KR20040067638A - Method for modifying surface of titanium for implant using proteins - Google Patents

Abstract

PURPOSE: Provided is a surface-treated implant titanium which facilitates the adhesion of osteoblast to the bone-implant location as well as the adhesion of fibroblast to the gingival location, thereby exerting enhanced tissue healing effect. CONSTITUTION: A method of surface-treating the implant comprises the steps of: dipping implant titanium or titanium alloy in a hydroxyapatite paste, and drying it; heat-treating the implant titanium or titanium alloy at 800-900 deg.C for 1-2 hours under the atmosphere of argon or nitrogen gas; and dipping the heat-treated implant titanium or titanium alloy in a solution of type I collagen with a concentration of 0.005-0.5% or a fibronectin solution with a concentration of 0.0001-0.5% for 1-12 hours to thereby surface-coat it.

Description

Surface treatment method of implant titanium using protein {METHOD FOR MODIFYING SURFACE OF TITANIUM FOR IMPLANT USING PROTEINS}

The present invention relates to a surface treatment method of titanium or titanium alloy for implants using proteins.

Dental implants are used to permanently implant artificial teeth on the jaw bone of humans. The dental implants connect the jaw bones with the artificial teeth and handle and distribute the loads generated when chewing food. It also serves as a more stable tooth than conventional dentures. These implants are mechanically manufactured and must be made of biocompatible materials that are very stable against human tissues, free from side effects and other chemical and biochemical reactivity, and are also subject to repeated loading and instantaneous pressure implications. It is very important to choose a suitable material because the mechanical strength must be very high so as not to deform and destroy.

Various materials and alloys have been developed and tried as suitable materials for implants, and the materials of implants currently used are titanium (Ti) metals or alloys thereof [Larry L. Hench, "Bioceramics", J. Am. Ceram. Soc. 81 [7] 1705-28 (1998). Titanium or its alloys have the advantage of being easy to process and having high biocompatibility, high mechanical strength and bioinertness to human biological tissues. However, titanium and its alloys themselves have a long bonding time with the bone when implanted in the human body, and metal ions melt into the living body after a long time after transplantation.

In order to make up for these drawbacks, studies are being actively carried out on surface treatment of titanium and its alloys. Among them, the TiO ₂ oxide film present on the surface of titanium is stable in vivo, excellent in biocompatibility and positive in cell reaction. Oxidized implants have been reported to have superior bonds with bone at implantation compared to machined implants [Patrick J. Henry, Albert ES Tan, Brent P. Allan, Jan Hall and Carina Johansson]. , "Removal Torque Comparison of TiUnite and Turned Implants in the Creyhound Dog Mandible," Applied Osseointegration Research, 1 [1] 15-17 (2000)].

On the other hand, dental implants are divided into a fixed part and a circumferential part, which is applied to the human body to maintain its function by the healing of each tissue. First, the fixed part is responsible for chewing load by healing of bone tissue. Specifically, after the implant is embedded in bone, bone tissue is regenerated by attachment and proliferation of osteoblasts to perform its function as an artificial tooth. Can be. In addition, the zone of circumference is to maintain the health and function by preventing the penetration of microorganisms by the healing of soft tissues, the healing of connective tissue and epithelium of the soft tissues is important. In particular, the healing of connective tissue is the most important factor for the regeneration of soft tissues. Specifically, the attachment and proliferation of fibroblasts are the most important for healing.

Thus, the situation is required to develop the material of the implant that can improve the healing of bone tissue and soft tissue.

The present invention is to provide a surface treatment method of a dental implant that can improve the healing of bone tissue and soft tissue by applying a surface treatment to titanium or its alloy to solve the above problems.

Figure 1a shows a SEM photograph of the histological observation of the titanium metal surface before heat treatment in the present invention,

Figure 1b shows a SEM image of the histological observation of the surface of the titanium heat-treated for 1 hour at 800 ℃ in the present invention,

Figure 2a is a SAES graph showing the chemical composition of the surface of the titanium heat-treated at 800 ℃ in the present invention,

O: oxygen concentration distribution map,

Ti: Titanium atomic concentration distribution map,

P: distribution of phosphorus atom concentration.

Figure 2b is a graph showing the depth profiles (depth profiles) of phosphorus and calcium on the surface of the titanium metal heat-treated at 800 ℃ in the present invention,

P LVV2: atomic concentration distribution of phosphorus in titanium phosphide,

Ca: Distribution of atomic concentration of calcium.

Figure 3a is a SAES graph of the analysis of titanium metal after arcon sputtering for 20 seconds before heat treatment in the present invention,

3B is a SAES graph of analyzing titanium (after 80 seconds argon sputtering) metal heat-treated at 800 ° C in the present invention,

Figure 4a is a photograph showing an example of the scoring criteria of the results of centrifugal cell adhesion analysis,

Figure 4b is a photograph showing the cell adhesion in NHGF, human osteosarcoma cell lines MG63 and HOS to the protein of type I collagen or fibronectin in the present invention,

Figure 4c is a graph showing the cell adhesion level in NHGF, human osteosarcoma cell lines MG63 and HOS for protein of type I collagen or fibronectin in the present invention,

5 is an absorbance graph showing cell adhesion in NHGF, human osteosarcoma cell lines MG63 and HOS for titanium metal according to the conditions of the present invention.

In order to achieve the above object, the present invention

Depositing titanium or titanium alloy for implant in an apatite hydroxide paste and drying (step 1);

Heat-treating the titanium or titanium alloy for the implant (step 2); And

It provides a method for surface treatment of the implant consisting of the step (step 3) of coating the surface by dipping the heat-treated implant titanium or titanium alloy in a type I collagen or fibronectin solution.

That is, the surface treatment method of the implant of the present invention can further improve the surface hardness by treating the implant titanium or titanium alloy with hydroxide apatite,

In addition, the surface of titanium or titanium alloy is coated with type I collagen or fibronectin, which is a type of extracellular matrix protein, so that the bone embedded site promotes adhesion of osteoblasts, and the gingival site promotes adhesion of fibroblasts. Has the characteristics to improve the healing of tissues.

Hereinafter, the present invention will be described in detail.

First, the titanium or titanium alloy for implant is dipped in apatite hydroxide pate and dried.

Titanium for implants is characterized by easy processing and high biocompatibility, high mechanical strength and bioinertness to human biological tissues. Pure titanium metal or alloys thereof may be used in the present invention. The titanium alloy may be any titanium alloy that can be used for the implant, preferably Ti ₆ Al ₄ V may be used.

Apatite hydroxide has a structural formula of Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ , and is a kind of bioceramic containing bone inorganic components, which is prepared and used in the present invention in the form of a paste. At this time, the preparation of the paste can be performed by all methods conventional in the art.

After that, the heat treatment of the titanium or titanium alloy for implantation, through the thermal diffusion method by the heat treatment penetrates the calcium (Ca) and phosphorus (P) contained in the apatite hydroxide into the inside of titanium to improve the surface hardness of titanium or its alloys It serves to further improve. In consideration of this effect, the heat treatment is preferably carried out for 1 to 2 hours at 800 ~ 900 ℃ under an inert gas, wherein the inert gas is to prevent oxidation during heat treatment, argon gas or nitrogen gas Can be used.

Thereafter, the heat treated implant titanium or titanium alloy is immersed in a type I collagen or fibronectin solution to coat the surface. Specifically, the surface coating is performed by maintaining the implant in the solution for 1 to 12 hours.

Type I collagen is a type of extracellular matrix protein synthesized mainly by osteoblasts and fibroblasts in bone tissue, dermis, and tendons.It is involved in the durability of bone tissues and tendons, and also in the integrity of skin, major organs, and blood vessels. Plays an important role. Fibronectic is a type of extracellular matrix protein synthesized by osteoblasts and fibroblasts, and is a glycoprotein present in connective tissues, cell surfaces, plasma, and body fluids. Interact with cells through integrin or nonintegrin receptors.

The type I collagen and fibronectin play an important role in cell adhesion, migration and growth, and in the present invention, it is coated on the surface of the titanium or titanium alloy for implantation to promote osteoblast adhesion. In addition, the site located in the gingiva promotes adhesion of fibroblasts, thereby improving tissue healing.

In the present invention, in consideration of the above effects, type I collagen is used at a concentration of 0.005 to 0.5%, and fibronectin is used at a concentration of 0.0001 to 0.5%. When the concentration is less than the concentration range, there is a disadvantage that can reduce the cell adhesion of osteoblasts and fibroblasts to the titanium or titanium alloy for implant, if the concentration range is exceeded, for the preparation of protein solution and applying to titanium or titanium alloy It has a hard disadvantage.

In addition, the surface treatment method of the present invention further performs the washing step and the step of culturing by adding bovine serum albumin.

Specifically, after the surface treatment as described above, the added type I collagen or fibronectin is removed and carefully washed with phosphate-buffered saline (PBS) for 1 to several times. To block the untreated surface, bovine serum albumin is added and incubated at 37 ° C. After incubation, the bovine serum albumin is removed and carefully washed with PBS.

The implant applied by the surface treatment method is characterized in that the site embedded in bone promotes the attachment of osteoblasts, and the site located in the gingiva promotes the attachment of fibroblasts, thereby improving the healing of tissues. Due to these features, the surface treatment method of the present invention is an implant fixture that is in contact with bone, implant abutment and implant fixture in contact with gingiva and a one-piece implant (one piece implant) that is integral with abutment. It can be usefully applied.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Surface Treatment Method 1 of the Present Invention

Titanium (grade 2) having a diameter of 24 mm and a height of 1 mm was dipped in apatite hydroxide paste and dried in air.

The titanium was placed in a graphite crucible and subjected to heat treatment at 800 ° C. for 1 hour under argon gas after vacuum formation (10 ⁻² Torr).

The heat treated titanium was placed in a culture dish and 0.005% (50 μg / ml) type I collagen solution was added. Type I collagen was coated on the surface of the titanium by incubation for 12 hours at room temperature after addition.

The added Type I collagen was removed, washed carefully once with phosphate-buffered saline (PBS) and then bovine serum albumin 1 mg / ml was added and incubated at 37 ° C. for 30 minutes. Finally, the bovine serum albumin was removed and carefully washed with PBS.

Example 2 Surface Treatment Method 2 of the Present Invention

Titanium (grade 2) having a diameter of 24 mm and a height of 1 mm was dipped in an apatite hydroxide paste and then dried in air.

The titanium was placed in a graphite crucible and subjected to heat treatment at 800 ° C. for 1 hour under argon gas after vacuum formation (10 ⁻² Torr).

The heat treated titanium was placed in a culture dish and 0.005% (50 μg / ml) fibronectin solution was added. Fibronectin was coated on the surface of the titanium by incubation at room temperature for 12 hours after addition.

The added fibronectin was removed, washed carefully once with phosphate-buffered saline (PBS), and then bovine serum albumin 1 mg / ml was added and incubated at 37 ° C. for 30 minutes. Finally, the bovine serum albumin was removed and carefully washed with PBS.

Experimental Example 1 Surface Analysis

(1) SEM (Scanning Electron Microscopy)

For histological observation of the titanium material heat-treated by the present invention, the surface was analyzed by SEM (Hitachi X-650, Hiatachi, Japan). The sample used titanium before heat processing and titanium heat-processed at 800 degreeC for 1 hour. The results are shown in FIGS . 1A and 1B . Specifically, FIG. 1A analyzes the surface of titanium before heat treatment, and FIG. 1B analyzes the surface of titanium heat treated at 800 ° C. for 1 hour.

As shown in Figure 1a and Figure 1b , the heat treatment of titanium was found to grow needle-like surface texture. The surface texture of these needles grows as the heat treatment temperature increases, and the histological change of the titanium surface depending on the surface treatment temperature may significantly affect the diffusion of certain elements such as calcium and phosphorus from the apatite hydroxide. have.

(2) Scanning Auger Electron Spectroscopy (SAES)

For chemical composition analysis of the implant surface according to the present invention, surface analysis was performed using SAES (VGESCALAB 210, UK). The results are shown in FIGS . 2A- 3B .

As shown in FIGS . 2A and 2B , after forming a vacuum together with apatite hydroxide and heat-treating in an argon atmosphere, analysis of the chemical composition of the titanium material surface layer using SAES revealed that atoms of calcium and phosphorus diffused from the apatite into the titanium surface were penetrated. The concentration varied greatly with the heat treatment temperature. In the case of pure titanium, it was confirmed that the amount of P diffused up to 7% or more due to the "gettering effect" of P at Ca at 800 ° C. before the phase transformation temperature of titanium.

As shown in FIGS . 3A and 3B , the diffused phosphorus (P) was combined with titanium to observe that the kinetic energy of phosphorus in the SAES spectra changes from about 110 eV to about 120 eV, which indicates that phosphorus is in the phosphate state. It was confirmed that the titanium phosphide (titanium phosphide) was formed by changing from.

Experimental Example 2 Surface Hardness Experiment

The surface hardness of titanium which was immersed in an apatite hydroxide face and dried and then heat treated for 1 hour in an argon gas atmosphere was measured using a Vickers hardness tester. The results are shown in Table 1 below.

Materials Temp. (℃) Vickers Hardness (Hv) N.T 333.2 (20.3) ^* 800 443.5 (20.6) *: () Is the standard deviation

As shown in Table 1 , the viscous hardness before heat treatment at 480 ° C compared to 333.2 Hv, the hardness of the Vickers is 443.5 Hv after the heat treatment can be seen that the surface hardness increased by about 110 Hv. For this reason, it can be seen that the surface hardness of titanium or its alloy is further improved by treating the apatite hydroxide in the present invention.

Experimental Example 3 Centrifugal Cell Adhesion Assay

(1) Cell and Cell Culture

Primary normal human gingival fibroblasts (hereinafter referred to as "NHGF") were established from gingiva connective tissue excised from patients undergoing oral surgery. Cells proliferating out from explant cultures were continuously cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS).

In addition, human osteosarcoma cell lines were purchased from MG63 (ATCC CRL 1427) and HOS (ATCC CRL 1543) purchased from ATCC.

(2) Centrifugal cell adhesion assay

Centrifugal cell adhesion was measured according to the method of Hashimoto-Uoshima et al. (1997).

Specifically, 96-well U-bottom polyvinylchloride plastic microtiter plates (Falcon, Lincoln Park, NJ) were used as ECM elements type I collagen (50 μg / ml), fibronectin (1 μg / ml) or laminin (10 μg / ml)) was diluted in PBS and adsorbed at 4 ° C. overnight. After washing the wells, unbound sites were blocked with PBS containing 10 mg / ml BSA. Cells were detached with 10 mM EDTA (Gibco BRL) and a cell suspension containing 5 × 10 ³ cells was dispensed into each well and allowed to settle and attach for 15 minutes at 37 ° C. in 5% CO ₂ conditions. After the incubation period the plates were centrifuged at 1,000 g for 5 min at 4 ° C. (Sorval RT6000B centrifuge). The medium in the wells was carefully removed and changed to 17.5% formaldehyde containing 0.005% crystal violet in PBS for 2 hours for fixation. The effect of centrifugation on cell adhesion to the already coated ECM protein was determined by observing the cells in the round bottom wells and scored according to the method of Hashimoto-Uoshima et al . (1997). In addition, the center of each well was photographed. Mean and standard deviation were calculated from six independent experiments. The results are shown in FIGS . 4A- 4C . Specifically, Figure 4a shows an example of the scoring criteria, Figure 4b shows a cell adhesion assay picture, Figure 4c is a graph showing the cell adhesion level.

4A is an example of a scoring criterion, where 0 is fully agglomerated, 1 is centrally aggregated with a few cells resistant to desorption around, 2 is a cell resistant to desorption except a few clusters in the center, 3 represents cells resistant to desorption without precipitation or clusters in the center or around the 96 well U-bottom plates.

As shown in Figures 4b and 4c , it can be seen that type I collagen and fibronectin promote cell adhesion of NHGF and human osteosarcoma cell lines MG63 and HOS. Specifically, for NHGF, type I collagen promotes cell adhesion, while fibronectin does not. However, for human osteosarcoma cell lines MG63 and HOS, both type I collagen and fibronectin promoted cell adhesion.

Experimental Example 4 Cell Attachment Analysis

The titanium plate prepared in Example was placed in a 6 well plate, and 1.5 ml of Type I collagen (50 µg / ml) or fibronectin (1 µg / ml) was added and incubated overnight at room temperature to coat the ECM protein. 1.5 ml of PBS was added as a control and treated the same. The coated titanium plate was gently washed once with 2 ml of distilled water and then dried in air for 20-30 minutes.

Each cell was aliquoted at an amount of 5 × 10 ⁵ cells (2.5 mL) per well of the 6 well plate. Cell culture was performed for 30 minutes in a 37%, 5% CO ₂ incubator using DMEM + 10% FBS + 1% antibiotic as a culture medium. After the culture was removed, the cells were gently washed once with PBS, and then, 2 ml of 10% formalin in PBS was added thereto, and left at room temperature for 15 minutes. After washing twice with distilled water, 1% toluene blue in 2 ml of 10% formalin solution was added thereto, and left at room temperature for 1 hour.

The titanium plate was washed three times with distilled water. Shake each time to let the dye out of the bottom, and then lift and release the disc a little bit to get all the dye off the bottom of the plate. The titanium plate was transferred to a new 6 well plate and 1 ml of 2% SDS was added to lyse the cells for 15 minutes. 100 μl of the cell lysate was transferred to a 96 well plate (2 times each). Absorbance was measured at 595 nm with a plate reader (Micro plate reader Model 550, BIO-RAD). The results are shown in Fig.

As shown in FIG. 5 , it can be seen that the titanium surface-treated with type I collagen or fibronectin after heat treatment promotes cell adhesion of NHGF and human osteosarcoma cell lines MG63 and HOS, and is preferably surface treated with type I collagen after heat treatment. It can be seen that one titanium further promotes cell adhesion.

As described above, the implant surface treatment method of the present invention can further improve the surface hardness by treating the titanium or titanium alloy for implants with hydroxide apatite, and also the type of extracellular matrix protein to the titanium or titanium alloy surface I-coated implants, which are coated with collagen or fibronectin, promote the adhesion of osteoblasts to the bone-embedded area and promote the adhesion of fibroblasts to areas of the gingiva that can improve tissue healing. It can be usefully applied to implant abutments, implants that are in contact with the stagnation and gingival, and to one piece implants, which are integral to the implant fixture and abutments.

Claims (6)

Depositing titanium or titanium alloy for implant in an apatite hydroxide paste and drying (step 1); Heat-treating the titanium or titanium alloy for the implant (step 2); And The surface treatment method of the implant comprising the step of coating the surface by dipping the heat-treated implant titanium or titanium alloy in a type I collagen or fibronectin solution. The method of claim 1, wherein the concentration of the type I collagen solution of step 3 is 0.005 to 0.5% and the concentration of the fibronectin solution is 0.0001 to 0.5%. The method of claim 1, wherein the coating of step 3 is immersed in the type I collagen solution or fibronectin solution and maintained for 1 to 12 hours. The surface treatment method of an implant according to claim 1, wherein the heat treatment of step 2 is performed at 800 to 900 DEG C for 1 to 2 hours under an inert gas. The surface treatment method of an implant according to claim 1, wherein the inert gas is an argon gas or a nitrogen gas. The method of claim 1, wherein the method for treating the surface of the implant further comprises the step of washing after coating in step 3 and culturing by adding bovine serum albumin after washing.