantly reduced in group M compared to group T (Fig. 3 (Fig. 3). a)). In addition, it was confirmed that there was a significant increase in the dead bacteria relative fluorescence units ( p <0.05) (Fig. 3(b)). In the experimental group treated according to the present invention, group G, it was confirmed that the adhesion was small in both live and dead bacteria.

As a result of quantitative analysis of the adhesion of Porphyromonas gingivalis , it was confirmed that live bacteria relative fluorescence units were significantly reduced in group M compared to group T (Fig. 4). (a)). In addition, it was confirmed that there was a significant increase in dead bacteria relative fluorescence units ( p <0.05) (Fig. 4(b)). In the experimental group treated according to the present invention, group G, it was confirmed that the adhesion was small in both live and dead bacteria.

Cell activity evaluation

As a result of evaluation of adhesion and proliferation of gingival fibroblasts L-929 cells, it was confirmed that cell activity was significantly increased in group G, which is the experimental group treated according to the present invention ( p <0.05) ( FIG. 5 ).

As a result of evaluation of adhesion and proliferation of osteoblast MC3T3-E1 cells, it was confirmed that cell activity was significantly increased in group G, which is the experimental group treated according to the present invention ( p <0.05) ( FIG. 6 ).

In summary, according to the surface treatment method of the titanium implant according to the embodiment of the present invention, the adhesion of harmful bacteria present in the oral cavity, such as Streptococcus mutans or Porphyromonas gingivalis ) It was confirmed that there was an effect of advancing an effective and stable healing process after periodontal surgery by increasing the adhesion and proliferation of periodontal tissues such as gingival fibroblasts or osteoblasts while suppressing them.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (19)

Forming a nitrided surface by supplying _{nitrogen (N 2} ) gas to a substrate including titanium or a titanium alloy; and
A method for surface treatment of a titanium substrate, comprising the step of anodizing the substrate on which the nitrided surface is formed in _{an electrolyte solution containing phosphoric acid (H 3} PO _{4 ) and hydrofluoric acid (HF) to form a porous layer,}
The step of forming the nitrided surface is carried out under a temperature of 900 to 1100 ℃,
The step of forming the porous layer is performed for 40 to 80 minutes by applying a voltage of 10 to 30 V,
The porous layer is formed including large pores and small pores,
The average size of the air pores in the porous layer is 50-100㎛,
The average size of the small pores in the porous layer is 25-30nm, the titanium substrate surface treatment method. The method of claim 1,
The step of forming the nitrided surface is performed while supplying nitrogen gas to the substrate including the titanium or titanium alloy under a vacuum atmosphere, the surface treatment method of a titanium substrate. delete The method of claim 1,
The step of forming the nitrided surface is performed for 1 to 12 hours, a method for treating a surface of a titanium substrate. delete The method of claim 1,
After the step of forming the porous layer, the method for treating a surface of a titanium substrate further comprising the step of treating the plasma on the substrate on which the porous layer is formed. 7. The method of claim 6,
The plasma is atmospheric pressure plasma, a method of treating a surface of a titanium substrate. 7. The method of claim 6,
The plasma is generated under a carrier gas atmosphere of methane, argon, or a mixture thereof, a method for treating a surface of a titanium substrate. 9. The method of claim 8,
wherein the carrier gas is injected at a flow rate of 2 to 10 L/min. 7. The method of claim 6,
The plasma is treated for 2 to 10 minutes, a method of treating a surface of a titanium substrate. 7. The method of claim 6,
After the plasma treatment, the method for treating a surface of a titanium substrate further comprising coating graphene or a graphene derivative on the plasma-treated substrate. delete delete delete The method of claim 1,
The porous layer is 10000nm ^{2 A} method of treating a surface of a titanium substrate comprising 2 to 8 small pores per unit area. The method of claim 1,
The substrate comprising titanium or a titanium alloy is for dental implants, a method of surface treatment of a titanium substrate. A titanium substrate treated with the method for surface treatment of a titanium substrate according to any one of claims 1, 2, 4, 6 to 11, and 15 to 16. 18. The method of claim 17,
The titanium substrate is for dental implants, a titanium substrate. A dental implant comprising a titanium substrate treated with the method for surface treatment of a titanium substrate according to any one of claims 1, 2, 4, 6 to 11, and 15 to 16.

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